Intestinal microRNA Response to Citrobacter rodentium in the Presence and Absence of

Bifidobacterium bifidum MIMBb75

by

Bijun Wen

A thesis submitted in conformity with the requirements for the degree of Mater of Science

Graduate Department of Nutritional Sciences University of Toronto

© Copyright by Bijun Wen 2015

ii

Intestinal microRNA Response to Citrobacter rodentium in the

Presence and Absence of Bifidobacterium bifidum MIMBb75

Bijun Wen

Master of Science

Graduate Department of Nutritional Sciences

University of Toronto

2015

Abstract

The continuous crosstalk between gut microbiota and the host epithelium is essential for

intestinal homeostasis. Presence of harmful or beneficial bacteria can impinge host gene

expression and affect homeostasis. Yet, the molecular basis underlying host-bacterial crosstalk is

unclear. The objectives of this project were to determine if pathogen infection impacts

microRNA-mediated posttranscriptional regulation of gene expression in mouse intestine and if

probiotic treatment improves pathogen-induced pathologies and microRNA alterations. It was

found that Citrobacter rodentium infection alters murine colonic microRNA signature with

implications in modulating host gene expression involved in the apoptosis pathway, contributing

to the epithelial hyperplasia in response to Citrobacter rodentium pathogenicity. Probiotic

Bifidobacterium bifidum MIMBb75 supplementation did not attenuate intestinal pathology nor

normalize microRNA alterations. Overall, these findings indicate that host-bacterial crosstalk is

potentially mediated by microRNA modulation, and this particular strain of Bifidobacterium

bifidum did not confer apparent benefits in Citrobacter rodentium infection.

iii

Acknowledgments

I would like to express my sincere gratitude to many individuals for their help and support

throughout my Master’s study.

First and foremost, I would like to thank my supervisor, Dr. Elena Comelli, for giving me the

opportunity to work on this intriguing research project. The knowledge and experience that I

have gained from this project are invaluable for a lifetime. All of these would not have been

possible if it were not for Elena’s continuous support. She has immense knowledge that helped

guide me throughout this research study. Elena, I would like to thank you for being such an

understanding, attentive and inspiring supervisor. I am especially grateful for the countless time

you spent providing prompt and insightful feedback on my questions, writings and presentations

even when you had a busy schedule. Although English is not my first language, you have always

been patient listening to my ideas and believed in my ability to present our data whether in your

undergraduate class or conferences. This is really motivating and important for me to build up

confidence for public speaking. I would also like to thank you for encouraging me to stay calm

and positive when I felt nervous and overwhelmed.

I would also like to thank my advisory committee members, Dr. Ward and Dr. Kim. Their advice

and enlightening questions at each committee meeting had provided me with new perspectives

and helped me in advancing my understanding about the project.

This project would not have been possible without the help from my lab members and I really

treasure the friendships that I have made with them. I would like to give a special thanks to Jim

Chen for helping me with the implementation of the intestinal permeability test and teaching me

how to make histology slides step by step. In addition, his kindness in offering suggestions

related to practical issues based on his profound experience in conducting animal studies will not

soon be forgotten. I would also like to thank our postdoctoral fellow, Amel Taibi, for teaching

me everything that I needed to know about microbiology and performing gene expression

experiments. She was such an attentive and caring person offering help whenever needed on her

own accord. I am especially delighted to have made a great friendship with Christopher Villa

over the years and I really appreciate his kind advice and comforting words when I was going

through the ups and downs of a Master’s student. His help with my project, improving my

iv

English, as well as setting up the computer before my seminars and presentations just reinforces

his caring and generous nature. I would also like to thank Monica Ponta, Sofia Sagaidak and

Alex Lee for their help on the project and being good friends when I needed it most. Moreover, I

would like to thank the department staff, Louisa and Emeliana for helping me with

administrative issues.

Lastly, I would like to take this opportunity to thank my family. Thank you, mom and dad, for

being so understanding and supportive throughout my life, especially for giving me all the

essentials required to ensure my successful completion of my education and career goals. The

unconditional love that my parents and my sister have given me has always contributed to my

desire of becoming a better person. I cannot use words to express how grateful I am and how

much I love you all.

v

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ............................................................................................................................ v

List of Tables ................................................................................................................................ vii

List of Figures .............................................................................................................................. viii

List of Abbreviations ..................................................................................................................... ix

Chapter 1 Introduction .................................................................................................................... 1

1 Introduction ................................................................................................................................ 1

Chapter 2 Literature review ............................................................................................................ 4

2 Literature Review ....................................................................................................................... 4

2.1 Intestinal Homeostasis ........................................................................................................ 4

2.1.1 Intestinal Epithelium Turnover ............................................................................... 5

2.1.2 Physical and Chemical Barriers .............................................................................. 7

2.1.3 Immune Barrier ....................................................................................................... 7

2.1.4 Gut Microbiota ........................................................................................................ 8

2.2 Foodborne Enterohemorrhagic E. coli .............................................................................. 11

2.2.1 EHEC Infection ..................................................................................................... 11

2.2.2 Citrobacter rodentium .......................................................................................... 12

2.2.3 EHEC in IBD ........................................................................................................ 14

2.2.4 EHEC in CRC ....................................................................................................... 15

2.3 Probiotics .......................................................................................................................... 16

2.3.1 Bifidobacterium bifidum ....................................................................................... 19

2.3.1.1 Adhesive Factors and Interaction with the Host ..................................... 20

2.3.1.2 Health Benefits ....................................................................................... 22

2.4 MicroRNA ........................................................................................................................ 31

vi

2.4.1 Response of Intestinal miRNA to Bacteria ........................................................... 33

2.4.2 MiRNA deregulations in IBD and CRC ............................................................... 42

2.5 Animal Model ................................................................................................................... 44

Chapter 3 Rationale, Hypothesis and Objectives .......................................................................... 46

Chapter 4 Study 1- Citrobacter rodentium Infection Alters Murine Colonic microRNA

Signature .................................................................................................................................. 48

4.1 Abstract .............................................................................................................................. 49

4.2 Introduction ........................................................................................................................ 50

4.3 Materials and Methods ....................................................................................................... 51

4.4 Results ................................................................................................................................ 56

4.5 Discussion .......................................................................................................................... 79

Chapter 5 Study 2- Effects of Bifidobacterium bifidum on Citrobacter rodentium Infection

via microRNA Modulation ...................................................................................................... 85

5.1 Abstract .............................................................................................................................. 86

5.2 Introduction ........................................................................................................................ 87

5.3 Materials and Methods ....................................................................................................... 88

5.4 Results ................................................................................................................................ 92

5.5 Discussion .......................................................................................................................... 99

Chapter 6 General Discussion ..................................................................................................... 103

6.1 Strengths, Limitations and Future Directions .................................................................. 105

6.2 Implications ...................................................................................................................... 108

6.3 Conclusions ...................................................................................................................... 110

References ................................................................................................................................... 111

vii

List of Tables

Chapter 2

Table 2.1 Clinical Health Benefits of B. bifidum .......................................................................... 26

Table 2.2 Impacts of Gastrointestinal Pathogens on Host microRNA Expression ....................... 36

Table 2.3 Impacts of Probiotics on Host Intestinal microRNA Expression ................................. 40

Chapter 4

Table 4.1 Differentially Expressed miRNAs ................................................................................ 60

Table 4.2 GO Biological Process (total number of genes: 824; total number process hits: 2158).

....................................................................................................................................................... 63

Table 4.3 GO Molecular Function (total number of genes: 824; total number process hits: 1062).

....................................................................................................................................................... 64

Table 4.4 GO Cellular Component (total number of genes: 824; total number process hits: 487).

....................................................................................................................................................... 65

Table 4.5 Panther Pathways (total number of genes: 824; total number process hits: 895). ........ 66

viii

List of Figures

Chapter 2

Figure 2.1 Colonic Cell Turnover and Transmissible Murine Hyperplasia .................................... 6

Figure 2.2 Models of miRNA-dependent Regulatory Network. ................................................... 32

Chapter 4

Figure 4.1 Study Design ............................................................................................................... 52

Figure 4.2 C. rodentium infection kinetics and effect on mouse body and organ weights. .......... 57

Figure 4.3 C. rodentium induced intestinal lesions, crypt hyperplasia and barrier dysfunction. .. 58

Figure 4.4 Loss of barrier integrity of C. rodentium infected mice on day 10 p.i. ....................... 59

Figure 4.5 C. rodentium infected mice exhibit distinct miRNA signature. .................................. 61

Figure 4.6 Enriched signaling pathways among miRNA-regulated gene targets. ........................ 73

Figure 4.7 Putative regulatory network of selected miRNAs. ...................................................... 75

Figure 4.8 Expression of selected genes in distal colon of sham and infected mice. ................... 76

Figure 4.9 Action of 11 differentially expressed miRNAs on Bim. ............................................. 78

Chapter 5

Figure 5.1 Study Design. .............................................................................................................. 89

Figure 5.2 C. rodentium infection kinetics and effect on mouse body and organ weights. .......... 93

Figure 5.3 Fecal B. bifidum load before and after infection. ........................................................ 94

Figure 5.4 B. bifidum effects on intestinal crypt hyperplasia and tissue damage at day 10 p.i.. .. 96

Figure 5.5 B. bifidum effects on intestinal barrier at day 10 p.i. ................................................... 97

Figure 5.6 Distal colon miRNA expression at day 10 p.i.. ........................................................... 98

ix

List of Abbreviations

A/E lesions-attaching and effacing lesions

ABC-ATP-binding cassette

Abcc3-ATP-Binding Cassette, Sub-Family C, Member 3

AIEC-adherent-invasive Escherichia coli

AJ-adherence junctions

B. bifidum-Bifidobacterium bifidum

Bim-B cell leukemia/lymphoma-2 interacting mediator

BopA-bifidobacterial outer protein

C. rodentium-Citrobacter rodentium

C1galt1-core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-

galactosyltransferase 1

CD-Crohn’s disease

Cdkn1a-cyclin-dependent kinase inhibitor

CFU-colony forming units

CRC-colorectal cancer

CT- cycle threshold

Cxcl1/2-chemokine (C-X-C motif) ligand 1/2

DMH-1,2-dimethylhydrazine

DSS-dextran sodium sulfate

E. coli-Escherichia coli

EAEC-enteroaggregative E. coli

EHEC-enterohemorrhagic E. coli

EIEC-enteroinvasive E. coli

EMT-epithelial–mesenchymal transition

Epas1-endothelial PAS domain protein 1

EPEC-enteropathogenic E. coli

EPK-Extracellular signal-regulated kinases

ETEC-enterotoxigenic E. coli

FASEB-Federation of American Societies for Experimental Biology

x

FOS-fructooligosaccharides

Gb3-globotriaosylceramides

GOS-galactooligosaccharides

GRO-growth-related oncogene

H&E-hematoxylin and eosin

H. pylori -Helicobacter pylori

HMOs-human milk oligosaccharides

IBS-irritable bowel diseases

IgE-immunoglobulin E

IL- interleukin

IBD-inflammatory bowel disease

IRAK1 – interleukin-1 receptor-associated kinase 1

L.-Lactobacillus

LB-Luria-Bertani

LEE-Locus of Enterocyte Effacement

LPS-lipopolysaccharide

M-CSF-macrophage colony-stimulating factor

MAMPs-microbial associated molecular patterns

MAPK-Mitogen-activated protein kinase

miR-microRNA (mature form)

miRNA-microRNA

mRNA-messenger RNA

NEC-necrotizing enterocolitis

NLRs-Nod-like receptors

p.i.-post-infection

PDCD4 -programmed cell death 4

pre-miRNAs-precursor miRNAs

pri-miRNAs-primary miRNAs

Prkcz -protein kinase C zeta isoform a

PRRs-pattern recognition receptors

PTEN -phosphatase and tensin homolog

RAC2-Ras-related C3 botulinum toxin substrate 2

xi

RCTs-randomized controlled trials

REST-Relative Expression Software Tool

RhoB -Ras Homolog Family Member B

RISC-RNA-induced silencing complex

Rnd3- Rho family guanosine triphosphate-ase 3

S.-Saccharomyces

SCFAs-short chain fatty acids

Ship1 -Src homology2 domain-containing inositol phosphatase

Stx-Shiga toxin

T3SS-type III secretion system

Tab2 -TGF-beta activated kinase 1/MAP3K7 binding protein 2

Tad-tight-adherence

TA-transit amplifying

Th-T helper

Tir-translocated intimin receptor

TJ-tight junctions

TLRs-Toll-like receptors

TMCH-transmissible murine colonic hyperplasia

TNBS-trinitrobenzene sulfonic acid

TRAF6-Tumor necrosis factor receptor-associated factor 6

Treg-regulatory T

UC-ulcerative colitis

UTR-untranslated region

Wnt-Wingless

Zeb1/2 -zinc finger E-box binding homeobox 1/2

1

Chapter 1

Introduction

1 Introduction

Gastrointestinal diseases, including both acute and chronic conditions such as infectious diarrhea

and inflammatory bowel disease (IBD), account for considerable economic and healthcare

burden in Canada. According to the Canadian Communicable Disease Report, there are

approximately 1.3 cases per-person year of acute gastrointestinal diseases , with symptoms of

vomiting and diarrhea, translating to an annual economic cost of 3.7 billion dollars [1].

Foodborne enteric pathogen infection is the major cause of acute gastrointestinal diseases with

the estimated occurrence of 11 million episodes every year in Canada [1]. Verotoxigenic

Escherichia coli (E. coli), Salmonella, and Campylobacter are the most common bacterial agents

responsible for foodborne diseases [2]. In contrast, IBD is a chronic recurrent condition affecting

over 250,000 Canadians, which represents one of the highest prevalence rates around the world

[3]. In 2012, the economic costs of IBD were about 2.8 billion dollars in Canada. Patients suffer

from both direct and indirect long-term consequences, including work losses and lower quality of

life compared to the general population [4]. Notably, enteric infection episodes and IBD are

linked to the development of colorectal cancer (CRC) [5-7], which is the third most common

diagnosed cancer and the fourth most common cause of cancer death worldwide [8, 9].

The gut microbiota is a complex community of trillions of microorganisms. Alteration of the gut

microbiota composition (dysbiosis) and disruption of its crosstalk with the host have pivotal

implications in these diseases. Fecal microbiota analysis revealed that IBD patients experience

dysbiosis with increased abundance of Proteobacteria and decreased abundance of Bacteroidetes

at the phylum level [10, 11], as well as reduced counts of bacteria belonging to the

Bifidobacterium genus [12]. Pathogens have capacity to breach the host defense mechanisms and

disrupt the healthy host-microbial crosstalk. Enterohemorrhagic E. coli (EHEC) O157:H7 is one

of the most common foodborne enteric pathogens responsible for infectious diarrhea outbreaks

and acute hemorrhagic colitis around the world [13]. Citrobacter rodentium (C. rodentium), a

2

murine-specific pathogen adopts the same virulence mechanisms as EHEC and is commonly

used to model EHEC infection. Colonization of C. rodentium can cause prominent dysbiosis as

showed by the reduction of overall microbiota diversity. Up to 90% of the microbiota can be

replaced by the pathogen at the peak of infection [14, 15]. This is accompanied by intestinal

barrier dysfunction, robust immune responses, as well as colonic crypt hyperplasia, which is the

hallmark of C. rodentium infection. As these features resemble some of the pathological

manifestation of IBD and colonic tumorigenesis, C. rodentium infection has also been

extensively used to study IBD and colorectal cancer development [15].

Probiotics, live microorganisms which when administered in adequate amounts confer a health

benefit on the host [16], contribute to maintenance and restoration of intestinal homeostasis.

Bifidobacterium bifidum, an indigenous member of the human microbiota and a common

probiotic, was shown to confer health-promoting effects in many conditions upon consumption

including, but not restricted to, infectious diarrhea [17-19], necrotizing enterocolitis (NEC) [20],

and irritable bowel syndrome [21-23]. This withstanding, results have not been always consistent

across clinical studies hence recommendation in clinical practice cannot be made yet. Functional

studies also revealed that B. bifidum use can improve gut microbiota composition [24], enhance

barrier function [25-27], modulate host immunity [28-30] and exert antimicrobial activity. B.

bifidum has also been implicated in EHEC infection and IBD. B. bifidum ATCC 29521 has been

shown to interfere with EHEC attachment and colonization in vitro [31]; and oral administration

of B. bifidum S17 alleviated intestinal inflammation in mice with chemically-induced IBD [32].

However, no study has examined the effect of B. bifidum in C. rodentium infection.

Although the host-microbial crosstalk is essential for intestinal homeostasis, the molecular basis

underlying both harmful and beneficial bacteria-host interaction is not fully understood. It is

known that the interaction between host and microorganisms can significant impact host gene

expression [33]. Genome-wide transcriptome analyses revealed that both C. rodentium infection

and B. bifidum colonization can impinge host gene expression in the intestine [30, 34, 35]. In

addition, recent reports show that bacteria can also affect the expression of intestinal microRNAs

(miRNAs). MiRNAs are non-coding RNA molecules, which function post-transcriptionally to

fine-tune gene expression. Currently, 2,588 miRNAs in humans and 1,915 miRNAs in mice have

been identified (miRBase release 21, June 2014) [36]. Previous studies in our research group

have discovered that the presence of microbiota can impact murine miRNA with potential

3

implications in intestinal barrier gene regulation [37]. Administration of B. bifidum MIMBb75 to

mice for two days also affected miRNA expression in the caecum. Meanwhile, recent studies

have demonstrated that various pathogen infections, such as Listeria monocytogenes [38]

Salmonella [39] and Campylobacter concisus [40], can impinge host mucosal miRNA signature.

Interestingly, a study used sterile (germ-free) and conventional (harboring a normal microbiota)

mice and showed that the presence of the microbiota influences the intestinal miRNA response

to Listeria infection [38]. This suggests that the interaction between host and bacteria in the gut

is regulated by miRNAs. However, molecular mechanisms of host-bacteria crosstalk via miRNA

modulation have not been comprehensively investigated in the frame of pathogen-host and

probiotic-host interaction.

The aim of the present study was to investigate if intestinal miRNA are altered in response to C.

rodentium infection and if this response is modulated by probiotic B. bifidum MIMBb75

resulting in mitigation of intestinal inflammation.

4

Chapter 2

Literature review

2 Literature Review

2.1 Intestinal Homeostasis

The intestinal tract comprises the small (duodenum, jejunum, ileum) and large (caecum, colon,

rectum) intestine and is the primary site of nutrient, water, and electrolyte transport; this entails a

series of processes including digestion, absorption, motility, and secretion. The intestinal tract

wall consists of four layers, including the mucosa, submucosa, muscularis externa, and serosa

along the transversal axis. The intestinal mucosa comprises three layers, the epithelium

monolayer facing the lumen, the subepithelial lamina propria and the muscularis mucosae.

Numerous folds are formed in the inner surface of the mucosa along the lumen. Villi (finger-like

luminal protrusions) and microvilli (finger-like structure on brush border membrane of epithelial

cells) are formed in the small intestine, which enhances effectiveness of nutrient absorption;

crypts (flask-shaped submucosal invaginations) are formed in both the small and large intestine

and function as proliferative and secretory compartments of the intestinal epithelium. The

submucosa is a layer of connective tissue, where nerves, blood and lymph vessels are located.

The muscularis externa and serosa are made up of smooth muscle and connective tissue

constituting the outer wall of the intestinal tract, which is responsible for motor activity. Besides

its role in nutrient absorption, the intestinal tract is also the largest immune organ of the body.

The intestinal tract is constantly confronted with dietary and bacterial antigens from the external

environment and is in a symbiotic relationship with the resident microbiota. In order to maintain

homeostasis, the intestinal tract has developed the capacity to distinguish self from non-self and

plays a critical function in preventing harmful compounds or pathogens from entering the

internal system. This is achieved through a complex line of defense that includes epithelium

turnover as well as physical, chemical, and immunological barriers.

5

2.1.1 Intestinal Epithelium Turnover

The intestinal epithelium has a turnover rate of about 5 days in humans and 3 days in mouse,

which is thought to be essential for the timely removal of harmful substances and pathogens [41].

This continuous self-renewal is sustained by proliferation and differentiation of multipotent stem

cells located at the base of the crypt (Figure 2.1, panel A). Upon stimulation, multipotent stem

cells divide into daughter cells known as transit amplifying (TA) cells, which can proliferate

rapidly. Proliferation is driven by the Wingless (Wnt)/β-catenin signalling pathway, which is

initiated by the ligand binding of the secreted glycoprotein Wnt to its membrane-bound

receptors. The activation of this signalling cascade leads to accumulation of β-catenin in the

cytoplasm by sequestering proteins responsible for β-catenin degradation, including APC and

Auxin. As cytoplasmic β-catenin increases, it translocates into the nucleus acting as a

transcriptional factor that promotes proliferation [41]. After a limited number of divisions, the

TA cell population expands to the midway of the crypt and gradually differentiates into epithelial

cells. Absorptive enterocytes represent 90% of the differentiated cells; other cell types include:

goblet cells (secreting mucus), paneth cells (producing antimicrobial peptides and enyzmes),

enteroendocrine cells (secreting hormones), and microfold cells (M cells, sensing antigens). The

distribution and abundance of the diverse cell types is region specific along the intestine. For

example, paneth cells and M cells are predominately found in the small intestine, whereas goblet

cells are abundant in the large intestine [42]. The self-renewal process is completed as

differentiated epithelial cells migrate toward the top of the villus (small intestine) / crypt (large

intestine), where programmed cell death and shedding take place. Therefore, epithelial turnover

requires a tight balance between proliferation, differentiation, apoptosis and exfoliation

processes, and ensures maintenance of epithelial barrier and absorptive functions, contributing to

intestinal defense and homeostasis.

6

Figure 2.1 Colonic Cell Turnover and Transmissible Murine Hyperplasia

This figure illustrates (A) cell turnover in the colon under homeostasis and (B) during C. rodentium infection. (A)

Stem cells, located at the base of the colonic crypt, divide into hyperproliferative TA cells under stimulation of the

signaling molecule Wnt. After a few divisions, TA cells differentiate into various epithelial cells such as absorptive

enterocytes and goblet cells, which produce a thick mucus layer separating the epithelium from the colonic lumen.

When epithelial cells migrate to the top of the crypt, they undergo programmed cell-death and are removed by cell-

shedding. (B) C. rodentium infection is marked by dramatic crypt hyperplasia in the colon known as transmissible

murine colonic hyperplasia (TMCH). The expression of the type III secretion system allows C. rodentium to form

tight attachment onto the host epithelial cell, resulting in host mucosal damage known as attaching and effacing

lesions (A/E lesions), characterized by effacement of microvilli on enterocytes and formation of pedestal-like

structure beneath the attachment site. A/E lesion prevents epithelial cells from shedding. Inflammation is triggered

by the activation of membrane-bound Toll-like receptors (Tlr2/4) and the cytosolic Nod-like receptors (Nod1/2),

which in turn activate the NF-kB signaling cascade, leading to proinflammatory cytokine production that drives Th1

and Th17-dominated responses. Although not completely clear, NF-kB activation leads to over-activation of Wnt

along with other uncharacterized signals, which inhibit hyperproliferative TA cells from differentiating. The

accumulation of undifferentiated cells at the base of the crypt and inhibition of cell-shedding at the top of the crypt

lead to dramatic crypt elongation. The increase of TA cell population eliminates goblet cells, impairing barrier

function of the mucus layer. The impairment of epithelial barrier integrity allows paracellular translocation of

bacterial cells in the system, modified from [15].

7

2.1.2 Physical and Chemical Barriers

In addition to epithelium turnover, several lines of defense to physically and chemically protect

the internal environment from foreign insults are in place to maintain homeostasis. The first is a

physical barrier created by the mucus covering the epithelium. This comprises two layers; an

inner layer, devoid of bacteria and adhering tightly to the epithelium, and an outer layer which is

thicker (830 vs. 116 μm in the human colon) and harbors commensal microorganisms [43].

Mucins produced by goblet cells are the main constituent of the mucus layers. They are heavily

glycosylated proteins that contribute to the viscoelastic properties of the mucus. Oligosaccharide

side chains on mucins may serve as attachment sites and carbon source for bacteria, especially

for commensal bacteria [44].

Intestinal epithelial cells are kept together by tight junctions (TJ), adherence junctions (AJ) and

desmosomes. TJs are intercellular protein complexes made up by transmembrane claudins,

occludin, tricellulin, and intracellular zona occludens and F-actin. They are anchored at the

apical side of the epithelium, sealing the gap between adjacent cells and preventing paracellular

translocation of luminal substances. A dynamic model of TJs has been proposed, indicating that

they can open and close upon stimulation [45]. AJs are made up of transmembrane E-cadherin,

and intracellular β-catenin and α-catenin. Both AJs and desmosomes are located beneath the TJ

and are responsible for strengthening cell-to-cell adhesion [46].

Chemical protection is provided by antimicrobial peptides secreted by paneth cells (for example,

α-defensins and lysozyme) and enterocytes (for example, β-defensins and cathelicidins). These

compounds have cationic regions that can interact with negatively charged phospholipids on the

membrane of both Gram-positive and Gram-negative bacteria, resulting in depolarization and

structural damage of the bacterial membrane [47].

2.1.3 Immune Barrier

Some bacteria can breach the physical and chemical barriers and come in close contact with the

epithelium. The intestinal epithelium has an immunological surveillance system to recognize and

respond to these challenges in order to maintain homeostasis. The activation of the innate

immune response via pattern recognition receptors (PRRs) plays a pivotal role in this

surveillance system [48]. PRRs are glycoprotein receptors specialized in recognizing

8

evolutionarily conserved structures of microbes, known as microbial associated molecular

patterns (MAMPs). Examples of MAMPs include lipopolysacharide (LPS) of Gram-negative

bacteria, flagellin, and peptidoglycans components of the bacterial cell wall [49]. There are two

classes of PRRs, the membrane-bound Toll-like receptors (TLRs) and the cytosolic Nod-like

receptors (NLRs). Ligand binding of MAMPs to PRRs activates NF-κB signalling cascades,

triggering production of proinflammatory cytokines and chemokines for bacterial elimination.

Interestingly, although MAMPs are common in both resident and pathogenic bacteria, the

epithelium has the ability to discriminate between the two. Unlike pathogenic bacteria, which

can invade deep into the host tissue, commensals are localized on the mucosal surface [48]. It has

been found that TLR5 (recognizing flagellin) expression is lower in the apical than the

basolateral membranes of the epithelial cells [50]; TLR2 (recognizing peptidoglycan) and TLR4

(recognizing LPS) are generally down-regulated in epithelial cells [51, 52]. Thus, direct contact

of commensal-derived MAMPs with PRRs is reduced. Nevertheless, immune response toward

commensal bacteria is critical for gut homeostasis. This is well-evidenced by studies showing

that inhibition of NF-κB activation in epithelial cells leads to spontaneous colitis in conventional

mice [53]; also dextran sodium sulfate (DSS)-treated mice deficient in TLR4 or Myd88, an

adaptor necessary for TLR activation, have reduced inflammatory response but increased colitis

and bacterial translocation across the epithelium [54]. NF-κB-induced cytokine production plays

an important part in the polarization of cell-mediated immune response, which involves

differentiation of naive T-lymphocytes into functionally distinct subtypes, such as

proinflammatory T helper (Th) 1, Th2, and Th17 cells, and anti-inflammatory regulatory T (Treg)

cells [55]. It is believed that constant interaction between commensal bacteria and host

epithelium promotes a balance of Treg versus Th1 or Th17 response [56-58]. Therefore, under

homeostasis the gut attains tolerance toward resident bacteria, and immunological readiness

against pathogenic attack.

2.1.4 Gut Microbiota

The human gastrointestinal tract harbours a complex community of microorganisms from all

three domains of life including bacteria, archaea and eukarya, collectively known as the gut

microbiota. The gut microbiota is a diverse and dynamic ecosystem, with approximately 1014

microbe cells encompassing over 100 times more genes (microbiome) than the human genome

[59, 60]. The gut microbiota is dominated by the phyla of Bacteroidetes (B), Firmicutes (F),

9

Proteobacteria (P), and Actinobacteria (A), and by species of the genera Bacteroides (B),

Clostridium (F), Lactobacillus (F), Escherichia (P) and Bifidobacterium (A) [60, 61]. Microbial

density and diversity vary along the horizontal and longitudinal gut axes. Specifically, bacterial

numbers, expressed as colony forming units (CFU)/gram of gut content, increase from 103 in the

stomach and 104-10

7 in the jejunum and ileum to 10

11-10

12 in the colon, which is the most

heavily colonized region of the gastrointestinal tract [60]. The upper gastrointestinal regions are

predominantly occupied by aciduric bacteria such as lactobacilli and streptococci, whereas

colonic regions are preferential sites for facultative and obligate anaerobes such as

Enterobacteria, Bacteroides and Bifidobacterium [62]. Despite the diverse and dynamic nature

of the gut microbiota, it is relatively resilient to changes. Alteration of microbiota, known as

dysbiosis, is associated with intestinal disorders, including IBD [12, 63] and CRC [64], although

it remains unclear whether dysbiosis is the cause of effect of these disorders. Specifically,

pyrosequencing analysis of fecal microbiome from twin pairs, where only one twin developed

IBD, disclosed significant microbial composition alterations in IBD (Crohn’s disease subtype)

patients, which showed an increase abundance of Firmicutes and Proteobacteria at the phylum

level compared to their healthy twin. The increase of Proteobacteria is primarily attributable to

increased representation of the Enterobaceriaceae family, which mainly consists of E. coli [10].

In line with this, presence of adherent-invasive E. coli (AIEC) was more frequently identified in

IBD patients than healthy controls [65]. Likewise, another twin-study revealed that IBD

(ulcerative colitis subtype) patients had a general reduction of mucosal microbiota diversity with

a higher proportion of Proteobacteria and less Bacteroidetes compared to their IBD-free twin

[11]. In CRC, the microbiota composition at genus level showed clear separation between CRC

patients and healthy controls, and the Bacteroides/Prevotella group was significantly increased

in patients [64].

Maintenance of a stable microbiota is essential for intestinal homeostasis. Indeed, the continuous

interaction between gut microbiota and the host imparts many beneficial effects, such as,

providing nutrients and vitamins [66], regulating host’s energy balance [67], protecting against

pathogens [68], and educating the intestinal immune system [69]. For example, bacterial

biosynthesis and fermentation can result in production of vitamins (folic acid, vitamin B and K)

as well as short chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, which can be

utilized by the host as nutrient and energy sources [66, 67]. Gut microbiota resilience is critical

10

for colonization resistance to pathogens, providing protection to the host intestine. It has been

shown that germ-free and antibiotic-treated mice were more susceptible to pathogen infections,

such as C. rodentium [70], Listeria monocytogenes [71], and Salmonella enterica [72] than

conventional mice. Commensal bacteria are able to exclude pathogens from the mucosa via

secretion of antimicrobial peptides, competition for nutrients and attachment sites, and quorum

sensing [73]. These same mechanisms are also utilized by probiotic (beneficial) bacteria. For

example, E. coli Nissle 1907 can compete with Salmonella for iron and reduce Salmonella

colonization in mice [74]. Microorganisms utilize quorum sensing to regulate gene expression

based on population density via the secretion of compounds referred to as autoinducers [75]. It

has been shown that autoinducers produced by resident Ruminococcus obeum reduce

colonization of a diarrhea-causing pathogen, Vibrio cholera, in gnotobiotic mice associated with

an artificial bacterial community resembling the healthy human microbiota [76]. Some pathogens

have evolved colonization strategies that take advantage of quorum sensing. The expression of

the pathogenicity island Locus of Enterocyte Effacement (LEE) of EHEC is activated by a

quorum sensing molecule produced by the microbiota resulting in the formation of tight

attachment to intestinal cells [77]. Furthermore, commensal bacteria are able to reinforce the host

defense barrier by constantly shaping and priming the mucosal immune system. It has been

shown that resident microbes have both proinflammatory and anti-inflammatory effects on the

host mucosa to keep activities of different subsets of T-cells in balance. For example, the

resident microbe Candidatus Savagella (also known as segmented filamentous bacteria),

stimulates a pro-inflammatory Th17 response, which is crucial in fighting against pathogen

infection and is associated with colitis when overly activated [78, 79]. In contrast, other

commensal bacteria, such as Bacteroides fragilis, can downregulate Th17 response and induce

Treg response, thereby promoting immune tolerance [80]. Hence, the gut microbiota is essential

in maintaining physiological inflammation of the host mucosa.

11

2.2 Foodborne Enterohemorrhagic E. coli

Pathogenic bacteria have evolved strategies to escape host defenses and come in close contact

with host epithelium in order to survive and replicate. Although E. coli is the predominant non-

pathogenic facultative anaerobe found in the normal gut microbiota, some strains of E. coli have

acquired pathogenic properties to cause diarrheal disease [81]. These diarrheagenic E. coli strains

are categorized into five classes depending on their virulence. These include enteropathogenic E.

coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive

E. coli (EIEC), and EHEC. E. coli O157:H7, a serotype belonging to EHEC, is a foodborne

enteric bacterial pathogen responsible for most cases of infectious diarrhea outbreaks around the

world [13]. This infection can also lead to hemorrhagic colitis, hemolytic uremic syndrome, and

even death due to renal failure in children [13]. It is highly transmissible between persons via the

oral-fecal route and is usually acquired through consumption of ruminant feces-contaminated

drinking water and food, such as undercooked ground beef, fruits and vegetables. The largest

outbreak in Canada took place in Walkerton, ON in 2000 as a consequence of contaminated

drinking water resulting in over two thousand cases of infection and 7 deaths [82]. It was found

that infected individuals had an increased risk of developing a chronic condition known as post-

infectious irritable bowel syndrome (IBS) two years after the Walkerton outbreak [83]. Although

no correlation was observed between EHEC infection and other chronic disorders such as IBD

and CRC, viral and bacterial gastrointestinal infection have been suggested to play a role in the

development of these chronic disorders [84, 85]. The following sections will focus on addressing

pathogenesis and animal model of EHEC infection and its potential links to IBD and CRC.

2.2.1 EHEC Infection

EHEC infection causes a characteristic damage to the host intestine, known as A/E lesions [86].

A/E lesions are characterized by the intimate bacterial attachment onto the host epithelium,

effacement of intestinal brush border microvilli and formation of pedestal-like structures beneath

the attachment site [15, 87]. Virulence genes controlling the establishment of A/E lesions of

EHEC are located on the pathogenicity island, called LEE, which encodes a type III secretion

system (T3SS) [87]. The T3SS allows the bacteria to secret effector proteins into the host cell via

the formation of a needle-like channel breaching through the host membrane. One of the central

secreted effectors is the translocated intimin receptor (Tir), which can be incorporated onto the

host membrane surface, serving as a receptor for the bacterial cell surface adhesin protein called

12

intimin. This ligand-receptor binding ensures firm adhesion between the bacteria and the host

epithelium [15]. Other effector proteins such as EspG, EspF and MAP stay in the epithelial

cytosol and are able to hijack the host cell machinery to cause damages to the host cell, including

actin-cytoskeleton rearrangement [88, 89], tight junction defects[90, 91], solute transport

dysfunction [92, 93], innate immunity activation [94, 95], and cell-cycle disruption [96-99]. The

rearrangement of actin-cytoskeleton allows formation of the pedestal-like structure beneath the

attachment site, increasing the proximity between the bacterial cell and host epithelium. The

consequences of these pathologic actions are manifested as increased intestinal permeability,

electrolytes imbalance, crypt hyperplasia (details in section 2.2.2), and inflammation (details in

section 2.2.3) [15]. Moreover, E. coli O157:H7 has the ability to release the potent hemolytic

Shiga toxin (Stx) upon infection [100]. Stx is an AB toxin originated from bacteriophages. The

globotriaosylceramides (Gb3) receptor expressed on the intestinal epithelial cell surface can bind

to the B subunit of Stx triggering epithelial internalization of the toxin. Once the A subunit is

inside the host cell, it inhibits protein synthesis, causing local cell death in the intestine and

kidney damages via blood circulation. It has been suggested that Stx can also enter the host

system in a Gb3-receptor independent manner [101]. Treatment of EHEC remains challenging,

since antibiotic treatment augments Stx release upon bacterial cell death.

2.2.2 Citrobacter rodentium

C. rodentium is a Gram-negative murine-restricted pathogen. With 67% shared genetic

similarities with EHEC, C. rodentium also harbours and expresses LEE, and thereby, adopts the

same virulence mechanisms as EHEC [102]. Although C. rodentium does not process the Stx

gene, a genetically engineered Stx-expressing C. rodentium strain was recently constructed

[103]. C. rodentium infection has been extensively used to model EHEC infection. The

establishment of this animal model is usually achieved by orally inoculation of mice with live

cells of C. rodentium, which results in a highly reproducible infection cycle in mice of the same

strain [104-106]. In C57Bl/6 mice, the initial site of C. rodentium colonization is in the caecum;

but about 2-3 days post-infection (p.i.) C. rodentium re-localizes and predominantly colonizes

the colon, especially the distal colon. The peak of bacteria load is reached by day 8-10 with over

109 CFU/g of feces. However, complete bacterial clearance takes about 2 – 3 weeks after

inoculation [104, 105]. Colonization of C. rodentium causes significant dysbiosis. C. rodentium

infected mice had a 3-fold reduction of resident bacteria counts and percentages of Bacteroides

13

and γ-Proteobacteria were significant decreased and increased, respectively, on day 7 p.i. [14].

However, infection kinetics can vary between different mouse strains. NIH Swiss and C57Bl/6

mice are resistant strains that can recover from the infection, while C3H/HeJ mice are a

susceptible strain that suffers 100% mortality by day 10 p.i [107]. Compared to resistant strains,

C3H/HeJ mice were rapidly and heavily colonized by C. rodentium (109 vs. 10

4 CFU/g on day 4

p.i.), with greater mucosal damage and bacterial systemic translocation. Recent studies suggested

that transferring microbiota from resistant mice to C3H/HeJ mice reduces C. rodentium

colonization, colonic pathology and mortality [108, 109]. Ghosh et al. reported that the

microbiota of C3H/HeJ had a lower abundance of Bacteroidetes compared to that of C57Bl/6

mice, and receiving C57Bl/6 microbiota significantly increased C3H/HeJ Bacteroidetes load and

attenuated infection pathology, implying that the protective effects of resistant microbiota might

be attributable to Bacteroidetes abundance [108]. Using similar study approaches, Willing et al.

suggested that the protective effects of resistant microbiota was associated with modulating host

immunity toward Th17 response, as transferring susceptible mouse microbiota to resistant host

reduced this defensive response [109]. One study on germ-free C57Bl/6 mice found that germ-

free mice had a 10-fold increase of peak C. rodentium load with a delayed bacterial eradication

until after 42 days p.i; however there is no significant difference in tissue pathology or mortality

between germ-free and convention mice [110]. Interestingly, another study found that antibiotic-

induced dysbiosis exacerbated colon pathology but has no impact on C. rodentium colonization

in conventional mice [70]. These findings indicate that microbiota is involved in regulating host

susceptibility to C. rodentium infection, but not necessarily via pathogen exclusion.

The hallmark of C. rodentium infection is TMCH (Figure 2.1, Panel B) [15]. During C.

rodentium infection, there is an excessive proliferation of undifferentiated enterocytes (ie. TA

cells) in the colonic crypt, as well as inhibition of cell shedding at the top of the crypt due to the

tight bacterial attachment, which together lead to dramatic colonic crypt elongation, goblet cell

depletion and mucosal thickening, collective known as TMCH [15]. The initiation of TMCH is

believed to be associated with NF-κB activation, as MyD88-deficient mice did not develop

TMCH upon C. rodentium infection [111, 112]. Indeed, microarray analyses of colonic

transcriptome revealed pronounced alterations in expression of genes related to immune response

and cell-cycle regulation in C. rodentium-infected mice compared to controls, indicating C.

rodentium pathogenicity can impact host gene expression [34, 35]. For example, Spehlmann et

14

al. found that C. rodentium infection resulted in a significant induction of genes encoding for

chemokines production in the colon, such as chemokine (C-X-C motif) ligand 1/2 (Cxcl1/2),

which are potent chemokines invoking mucosal neutrophil influx and contribute to host defense

against pathogens [35]. Borenshtein et al. found that Aquaporin 8, encoding for a water channel

protein, was one of the prominent downregulated genes during C. rodentium infection [34]; it

was showed before that this gene was highly expressed in differentiated epithelial cells but

weakly in colorectal tumor tissue[113].

2.2.3 EHEC in IBD

IBD is a chronic intestinal disorder, characterized by sustained bowel inflammation, intestinal

barrier dysfunction, abdominal pain and diarrhea [114]. There are two main forms of IBD,

Crohn’s disease (CD) and ulcerative colitis (UC), distinguished mainly by histopathological

features. Notably, CD is associated with a patchy and transmural intestinal inflammation

dominated by Th1 responses, while UC demonstrates a continuous and shallow inflammation

dominated by Th2 responses [115]. There is an increase incidence and prevalence of IBD

worldwide, especially in North America, where an estimated 1.4 million people are affected by

IBD [116]. Although genetic, immunologic, and environmental factors have been identified, IBD

is a multifactorial disorder of unknown etiology [115]. There is no direct evidence showing

association between EHEC infection and IBD; epidemiology studies have suggested a link

between acute enteric infections and IBD, where the risk of developing IBD is increased after

enteric bacterial infection episodes [84, 117]. In addition, Qiu et al. revealed that IBD patients

have a higher serum level of EHEC-derived bacterial toxins compared to healthy controls [118].

It has been hypothesized that enteric infection may act as an initiation factor triggering chronic

inflammation in people genetically susceptible to IBD [119].

Because IBD is characterized by intestinal inflammation, common models of IBD are established

by using genetic modification (IL-10 knock out) and chemical reagents, such as DSS and

trinitrobenzene sulfonic acid (TNBS) to induce colitis in mice [115]. C. rodentium infection is

also extensively used as a biological reagent to model IBD [120, 121]. Mice infected with C.

rodentium elicit a Th1 profile that resembles the immune response seen in CD [121].

Specifically, upon colonization of the intestine C. rodentium-derived MAMPs are recognized by

TLR2, TLR4, as well as NOD1 and NOD2, followed by activation of NF-κB signalling, which in

15

turn induces production of proinflammatory cytokines, TNF-α, IFN-γ, IL-12 that characterize the

Th1 response [120, 122, 123]. A robust Th1 response can recruit lymphocytes and macrophages

to the infection site for pathogen control, resulting in significant immune cell infiltration into the

host mucosa. Besides sharing immunologic similarities with IBD, dysbiosis caused by C.

rodentium infection is also comparable to IBD. For instance, the increase of the proportion of

Proteobacteria in the microbiota found in C. rodentium infected mice is commonly seen in IBD

patients [124-126]. In fact, it has been suggested that infection-induced dysbiosis may lead to

hyperactive T-cell mediated immune response in genetically predisposed individuals,

contributing to persistent inflammation [127, 128]. Intriguingly, Fattouh et al. showed that

knockout of Ras-related C3 botulinum toxin substrate 2 (RAC2), a gene associated with IBD in

humans, attenuates C. rodentium-induced colitis in mice, which implies a role of RAC2 in IBD

pathogenesis [129]. Thus, understanding C. rodentium-induced colitis may contribute to

elucidation of the pathogenesis of IBD.

2.2.4 EHEC in CRC

CRC is the third most common diagnosed cancer and the fourth most common cause of cancer

death worldwide [8, 9]. Similar to IBD, it is more prevalent in Western countries. Genetic

predisposition [130], diet [131], and history of IBD [5] have been identified as some of the risk

factors associated with CRC. Recently, enteric pathogen infections have been implicated in the

etiology of CRC [6, 7]. Being one of the most common causes of enteric infection, EHEC has

been shown to be associated with CRC pathogenesis [97, 132-135]. Two independent studies

demonstrated that E. coli O157:H7-derived components have genotoxic effects and can lead to

genomic instability [133, 134]. This is important because chromosomal instability is a

characteristic observed in about 80% of CRC [136]. Specifically, Tyrer et al. reported that

exposure of Caco-2 and HCT116 cell lines to E. coli O157:H7-derived components including Stx

causes DNA double-strand breaks associated with cell cycle arrest, leading to an increase of cell

proliferation [133]. Similarly the expression of a tumor suppressor gene p53 was increased in

heat-killed E. coli O157:H7 treated mouse intestine, suggesting an activation of repair

mechanisms of DNA double-strand break [134] and dysfunction of p53 is associated with cancer

development including CRC in humans [137]. However, currently there is a lack of human

studies substantiating a link between EHEC and CRC. In fact, C. rodentium has recently

emerged as a model for studying colonic tumorigenesis. It has been shown that C. rodentium

16

infection promotes tumorigenesis in carcinogen-treated and genetically susceptible (ApcMin/+

heterozygous) mice [138, 139]. As discussed above, the hallmark of C. rodentium infection is

TMCH, which is believed to be associated with NF-κB signaling activation with the precise

mechanisms uncharacterized [15, 111, 112]. Tight regulation of the Wnt/β-catenin signalling

pathway is critical to the equilibrium of epithelial cell proliferation and differentiation, where

over-activation contributes to CRC development [41]. Mutation in Apc gene, encoding protein

that inactivates β-catenin, is seen in 80% of sporadic CRC [140]. Recent study by Chandrakesan

et al. revealed that elevation of active β-catenin and reduction of APC levels coincide with

increase of crypt hyperplasia, while inhibition of β-catenin activity leads to decrease hyperplasia

in C. rodentium infected mice, indicating a central role of Wnt/β-catenin activation in bacteria-

driven hyperplasia [141]. Interestingly, the interplay between NF-κB and Wnt/β-catenin in

controlling TMCH has been evidenced, although not fully understood [141, 142]. Moreover,

intestinal hypoxia and activation of the Wnt/β-catenin are essential for triggering epithelial–

mesenchymal transition (EMT), which is a key feature of cancer metastasis. It has been

demonstrated that C. rodentium infection induces a hypoxic state, and transforms cultured

enterocyte into fibroblast-like mesenchymal cells, which undergo significant epigenetic profile

changes such as histone modification and chromatin remodeling [143]. In summary, C.

rodentium-induced hyperplasia is an important model for studying the molecular events

underlying tumorigenesis and the early-onset of EMT.

2.3 Probiotics

Probiotics are defined as “live microorganisms which when administered in adequate amounts

confer a health benefit on the host” by the World Health Organization and Food and Agriculture

Organization [16]. Based on this definition, in order for a microorganism to be considered as a

probiotic, it must be capable to survive through the gastrointestinal tract, colonize and proliferate

in the gut, be safe and effective in humans, as well as remain viable and effective during product

shelf-life [144]. Probiotics can be found in various foods, including some fermented products,

nutritional supplements, and medical foods constituting a considerable part of the functional food

market [145]. Bacteria of the Bifidobacterium and L. (Lactobacillus) genera are the most

commercially available probiotics for humans [146]. Bifidobacterium species including B.

adolescentis, B. animalis, B. bifidum, B. breve and B. longum, and L. species including L.

acidophilus, L. casei, L. fermentum, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L.

17

rhamnosus and L. salivarius with a dosage of 109 CFU per food serving are the species for which

general probiotics claims are currently accepted by Health Canada [147]. Other bacterial species

from Enterococcus, Escherichia, Streptococcus genera and yeast Saccharomyces (S.) boulardii

have also been used as probiotics. They are usually isolated from fermented foods, intestinal

contents of humans and animals, and more recently breast milk and non-fermented meat and

fruits [148]. In addition to general benefits of supporting healthy gut microbiota, digestive tract,

and immune system [149], probiotics use may confer strain-specific beneficial effects in a broad

spectrum of conditions. These include infectious diarrhea, antibiotic-associated diarrhea,

pouchitis, atopic eczema, and upper respiratory tract infections [150].

It is believed that probiotics may exert beneficial effects against pathogen infections via their

antimicrobial activity. L. salivarius UCC118 has been shown to protect mice from Listeria

monocytogenes infection via the production of antimicrobial bacteriocin; while the mutant strain

defect of producing bacteriocin failed to reduce pathogen translocation to liver and spleen [151].

Similar to the gut microbiota, probiotics can exert pathogen exclusion effects to protect the host.

Johnson-Henry et al. showed that pre-treating mice with the probiotic mixture of L. rhamnosus

R0011 and L. acidophilus R0052 reduced C. rodentium colonization of the intestine and

attenuated TMCH and colitis; they further suggested that these probiotic effects are as a result of

colonization resistance, as co-culturing the probiotics with C. rodentium inhibits C. rodentium

growth on human colonic epithelial cells in a time-dependent manner [152]. Attachment to the

epithelium is essential for the pathogenicity of enteric pathogens; probiotics may out-compete

pathogens from adhering to the epithelium. Using in vitro competition assays, L. casei DN-114

001 and E. coli Nissle 1917 were showed to be able to prevent epithelial adhesion and invasion

of AIEC that are abnormally abundant in CD patients [153, 154]. It was suggested that the strong

binding ability of E. coli Nissle1917 to epithelial cells may allow the establishment of a biofilm

that physically blocks pathogens from accessing the epithelium [154]. Moreover, probiotics use

may also provide protection against pathogens by enhancing mucosal barrier integrity and host

immunity. Increased intestinal permeability as a result of redistribution and reduction of TJ

proteins is commonly observed in active IBD patients [155, 156]. Administration of the probiotic

mixture VSL#3 (L. casei, L. plantarum, L. acidophilus, L. bulgaricus, B. longum, B. infantis, B.

breve, and Streptococcus salivarius subspecies thermophilus) for 7 days to mice with DSS-

induced colitis, has been shown to mitigate inflammation and restore barrier integrity via

18

stabilizing expression and apical localization of TJ proteins, occluding, ZO-1 and claudins [157].

In line with this, in vitro studies further demonstrated that this effect of VSL#3 is achieved by the

activation of Extracellular signal-regulated kinases (EPK) in the Mitogen-activated protein

kinase (MAPK) signaling pathway that regulates TJ proteins expression [158, 159]. Gareau et al.

found that C. rodentium infection resulted in a decrease Treg response and increase mortality in

neonatal mice, but administration of the probiotic mixture L. rhamnosus R0011 and L. helveticus

R0052 normalized Treg levels and prevent neonatal mice from death. However, these protective

effects were abolished in T-cell deficient neonatal mice, indicating the critical role of T-cell

response in mediating probiotic function [160]. Similarly, probiotic B. breve was found to be

able to modulate T-cell polarization toward a Th2 and Treg biased profile in DSS-induced IBD

model [161].

It is worth noting that although encouraging results have been obtained in conditions like

infectious diarrhea and IBD from animal studies, the efficacy of probiotic use remains

controversial in human studies. For instance, although early findings from randomized control

trials (RCTs) suggested that the use of probiotic yeast S. boulardii can prevent recurrent of

Clostridium difficile-induced diarrhea [162, 163], a more recent systematic review of eleven

clinical trials revealed that S. boulardii treatment is ineffective in preventing C. difficile-induced

diarrhea [164]. Similarly, insufficient evidence was observed in terms of beneficial effects of

probiotics in IBD, especially for CD. According to systematic review studies, the use of VSL#3

was showed to significantly promote remission of active UC patients compared to placebo [165,

166], and all four RCTs showed positive effective of VSL#3 in maintaining remission of

pouchitis [167-170]. However, there is a lack of evidence for the efficacy of probiotic use in CD

patients. Indeed, the uses of VSL#3 [171] and several other probiotics such as E. coli Nissle

1917[172] and L. rhamnosus GG [173, 174] in clinical trials were all shown to be ineffective in

preventing relapses of CD compared to placebo. To date, recommendation of probiotics use for

IBD in clinical practice can only be made for pouchitis [150].

The following sections will focus on a probiotic bacterium, B. bifidum, and its health-promoting

effects.

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2.3.1 Bifidobacterium bifidum

B. bifidum is a Y-shaped Gram-positive, anaerobic, non-motile, non-gas-producing bacterium

belonging to the Actinobacteria phylum. B. bifidum was first isolated from the feces of breast-fed

infants by Henri Tissier in 1899 [175] and it is one of the 47 species in the Bifidobacterium

genus [176].

B. bifidum is one of the first autochthonous inhabitants that colonize the human lower

gastrointestinal tract shortly after birth [177]. Although other bifidobacterial species are

widespread along the intestine, B. bifidum is believed to localize distally [178, 179]. Yet, the

colonization patterns of this bacterium are largely unknown. A study revealed that the number of

B. bifidum cells is greater in the proximal colon and the cecum than in the distal colon or feces in

mice administered with B. bifidum [180]; nevertheless, other studies detected similar abundance

of B. bifidum at about 108 – 10

9 CFU/g in both ceacal lumen and feces of human adults [181-

183]. In addition to the digestive system, B. bifidum is also the most frequently detected species

in breast milk [184].

Gut microorganisms are capable to metabolize complex carbohydrates that are not readily

digestible by the host enzymes in intestine. It was found that bifidobacterial genomes contain

about 30% more genes involved in carbohydrate metabolism than most of other gut microbes

[176]. Although bifidobacterial species can metabolize a variety of carbohydrates (such as

dextrin, maltose, and lactose), the fermentation ability is believed to be dependent on species and

strains [185]. Compared to other bifidobacteria, B. bifidum strains degrade a relatively small

number of carbohydrates, which implicates a strategy for efficient niche-specific colonization

and adaptation [186]. What sets B. bifidum apart from other bacteria of the genus is the ability to

utilize host-derived carbohydrates, especially mucin-associated glycans and human milk

oligosaccharides (HMOs). Early studies showed that B. bifidum was the only species of the 29

bifidobacteria tested that can metabolize porcine gastric mucin [187]. It is known that the

capacity of bifidobacteria to degrade intestinal mucin depends on the presence of genes, afcA and

engBF, encoding two different extracellular glycosidases. An in vitro study of different

representative strains of bifidobacteria species demonstrated that only strains of B. bifidum, L22

and D119, possess both genes with the highest degradation competence [188]. In contrast to

other species, B. bifidum strains, especially PRL2010, can grow on mucin-base medium by using

20

mucin as their sole carbon source [19]. Only Bifidobacterium and Bacteroides were reported to

have the capacity utilize HMOs. It was previously shown that exploiting its special enzymes a-

fucosidases and lacto-N-biosidase B. bifidum breaks down HMOs more efficiently than other

species such as B. adolescentis [189]. The capability to ferment HMOs implies a strong selective

advantage of this species in very early life (i.e. during breastfeeding), accounting for their

predominance in infant gut microbiota. Moreover, members of B. bifidum can also ferment plant-

derived oligosaccharides, such as fructooligosaccharides (FOS) and galactoologosaccharides

(GOS), which are recognized as bifidogenic factors. For example, it was shown that strain

NCIMB 41171 can produce four different β-galactosidases; oral intake of metabolites from these

enzymes significantly increased bifidobacterial counts in feces of healthy humans [190].

2.3.1.1 Adhesive Factors and Interaction with the Host

The ability to adhere to the intestinal epithelial cells may play a pivotal role for bacterial

establishment in the gut. It has been shown that B. bifidum strains may possess adherent

properties. For example, a probiotic strain, B. bifidum MIMBb75, was shown to bind most

tightly to Caco-2 cell monolayer than other probiotic species such as B. longum NCC2705 and L.

rhamnosus GG [29]. B. bifidum MIMBb75 can also adhere to mucus producing HT-29 epithelial

cells and display autoaggregating phenotype. The finding that strong autoaggregation takes place

under low pH conditions might indicate a protective strategy for gastric transit. It is plausible that

bacterial cells self-aggregate in the stomach hindering adhesins to interact with host epithelium,

whereas they disassociate in the intestine as pH rises, permitting attachment and colonization

[179]. Two surface adhesion-like molecules, bifidobacterial outer protein (BopA) and hair-like

appendages have been proposed to be involved in assisting B. bifidum to adhere to the

epithelium.

BopA is a cell wall-associated lipoprotein that is unique to the B. bifidum species [29]. It was

first identified in B. bifidum MIMBb75, where BopA was chromatographically purified from

bacterial cell wall extracts. Competitive adhesion assays showed that treatment of purified BopA

significantly inhibited adhesion of B. bifidum strains MIMBb75 to Caco-2 cells [29]. Likewise,

another study showed that adhesion of B. bifidum S17 was reduced after incubating T84 and

HT29 cells with purified BopA protein, suggesting BopA may compete with B. bifidum for

binding sites on epithelial cells [191]. Overexpressing BopA in B. longum resulted in a

21

prominent increase of adhesion to epithelial cells compared to wild-type B. longum, which does

not possess BopA and has weak adherent ability [191]. However, the role of BopA in B. bifidum

adhesion remains controversial. A more recent study revisited BopA and showed that blockage

of BopA did not cause significant reduction of bacterial adhesion, arguing that there may be

cooperative mechanisms involved [23]. It has been proposed that BopA is a moonlighting protein

for it is thought to exercise multiple biological functions besides being an adhesion factor [29,

191]. Gene and protein sequence analyses revealed that BopA is located on an operon encoding a

putative ATP-binding cassette (ABC) transporter, and the BopA protein contains a

tripeptide/oligopeptide solute-binding domain, implying its potential role in peptide nutrient

uptake [29, 191]. Interestingly, incubation of Caco-2 cells with B. bifidum MIMBb75 or purified

BopA resulted in elevation of IL-8 levels but did not impact IL-6 production, indicating that B.

bifidum MIMb75 and BopA may be able to modulate the host immune response stimulating

transient subclinical inflammation in the gut [29].

Like other bifidobacteria, B. bifidum also possesses surface hair-like appendages, known as pili

and fimbriae. They are considered as strong adhesion factors contributing to intimate interaction

between microbes and host epithelial cells [192]. For example, tight-adherence (Tad) pili

identified in B. breve UCC2003 were shown in vivo to be essential for colonization in the mouse

gut [193, 194]. The gene of Tad pili is strictly conserved within bifidobacteria, such that it is also

present in B. bifidum S17 and PRL2010 [195, 196]. However, the number and sequence of gene

clusters encoding for sortase-dependent pili differ among strains, which might confer strain

specific functions. For instance, it is known that B. bifidum PRL2010 displayed the highest

binding ability to extracellular matrix proteins, especially fibrinogen, than other bifidobacteria

possessing these sortase-dependant pili, such as B. breve and B. adolescentis [196]. The two

sortase-dependent pili identified on strain PRL2010 can induce TNF-α response in vivo; when

transgenically expressed these pili genes in the initially nonpiliated bacteria strain, TNF-α

response was also induced upon intestinal colonization. Stimulation of TNF-α response might be

an important feature of PRL2010 as a probiotic strain, since TNF-α associated cytokines not only

represent major proinflammatory mediators, but may also play a crucial role in exerting

antitumor and anti-infection effects [196-198]. These observations imply that sortase-dependent

pili not only promote adhesion but also modulate host immune responses. Indeed, exposing

human epithelial cell lines and mouse intestinal cells to PRL2010 resulted in significant changes

22

of intestinal cell transcriptome affecting an array of immune-related genes; this includes

pronounced downregulation of heat shock proteins and chemokines such as Cxcl2 and Cxcl3,

along with upregulation of antimicrobial β-defensins and TJ proteins [30]. Importantly, heat

shock proteins are usually expressed in response to cellular stress which may have implications

in tumorigenesis linked to infection and chronic inflammation [199]; overexpression Cxcl2 and

Cxcl3 have also been associated with colorectal carcinoma [200]. These suggest that B. bifidum

PRL2010 impacts host gene expression which may presumably enhance host innate defense

response.

2.3.1.2 Health Benefits

Probiotic effects of different B. bifidum strains have been implicated in various disease

conditions as summarized in Table 2.1. Most clinical studies use B. bifidum in a mixture with

other probiotic species from the Lactobacillus genus. Although beneficial effects observed from

a probiotic mix cannot be extrapolated to B. bifidum alone, these studies can still be interesting

because they suggest complementary effects of B. bifidum with other probiotics and provide

directions for mechanistic studies. In infants and children, the use of B. bifidum has shown

beneficial effects in infectious diarrhea and NEC. A large multicentre single-blind RCT showed

that oral administration of a probiotic mixture containing B. bifidum for 5 days significantly

reduced duration and severity of acute diarrhea in children aged 3-36 months compared to the

placebo [17]. Similar results were found in two other clinical trials in hospitalized infants and

children between 3 to 120 months old with different probiotic formulas containing B. bifidum

[18, 19]. Animal studies showed that B. bifidum strains, such as ATCC15696 and CIDCA5310,

improved diarrhea and enterocolitis caused by rotavirus and Clostridium difficile, which are two

common infectious agents responsible for hospital-acquired diarrhea in children [201-203]. The

use of B. bifidum in a probiotic mixture has been found to reduce incidence and severity of NEC

in very low birth weight preterm infants in a retrospective cohort study [20]; but two other

double-blind RCTs with different B. bifidum-containing probiotic mixtures found only reduction

in NEC incidence and morbidity, with no difference in severity [204, 205]. Feeding NEC

neonatal rats with B. bifidum strain OLB6378 attenuated NEC-induced mucosal apoptosis and

inflammation, via normalizing the expression and localization of TJs and AJs as well as

proinflammatory cytokine IL-6 level [25, 26, 206]. Furthermore, B. bifidum consumption has

also been evidenced to have systemic benefits outside of the intestinal system, such as in atopic

23

eczema and asthma among children, plausibly via reducing serum levels of antibody

immunoglobulin E (IgE) [207-209]. B. bifidum use in adults is mainly associated with beneficial

effects in IBS. IBS is a chronic gastrointestinal functional disorder, characterized by abdominal

pain and bloating in absence of detectable structural abnormalities or obvious inflammation

[210]. It has been shown by two clinical trials that B. bifidum BGN4 and NCIMB 30153 when

used in a mixture with Lactobacillus species improved IBS symptoms with no adverse effects

[21, 22]. Interestingly, a recent prospective RCT showed that the use of B. bifidum MIMBb75 for

4 weeks was able to significantly alleviate pain, discomfort and digestive disorder in IBS patients

compared to the placebo controls [23].

Moreover, B. bifidum has also been implicated in pathogen infections, IBD and CRC. Ingestion

of fermented milk containing strain BF-1 for 12 weeks resulted in reduction of serum biomarkers

associated with H. pylori (Helicobacter pylori) infection compared to baseline in H. pylori-

positive patients [211]. Bayoumi et al. recently showed that B. bifidum ATCC 29521 was able to

interfere with EHEC and Salmonella enterica serovar Typhimurium attachment and colonization

by downregulating the pathogens’ virulence factors intimin expression in vitro, thereby attaining

pathogen exclusion effects [31]. Although there is insufficient clinical evidence showing B.

bifidum efficacy in IBD to date, beneficial effects of B. bifidum have been implicated in animal

and cell culture studies in intestinal inflammation. For instance, Kim et al. reported that dietary

supplement of B. bifidum BGN4 alleviates lymphocytes infiltration and Th1-type cytokines

production in mice with induced IBD [212]. Similarly, ingestion of strain S17 before and after

induction of TNBS-colitis reduced Th1-driven intestinal inflammation [32]. One study using

non-invasive ETEC to induce Th1 inflammation in rats demonstrated that the supplementation of

a probiotic mix composed of B. bifidum R0071, B. longum subsp. infantis R0033 and L.

helveticus R0052, modulated Th1 immune response by supressing expression of

proinflammatory cytokines IL-6 and TNF-α [213]. In DSS-model of colitis, the probiotic mixture

LaBb Dahi containing L. acidophilus LaVK2 and B. bifidum BbVK3 was found to be able to

inhibit activity of β-glucuronidase, which is an intestinal enzyme elevated in colitis and

associated with carcinogenic and cytotoxic metabolites production [214]. As a matter of fact,

links have been drawn between B. bifidum and CRC. Specifically, an early in vitro study

demonstrated that in a screen of 30 different bifidobacterial strains, B. bifidum BGN4 elicited the

highest inhibitory effect on colon cancer cells HT-29 growth; treatment of BGN4-derived

24

polysaccharides inhibits DNA synthesis of various colon cancer cell lines in a dose-dependent

manner, suggesting their antitumor effects via repressing cellular proliferation [215]. A recent

study showed that feeding LaBb Dahi for 32 weeks in the 1, 2-dimethylhydrazine (DMH)

induced colorectal carcinogenesis rat model was able to protect rats from developing CRC

evidenced by significant attenuation of pre-neoplastic lesions formation in treated rats [216]. It

was postulated by the investigators that the molecular basis underlying the anti-carcinogenic

effects of LaBb Dahi could be attributed to upregulation of carcinogen-detoxifying activities,

downregulation of β-glucuronidase activity, and decrease expression of anti-apoptotic bcl-2 and

c-myc genes in colon tissue [216].

Most studies to date have focused on examining beneficial effects of B. bifidum in diseases

states, while only a few explored health maintaining effects of B. bifidum. These include two

RCTs reporting that the use of B. bifidum in healthy children and adults for over 3 months was

linked to a reduction and shortening of common cold episodes [217, 218]. An early study by

Schiffrin et al. demonstrated that intake of B. bifidum Bb12 was able to increase phagocytosis

activity against E. coli species in healthy adult blood, suggesting an enhancement of host anti-

infectious mechanism in response to B. bifidum [219]. Immunomodulation effects were also seen

in studies with healthy animals, where colonization of B. bifidum given in a probiotic mixture

increased anti-inflammatory cytokine IL-10 production of the large intestine [220]. Another

beneficial effect of B. bifidum is associated with improvement of gut microbiota composition.

Specifically, one small clinical trial showed that ingestion of B. bifidum containing probiotics

significantly increased the abundance and diversity of fecal bifidobacterial population compared

to baseline in healthy elderly subjects [24]. Inoculation of healthy mice with B. bifidum

MIMBb75 for two weeks, altered microbiota composition at different intestinal loci including

reduction of Clostridium coccoides in the ceacum, increase of total bacteria in the proximal

colon and increase of bifidobacteria in the proximal and distal colon [180]. Moreover,

colonization of B. bifidum may impart nutritional benefits to healthy individuals. Fermentation of

non-digestible oligosaccharides by B. bifidum can result in production of SCFAs such as

butyrate, which is a principle energy source supporting colonocyte growth [221-223]. Another

nutritional benefit of B. bifidum springs from its biosynthesis of vitamins that cannot be

endogenously generated by human, especially B group vitamins. In fact, B. bifidum is one of the

25

most effective folate producers within the genus [224, 225], and administration of B. bifidum was

found to significantly improve folate status in animals and humans [226-228].

26

Table 2.1 Clinical Health Benefits of B. bifidum

Conditions Subjects Study Design Agents and Administration

(strains are indicated when provided)

Main Findings Ref.

Disease-related

Acute diarrhea

Hospitalized

infants

Double-blind RCT1,

n=55.

B. bifidum and S. thermophilus

supplemented in infant formula; once

daily for 4447 patient-days

Reduce the incidence of acute

diarrhea.

[18]

Hospitalized

children

Prospective, single-

blind, multicenter

trial, n=209.

B. bifidum, B. longum, L. acidophilus, L.

rhamnosus, Enterococcus faecium, with

fructooligosaccharides; once daily for 5

days.

Reduced duration of diarrhea and

hospitalization.

[229]

Children Single-blind,

multicentre RCT,

n=571.

B. bifidum, L. delbrueckii var bulgaricus,

Streptococcus thermophilus, and L.

acidophilus, once daily for 5 days.

Reduced duration and severity of

diarrhea.

[17]

Pathogen

Infection

Candida infection

in young patients

with broad

spectrum

antibiotics therapy.

Double-blind RCT,

n=150

B. bifidum, B. longum, L. acidophillus, L.

rhamnosus, Saccharomyces boulardi,

Saccharomyces thermophilus, with

fructooligosaccharides; once daily for 7

days.

Reduced Candida albicans and

Candida tropicalis colonization in

the intestine and candiduria.

[230]

27

Children infected

by H. pylori

RCT, n=100

B. bifidum-12 and L. acidophilus; once

daily for 6 weeks.

Improved H. pylori eradication

and restored altered gut

microbiota; ineffective in

reducing eradication therapy-

associated adverse effects;

[231]

Adult patients with

H. pylori infection

Double-blind RCT,

n=107

B. bifidum, L. acidophilus, L. rhamnosus,

and Streptococcus faecium; once daily for

30 days.

Ineffective in improving efficacy

or reducing adverse effects of

eradication therapy

[232]

Adult patients with

H. pylori infection

Double-blind RCT,

n=30.

B. bifidum CUL17 and Rhodia and

L.acidophilus; once daily starting 7 days

before H. pylori eradication therapy

eradication therapy for 15 days.

A trend to improve microbiota

composition altered by antibiotic

treatment, but not statistically

significant.

[233]

Functional

gastrointestinal

disorders

Adult patients with

IBS2

Prospective, double-

blind, multi-centre

RCT, n=122.

B. bifidum MIMBb75; once daily for 4

weeks.

Improved the IBS symptoms and

digestive disorder.

[23]

Adult patients with

IBS

Double-blind

placebo-controlled

trial, n=52.

B. bifidum CUL20, B. lactis CUL34, L.

acidophilus CUL60 and L. acidophilus

CUL21; once daily for 8 weeks.

Improved IBS symptoms. [22]

Adult patients with Prospective, double- B. bifidum BGN4, B. lactis AD011, L. Improved IBS symptoms with no [21]

28

IBS blind RCT, n=70

acidophilus AD031, and L. casei IBS041;

twice daily for 8 weeks.

adverse effects.

Adult patients with

gastric and lower

abdominal

symptoms

Double-blind,

controlled, crossover

trial, n=27

B. bifidum YIT 10347 and Streptococcus

thermophilus YIT 2021 fermented milk;

once daily for 2 weeks, followed by

crossover for 3 weeks after washout

period of 3 weeks.

Reduced gastric symptoms. [234]

Children and adult

patients with

functional

gastrointestinal

disorders

Open-label clinical

trial, n=37

B. bifidum YIT 10347 and Streptococcus

thermophilus YIT 2021 fermented milk;

once daily for 4 weeks.

Improved gastrointestinal

symptoms and reduced

psychological stress.

[235]

NEC3

Preterm infants

with very low birth

weight

Prospective, double-

blind, multicenter

RCT, n=434.

B. bifidum NCDO 1453 and L.

acidophilus NCDO 1748; twice daily for

6 weeks.

Reduced incidence of death and

NEC.

[204]

Preterm infants

with very low birth

weight

Double-blind RCT,

n=145.

B. bifidus, B. infantis, and Streptococcus

thermophilus; once daily until 36 weeks

postconception.

Reduced both the incidence and

severity of NEC.

[20]

Preterm infants

with very low birth

weight

Prospective, double-

blind RCT, n=186.

B. bifidum, B. infantis, B. longum and L.

acidophilus; twice daily.

Reduce NEC-associated

morbidity and duration of

hospitalization, no significant

difference in NEC severity.

[205]

Allergy

Pediatric patients

with atopic

Double-blind RCT,

n=40.

B. bifidum, L. acidophilus, L. casei, and L.

salivarius; once daily for 8 weeks.

Reduced atopic dermatitis

severity and serum IL-5, IL-6,

[207]

29

dermatitis IFN-γ, and IgE levels.

Infants born to

mothers with a

family history of

allergic diseases

Double-blind RCT,

n=68.

B. bifidum BGN4, B. lactis AD011, and L.

acidophilus AD031; once daily to

pregnant women starting at 4–8 weeks

before delivery and continuing until 6

months after delivery.

Reduced incidence of eczema in

infants at 1 year-old; no

significant difference in serum

IgE level.

[209]

Children with mild

to moderate

asthma

Placebo-controlled

trial, n= 46.

B. bifidum, L. acidophilus, and L.

delbrueckii subsp. bulgaricus; once daily

for 12 weeks.

Reduced episodes of asthma

exacerbations and improved lung

function.

[208]

Others

HIV-infected

children

Double-blind RCT,

n=77.

B. bifidum and Streptococcus

thermophilus; once daily for 2 months. Ineffective in improving diarrhea

episodes.

[236]

Adult patients with

non-alcoholic

steatohepatitis

Open-label RCT,

n=20

B. bifidum, L. plantarum, L. acidophilus,

L. rhamnosus; once daily for 6 months.

Reduced liver fat (ie. intrahepatic

triglyceride content), and serum

aspartate aminotransferase level.

[237]

Institutionalized

elderly

Open-label RCT,

n=25

B. bifidum; once daily for 28 days.

Reduced colonic inflammatory

infiltration, chronic inflammation

in the sigmoid colon.

[238]

30

Health

Pathogen

Infection

(prevention)

Healthy

schoolchildren

Double-blind RCT,

n=80.

B. bifidum and L. acidophilus; twice daily

for 3 months.

Reduced episodes of common

cold and related symptoms (fever,

cough, rhinorrhea), but no

significant benefit on diarrhea and

vomiting.

[217]

University students

with academic

stress

Prospective, double-

blind RCT, n=583

B. bifidum R0071

B.longum ssp. infantis R0033,

Lactobacillus helveticus R0052; once

daily for 6 weeks.

Reduced cold/flu episodes during

acute stress.

[239]

Healthy adults Double-blind RCT,

n=179.

B. bifidum MF, B. longum SP, and L.

gasseri PA; once daily for 3-5 months.

Reduced the severity of symptoms

and shortened common cold

episodes.

[218]

Healthy adult

volunteers

Double-blind RCT,

n=80.

B. bifidum BF-1 and Streptococcus

thermophilus YIT 2021 fermented milk;

once daily for 12 weeks.

Reduced values of biomarkers for

inflammation associated with H.

pylori infection and improved

upper gastrointestinal health.

[240]

1RCT, randomized controlled trial;

2IBS, irritable bowel syndrome;

3NEC, necrotizing enterocolitis.

31

2.4 MicroRNA

MicroRNAs (miRNAs) are 20-22 nucleotides long, single-stranded, non-coding RNA molecules

involved in posttranscriptional gene regulation by targeting mRNA transcripts [241]. In animals,

genes encoding miRNAs are transcribed by RNA polymerase II into primary precursor miRNAs

(pri-miRNAs), which are then processed by the enzyme Drosha into hairpin-shaped precursor

miRNAs (pre-miRNAs). Through Exportin-5, pre-miRNAs are then exported to the cytoplasm,

where they are further cleaved by the enzyme Dicer into a miRNA duplex. Only one of the

strains of the duplex becomes a mature miRNA (miR). miR can be incorporated into the RNA-

induced silencing complex (RISC) and recognize complementary binding sites on the 3’

untranslated region (UTR) of target messenger RNA (mRNA) transcripts. Depending on the

degree of sequence complementarity, this may lead to mRNA translational repression or

degradation [242]. To date, 2,588 miRNAs have been identified in humans and 1,915 miRNAs in

mice (miRBase release 21, June 2014) [36]. In fact, up to 60% of the human transcriptome is

regulated by miRNAs [243]. This potent impact of miRNA in regulating gene expression is

attributable to the fact that each miRNA can have hundreds of mRNA targets while the same

target can be regulated by multiple miRNAs. Different regulatory network models have been

proposed to illustrate how miRNAs may exert cooperative actions to fine-tune gene expression

[244]. These include the linear model where one miRNA determines one phenotype, the

divergent model where one miRNA regulates genes involved in various functions, and the

network model where multiple miRNAs can target multiple genes in a cooperative manner to

determine one physiological outcome (Figure 2.2) [244]. A wide range of biological processes,

such as cell cycle [245] and immune responses [246], can be influenced by miRNA regulation.

Given the important role of miRNAs, it is not surprising that the expression of miRNA itself is

tightly regulated in the context of tissue type and developmental stage [242]. It is also known

that various stimuli can influence miRNA expression in a rapid and time-dependent fashion [247,

248]. For instance, exposing mouse lung tissue to the proinflammatory endotoxin LPS resulted in

transient global miRNA expression changes, which peaked at 3 hours and drops at 6 hours post-

LPS exposure and correlated with the intensity of the inflammatory response [248]. The

importance of miRNAs in intestinal homeostasis was demonstrated in Dicer1-deficient mice,

32

which exhibited significant intestinal barrier dysfunction and inflammation evidenced by the

decrease of goblet cell and increase of crypt apoptosis and lymphocyte infiltration compared to

the wild-type [249]. It is known that various gut bacteria and pathogens can impinge miRNA

expression, and deregulation of miRNA expression is associated with different pathological

conditions including IBD and colonic malignancy, which will be discussed in the following

sections.

Figure 2.2 Models of miRNA-dependent Regulatory Network.

A representation of different miRNA-mediated regulatory models proposed including the linear model

(miRNA1:Target1, determining phenotype 1), the divergent model (miRNA1:Target 1,2…n, determining

phenotype1,2,3), and the network model (miRNA1,2…n:Target2,3…n, determining phenotype2), modified from

[244].

33

2.4.1 Response of Intestinal miRNA to Bacteria

Two recent studies have demonstrated that the resident microbiota impacts the intestinal miRNA

signature. Singh et al. found that the presence of the gut microbiota influences the mouse caecal

miRNA signature with 16 miRNAs being differentially expressed between germ-free and

conventional mice. These include higher expression of miR-148a and reduced expression of

miR-150 in conventional versus germ-free mice. Putative targets of these microbiota-modulated

miRNAs were found to be involved in the regulation of the barrier function [37], which is in line

with the Dicer1-deficient study discussed above. For example, barrier related genes, C1galt1

(core 1 synthase N-acetylgalactosamine 3-beta-galactosyltransferase 1) and Prkcz (protein kinase

C zeta isoform a), which are putative targets of miR-148a, were deregulated in conditional

Dicer1 knockout mice, suggesting a potential role of microbiota-dependent epigenetic regulation

of host barrier function [37, 249]. Dalmasso et al. found 1 and 8 differentially expressed

miRNAs in the ileum and colon, respectively, of germ-free mice following colonization with

microbes derived from pathogen-free mice; by crossing gene expression data with potential

targets of these miRNAs, the upregulated Abcc3 (ATP-Binding Cassette, Sub-Family C, Member

3), a multidrug resistance-associated gene regulating xenobiotics and endogenous toxins

metabolism, was identified to be a direct target of miR-665, which was downregulated in

response to microbiota colonization in the colon [250]. Together, these studies show that the gut

microbiota is an important contributor to the homeostatic intestinal miRNA signature. Further

substantiation to this concept comes from a study performed with the pathogen Listeria

monocytogenes in germ-free and conventional mice. By comparing miRNA profiles between

germ-free and conventional mice challenged with Listeria monocytogenes, Archambaud et al.

identified 5 microbiota-dependent miRNAs alterations; namely, the decrease of miR-143, miR-

148a, miR-200b, miR-200c, and miR-378 were only observed in the ileum of conventional but

not in germ-free mice infected with Listeria monocytogenes [38]. Crossing bioinformatically

predicted targets of miR-143 and miR-378 with differentially expressed genes altered in response

to Listeria monocytogenes, led to the identification of a miRNA-mRNA network, which infers

possible miRNA-mediated regulation of host gene expression in response to pathogen infection.

Other gastrointestinal pathogen infections have been reported to alter host miRNA expression

including Helicobacter, Salmonella, and Campylobacter, as summarized in Table 2.2.

34

Many of these pathogen-induced miRNA alterations are involved in regulating host immune

responses, especially miR-146a and miR-155. These two miRNAs are consistently upregulated

upon Salmonella [39], Listeria [251], Campylobacter [40], and Helicobacter infection [252,

253], and are recognized as important negative regulators of innate immune response. It is

known that both miR-146a and miR-155 are induced upon activation of NFκB signalling

pathway as LPS-stimulated TLR4 activation resulted in increase expression of these miRNAs in

human monocytes [254]. MiR-146a was later found to be important in promoting immune

tolerance via targeting IRAK1 (IL-1R-associated kinase 1), which is an important adaptor

protein of MyD88, to repress TLR-induced NFκB activity [255-257]. Low IRAK1 protein

expression is essential for the mucosa to develop tolerance to initial colonization of resident

microbe during neonatal life. Chassin et al. showed that oral administration of miR-146a

inhibitor to vaginally delivered neonatal mice rescued mucosal IRAK1 protein expression and

resulted in increased susceptibility to epithelial apoptosis caused by bacterial colonization [257].

MiR-155 has been shown to have both pro- and anti-inflammatory effects as it targets both

suppressors such as Ship1 (Src homology2 domain-containing inositol phosphatase) and inducers

such as Tab2 (TGF-beta activated kinase 1/MAP3K7 binding protein 2) of inflammatory

signalling in immune cells [258-260]. The biological function of miR-155 augmentation during

pathogen infection is unclear but miR-155 deficiency has been shown to compromise host

immunity against infection. Specifically, miR-155 knockout mice were unable to develop

vaccine-induced immunity against H. pylori due to impaired Th1 and Th17 response [261]. In

addition to miR-146a and miR-155, a few other miRNAs were also constantly deregulated

during pathogen infections. Particularly, miR-16 and miR-200a/b/c were found to be consistently

up and down-regulated, respectively, in response to both H. pylori and Listeria infections [38,

251, 262-265]. The increase of miR-16 has recently been suggested to play a role in attenuating

TNBS-induced damages by suppressing TNF-α and Th1/Th17 responses [266], while the

decrease of miR-200a/b/c expression is linked to epithelial-to-mesenchymal transition [267].

Collectively, these findings may depict a plausible consensus of miRNA alterations in various

gastrointestinal pathogen infections. Nevertheless, there is only a few studies that looked at

miRNA alterations in response to pathogen infection, and they are limited to pathogens of the

stomach (H. pylori) and small intestine (Salmonella, Listeria, Campylobacter). Currently, no

data is available with respect to pathogen infections of the colon, such as EHEC. Interestingly,

one study showed that miR-155-deficient mice experienced a prolonged eradication of C.

35

rodentium and had impaired humoral immune response [268], unveiling the potential role of

miRNAs in regulating host response to the infection of this colonic pathogen.

Even fewer studies have looked at the effect of probiotic bacteria on miRNA expression (Table

2.3). One study showed that administration of two Lactobacillus species (L. paracasei and L.

casei) to Listeria monocytogenes infected mice attenuated infection severity as well as reductions

of miR-192, miR-200b and miR-215 levels caused by Listeria monocytogenes [264].

Interestingly, after exposing human dendritic cells to L. rhamnosus GG for 12 hours, expression

levels of miR-155 level was increased while miR-146a was decreased, implying

immunomodulation effects of this probiotic strain [269]. Very recently, a study demonstrated

that supplementing L. rhamnosus GG culture supernatant to mice increases TJ protein occludin

expression by inhibiting miR-122a expression, which protects mice from alcohol-induced

intestinal permeability augmentation and liver injury [270]. Another in vitro study showed that

incubating T84 cells with probiotic E. coli Nissle 1907 enhanced TJ protein expression by

downregulating miR-203, miR-483 and miR-595 [271]. Moreover, a previous study from our

group found that supplementation of probiotic strain B. bifidum MIMBb75 for two days resulted

in significant miRNA signature changes with an increased level of miR-148a observed in mouse

ceacum [272]. Overall, the current data suggests that probiotic bacteria may confer beneficial

effects in restoring pathogen-induced miRNA perturbation and enhancing host defense

mechanism presumably by shaping miRNA expression.

36

Table 2.2 Impacts of Gastrointestinal Pathogens on Host microRNA Expression

Bacterium Study Design Altered miRNA Physiological Outcome Ref.

H. pylori miRNA microarray profiling

of endoscopic gastric mucosa

biopsies from H. pylori-

positive patients and controls

(n=N/A)

↑: miR-223

↓: let-7a/b/d/e/f, miR-101, miR-103, miR-

106b, miR-125a, miR-130a, miR-141, miR-

200a/b/c, miR-203, miR-204, miR-210, miR-

214, miR-31, miR-32, miR-320, miR-375,

miR-377, miR-379, miR-429, miR-455, miR-

491-5p, miR-500, miR-532, miR-652

miRNA alterations are associated

with the degree of neutrophil and

mononuclear cell infiltration with

implications in chronic

inflammation.

[262]

Gastric biopsy from H.

pylori-positive patients and

controls (n=30)

↑: miR-155 miR-155 modulates inflammatory

response during the infection.

[263]

H. pylori 26695 infected with

various gastric epithelial cell

lines (GES-1, AGS and

MKN45) for 24 hours

↑: miR-155 (GES-1, AGS, MKN45); miR-

146a, miR-16 (GES-1)

miR-155 suppresses inflammation

by reducing IL-8 and growth-

related oncogene (GRO)-α

production.

[263]

Gastric biopsy from H.

pylori-positive patients and

controls (n=48)

↑: miR-146a miR-146a modulates

inflammatory response during

infection.

[273]

37

gastric epithelial cell lines

(MNK-45, GES-1, HGC-27,

AGS) infected with H. pylori

26695 for 24 hours

↑: miR-146a miR-146a represses NF-κB

activity by targeting IRAK1 and

TRAF6.

[273]

Listeria

monocytogenes

C57BL/6J female 9-12 week-

old conventional mice

infected with Listeria

monocytogenes EGDe for 24

and 72 hours (ileum)

↓: miR-143, miR-148a, miR-194, miR-200b/c,

and miR-378

Deregulations of miR-143, miR-

148a, miR-200b, miR-200c, and

miR-378 are microbiota-

dependent, in which miR-143 and

miR-378 are involved in a

putative regulatory network

controlling genes associated with

immune response, cell

differentiation, and enzymatic

activities.

[38]

C57BL/6J female 9-12 week-

old humanized mice infected

with Listeria monocytogenes

EGDe for 24 hours (ileum)

↓: miR-192, miR-200b, miR-215 miRNA alterations are involved in

a putative regulatory network

encompassing hundreds of

differentially expressed genes

upon the infection and may play

a role in epithelial cell

differentiation.

[264]

38

Caco-2 cells infected with

Listeria monocytogenes

EGDe for 1 hour

↑: miR-146b, miR-16, miR-155

↓: let-7a, miR-145

miRNA alterations are involved in

modulating inflammatory

response.

[251]

Mouse bone marrow derived

macrophages infected with

Listeria monocytogenes

EGDe for 6 hours

↑: miR-155, miR-146a, miR-125a-3p/5p, miR-

149, miR-147, miR-191, miR-132, miR-497,

miR-200c, miR-139-5p

miRNA alterations are involved in

innate immune defense against the

pathogen.

[265]

Salmonella

enterica

Phagocytic (Mouse RAW

264.7) and non-phagocytic

(Human HeLa) cell lines

infected with Salmonella

enterica serovar

Typhimurium SL1344 for 24

hours

↑: miR-146a/b, miR-155, miR-21(RAW264.7);

miR-1308 (Hela)

↓: let-7a/c/d/f/g/i, miR-98 (RAW264.7); let-

7a/c/d/f/g/i (Hela)

miRNA alterations are involved in

inflammatory response;

downregulation of let-7 family

induces IL-6 and IL-10

production in macrophages and

epithelial cells.

[39]

Human HT-29 infected with

Salmonella enterica serovar

enteritidis SE2472

↑: miR-128, miR-196a, miR-330-3p, miR-20a miR-128 suppresses macrophage

recruitment to infection site by

inhibiting the expression of

macrophage colony-stimulating

factor.

[274]

39

Campylobacter

concisus

Human monocytic leukemia

THP-1 cell line infected with

Campylobacter concisus for

6 hours

↑: miR-146a, miR-29b, miR-3687, miR-3648,

miR-7162, miR-6080, miR-3916, miR-221

↓: miR-4477b, miR-621

miRNA alterations are involved in

immune response and

tumorigenesis.

[40]

40

Table 2.3 Impacts of Probiotics on Host Intestinal microRNA Expression

Bacteria Study Design Regulated miRNA Physiological Outcome Ref.

L. rhamnosus

GG

supernatant

C57BL/6N male 8-10 week-old

mice administered with alcohol and

bacteria-free L. rhamnosus GG

supernatant

↓: miR-122a Downregulation of miR-122a

potentiates TJ protein expression,

improving alcohol-induced barrier

dysfunction.

[270]

L. rhamnosus

GG

Human dendritic cells incubated

with heat-killed Lactobacillus

rhamnosus GG for 12 hours

↑: miR-155

↓: miR-146a

miRNA alterations are involved in

modulating inflammatory response.

[269]

L casei BL23 C57Bl/6J female 9-12 week-old

humanized mice infected with

Listeria monocytogenes EGDe for

24 hours (ileum) after orally treated

with Lactobacillus for 3 days

↑: miR-192, miR-200b, miR-215 Lactobacillus treatments normalize

aberrant miRNA expression induced

by Listeria infection.

[264]

L. paracasei

CNCM I-3689

C57Bl/6J female 9-12 week-old

humanized mice infected with

Listeria monocytogenes EGDe for

24 hours (ileum) after oral probiotic

administration for 3 days

↑:miR-192 Lactobacillus treatments normalize

aberrant miRNA expression induced

by Listeria infection.

[264]

41

E. coli Nissle

1917

Intestinal epithelial cell line T84

incubated with E. coli Nissle 1917

for 3 hours

↓: miR-203, miR-483-3p, miR-595 miRNA downregulations improve

barrier function by modulating the

expression of regulatory and

structural components of TJs.

[271]

B. bifidum

MIMBb75

C57Bl/6J male 7 week-old mice

administered with B. bifidum

MIMBb75 for 2 days (Ceacum)

↑: miR-148a, miR-455 miRNA modulations may have

implications in improving barrier

function.

[272]

42

2.4.2 MiRNA deregulations in IBD and CRC

MiRNA deregulation has also been linked to chronic intestinal disorders. Alterations in miRNA

expression are common in the colon tissue of IBD patients. For example, by comparing miRNA

profiles between active UC and healthy subjects, Wu et al. identified 11 differentially expressed

miRNAs including the increase of miR-21 and decrease of miR-192 [275]. More miRNA

alterations were identified in other studies in colonic biopsy of IBD patients, such as

upregulation of miR-146a in CD [276] and miR-155 in UC [277] and downregulation of miR-

200b in both UC and CD patients [278]. Many of these miRNAs have been shown to play a role

in regulating immune response and barrier function. As mentioned before, NLRs are important

activator of NFκB innate immune response and NOD2 has been shown to be a strong genetic

locus contributing to IBD susceptibility [279]. It was found that miR-192, which is

downregulated in IBD [275], targets NOD2 while overexpressing miR-192 in HCT116 cells was

able to suppress NFκB activation and subsequent proinflammatory cytokine production [280].

MiR-192 has also been found to target macrophage inflammatory peptide-2 alpha, which is a

chemokine expressed by epithelial cells and has elevated levels in IBD [275]. MiRNAs may also

contribute to T-cell differentiation. For example, members in the miR-17-92 cluster (miR-17,

miR-20, and miR-18a, miR-19a, miR-19b and miR-92a) were shown to promote Th1 while

suppress Treg differentiation [281]. Moreover, alteration of miRNAs may contribute to intestinal

barrier dysfunction, a common feature in IBD [282]. The increase of miR-21 has been validated

by several studies on IBD patient colon biopsy [275, 277, 283]. Shi et al. showed that miR-21

knockout mice have improved severity of inflammation and survival rate in DSS-induced colitis

[284]. They further illustrated that overexpressing miR-21 in Caco-2 cells impaired TJ protein

expression accompanied with increased barrier permeability by directly targeting RhoB (Ras

Homolog Family Member B), which is a GTPase important in regulating actin cytoskeleton

organization [285]. In addition, one study found that miR-150 was upregulated in active UC

patients, and overexpression of miR-150 in HT29 cells potentiates apoptosis via inhibiting c-

Myb expression. C-Myb is an important regulator of cell cycle progression, and its

downregulation can lead to cell death, which may account for the loss of epithelium integrity

seen in IBD [286]. However, a recent study found that miR-21 expression varies in different

models of IBD, and miR-21deletion resulted in exacerbation of colitis in TNBS-treated mice.

43

Further work is needed to unravel these divergent functions of miR-21 in different IBD models

[287].

Aberrant miRNA expression patterns have been described in various cancers including CRC, as

evidenced by screening data of miRNA expression profile between tumor and nontumor tissues

using microarray or deep sequencing [288-290]. A recent review of over twenty studies on

miRNA expression in CRC concluded that there is a total of 160 miRNAs differentially

expressed in CRC, highlighting the downregulation of miR-143 and miR-145, and upregulation

of miR-20a and miR-92a as potential biomarkers for CRC [291]. Functional validations of these

findings revealed that miRNAs exert both oncogenic and tumor-suppressive effects in CRC

development and progression of CRC by modulating gene expression. MiRNAs that process

oncogenic effects are commonly denoted as oncomirs [292]. MiR-21 is an oncomir in CRC and

its expression level has been shown to increase in accordance with cancer development stages,

with greater degrees of elevation in advanced carcinomas than precancerous adenomas based on

in situ hybridization results [293]. Increased expression of miR-21 has been functionally verified

to inhibit tumor-suppressor gene expression, such as PTEN (phosphatase and tensin homolog)

[294] and PDCD4 (programmed cell death 4) [295]. Members in the miR-17-92 cluster have also

been shown to be consistently upregulated during the transition from adenomas to carcinomas

and are collectively referred to as oncomir-1 [296, 297]. In CRC, oncomir-1 and its homolog

miR-106a/b are overexpressed and intriguingly, elevations of these miRNAs in fecal samples

containing exfoliated colonocytes have been shown to be sensitive biomarkers for distal colon

cancer detection [298]. It is known that the activation of oncogene c-myc induces miR-17-92

expression, which in turn suppresses the expression of an array of anti-proliferative genes, such

as the cyclin-dependent kinase inhibitor (Cdkn1a) that controls cell cycle progression [299],

Bcl2-like 11 (Bim) [300] that promotes apoptosis and Rho family GTPase 3 (Rnd3) that controls

cytoskeletal dynamics [301]. Hence, increase expression of these oncomirs potentiates

colonocyte proliferation. On the contrary, miRNAs exerting tumor suppressive effects are

constantly downregulated during malignancy. EMT is an important process in the maintenance

of stemness, which allows epithelial cells to acquire mesenchymal cell phenotypes including

losses of cell-cell adhesion, cellular polarity and epithelial cell marker E-cadherin expression

accompanied by increased capacity to migrate and proliferate. However, uncontrolled activation

of EMT is a trigger of cancer progression and metastasis [302]. Low expression of miR-200

44

family (including miR-200a/b/c, miR-141 and miR-429) has recently been demonstrated to be

crucial in triggering CRC metastasis [303-305]. It has been functionally validated in many cell

types that miR-200s target Zeb1/2 (zinc finger E-box binding homeobox 1/2), two potent

transcription factors that promote EMT by repressing E-cadherin transcription [267]. Chen et al.

showed that transfecting miR-200c mimics into SW620 colon cancer cells resulted in lower

expression of Zeb1 mRNA and protein levels, as well as reduced number of migrating cells

[303]. Nonetheless, the upstream events causing the downregulation of miR-200s remain

unclear, but studies revealed that the expression of Zeb1 reciprocally suppresses miR-200s in

colon cancer cell lines. These findings imply that the balance expression between miR-200s and

Zeb1/2 could be a critical switch for metastasis [267]. Many other miRNAs have been

implicated in regulating various aspects of CRC development, and complex miRNA regulatory

networks are likely in place to fine regulate the phenotype. Hence, it is crucial and biologically

relevant to study miRNA fine-tuning effects in the context of a regulatory network.

2.5 Animal Model

The murine model is one of the most commonly used for gastrointestinal studies, especially

when investigating microbiota, pathological disorders and using functional genomics approaches

[199, 306, 307]. There are some anatomical and physiological differences between mice and

humans. Although the mouse gastrointestinal tract is relatively smaller in size than humans’, the

relative size of mouse caecum is larger than humans; the ceacum is the major site of fermentation

in mouse, but for humans fermentation takes place in the colon [308, 309]. In addition, gastric

pH is higher in mouse (about 4.0 in fasted animals vs 2.0 in humans) there is also more water

content per kg body weight in the mouse gastrointestinal tract than in humans [310]. These

differences may have implications in the transit time of food digestion and therefore duration of

interaction with the microbiota. With respect to microbiota composition, both man and mouse

have the same dominant bacterial phyla: Firmicutes, Bacteroidetes and Actinobacteria [311].

This withstanding, at a lower taxonomical level, the genus of Mucispirillum and some selected

bacterial species are exclusive to mice, such as Lactobacillus murinus, while others are of human

origin such as B. bifidum and B. breve [312]. By inoculating a human microbiota into germ-free

mice the humanized gnotobiotic mouse model has been developed and has been shown to

45

recapitulate about 100% and 88% of the human gut microbiota at phylum and genus level,

respectively [309]. With respect to pathological diseases like IBD and infectious diseases, upon

induction mice undergo some degrees of microbiota shift and share similar histological features

and inflammatory profiles as humans, as discussed in previous chapters [307, 313]. Moreover,

mouse and human share significant genetic homology. The protein-coding regions of human and

mouse genomes share approximately 85% similarity [314]. These regions are evolutionarily

conserved for their biological functions. Likewise, the majority of miRNA genes are also

conserved between human and mouse [315]. In fact, evolutionarily conserved miRNA homologs

across species are assigned to the same numerical identifier following the prefixes that

differentiate species such as, hsa-miR-21 in human and mmu-miR-21 in mouse [316].

C57Bl/6J is the most extensively used inbred mouse strain with its complete genome sequenced

[314, 317]. Given that inbred mice exhibit less genetic variability, most of the genetically

engineered mouse models are generated in the C57Bl/6 background. For example, the IL-10-

knock out colitis model was constructed using C57Bl/6 mice [318]. This would be an advantage

for the future studies for implementing genetically manipulated models. More importantly, with

respect to the present study, this strain of mice is resistant to C. rodentium infection compared to

C3H/HeJ [107]. Infection in this strain does not result in extensive mortality and the mice

recover within 3 weeks. This moderate degree of disease severity may help detecting potential

probiotic effects of B. bifidum and would allow studies of C. rodentium clearance. In fact, C57

Bl/6 mice have been used in the literature for probiotic studies in the context of C. rodentium

infection, as described previously [152, 160, 319-323].

46

Chapter 3

Rationale, Hypothesis and Objectives

Rationale:

The continuous crosstalk between the host epithelium and gut microorganisms is essential for

intestinal homeostasis. Presence of harmful bacteria can significantly disrupt host homeostasis.

C. rodentium is a murine-specific enteric pathogen used to model EHEC infection, IBD and CRC

in humans [15]. Colonization of C. rodentium peaks at day 10 post-infection in mouse distal

colon resulting in dysbiosis, intestinal barrier dysfunction, colitis and colonic crypt hyperplasia

[15]. In contrast to pathogen-host interaction, beneficial bacteria-host interaction contributes to

maintenance and restoration of intestinal homeostasis. B. bifidum is an indigenous member of the

human microbiota and a common probiotic. In vitro and in vivo studies have revealed that some

strains of B. bifidum may be beneficial in EHEC infection and IBD. Specifically, B. bifidum

ATCC 29521 limits EHEC attachment and colonization in intestinal cell culture [31]. B. bifidum

S17 [32] and BGN4 [212] were shown to be able to attenuate intestinal pathology in mice with

chemically- and immunologically-induced IBD, respectively . However, no study currently has

looked at the effect of B. bifidum on C. rodentium infection. Based on genome-wide

transcriptome microarray analyses, both host-C. rodentium and host-B. bifidum interaction can

impact of host gut physiology by modulating gene expression [30, 34, 35]. MiRNAs are potent

post-transcriptional regulators of gene expression. Increasing evidence has suggested that host-

microbial interaction can influence mucosal miRNA response. For instance, various enteric

pathogen infections can alter host gastric (H. pylori [262]) and small intestinal (Listeria [38,

264], Salmonella [39], and Campylobacter concisus [40]) miRNA signature. Our research group

previously demonstrated that conventional mice supplemented with B. bifidum MIMBb75

exhibit a different miRNA signature in the caecum [272]. The strain B. bifidum MIMBb75 has

been shown to have strong adhesive ability [29, 179] and improve IBS symptoms in a clinical

trial [23]. Nevertheless, whether the presence of this probiotic strain confers beneficial effects

and modulates miRNA response in the context of C. rodentium infection has yet to be explored.

47

Overall hypothesis:

B. bifidum MIMBb75 administration attenuates C. rodentium infection and modulates the

intestinal microRNA response associated with the infection in mouse.

Objectives:

(1) To determine if C. rodentium infection alters murine colonic miRNA signature and if the

alterations are involved in the host response to the infection (Study 1).

(2) To examine if B. bifidum MIMBb75 administration alleviates intestinal damage and

normalizes miRNA alterations associated with C. rodentium infection (Study 2).

48

Chapter 4

Study 1- Citrobacter rodentium Infection Alters Murine Colonic

microRNA Signature

Study Contributions:

All aspects of the in vivo study and analyses were conducted by the author.

This chapter has been partially reported through poster and oral presentations at the Federation of

American Societies for Experimental Biology (FASEB) Annual Conference (Boston, MA,

March 28-30, 2015), and at the Microbiology and Infectious Diseases Research Day at the

University of Toronto (June 15, 2015).

49

4.1 Abstract

MicroRNAs have been suggested to play a part in the interaction between pathogenic bacteria

and host cells. Citrobacter rodentium is a murine pathogen causing transmissible colonic

hyperplasia and colitis with similar pathogenicity as the foodborne enterohaemorrhagic

Escherichia coli O157:H7 in humans. This study aimed to examine if colonic miRNA signature

is altered during C. rodentium infection. C57Bl6/J male mice were randomized to C. rodentium-

infected or control group, and sacrificed at the peak of infection (10 days post-infection). Crypt

hyperplasia and intestinal inflammation were confirmed by histology and in vivo permeability

test. Colonic RNA was used to profile 578 miRNAs by NanoString technology. Gene targets of

the differentially expressed miRNAs were identified in silico by prediction algorithms and cross-

matching with experimentally verified targets databases. Ninety-one miRNAs were differentially

expressed (p<0.05), with 42 downregulated and 49 upregulated versus control (0.2-8.6 fold), and

infected samples clustered together and separately from the controls based on unsupervised

hierarchical clustering. Moreover, prediction analysis revealed that 865 genes targeted by these

miRNAs are mainly involved in cell cycle and immune response. Key genes relevant to C.

rodentium pathogenesis were examined and an inverse correlation was found between miR-17-

92 related clusters and their experimentally validated gene target Bim, which is an apoptosis

facilitator. This study shows that C. rodentium infection alters the intestinal miRNA signature;

differentially expressed miRNAs may be involved in host hyperplasia response during the

infection.

50

4.2 Introduction

Citrobacter rodentium is a murine-specific enteric pathogen. Similarly to foodborne EHEC in

humans, C. rodentium causes A/E lesions in the host. During infection, mice develop TMCH and

colitis, which are believed to be triggered by the activation of the Wingless (Wnt)/β-catenin and

the Tlr signalling pathways, respectively [15]. Because of the pathological similarities, including

crypt hyperplasia and Th1-dominated inflammation, C. rodentium infection is also used to model

colon tumorigenesis and IBD [15]. The host responds to C. rodentium infection via alteration of

gene expression, which is triggered by the recognition of the pathogen-associated molecular

patterns. Microarray studies found that genes involved in ion transport, epithelial cell

development, and inflammatory responses are altered, such as the decrease of solute carrier

family 26 member 3 (Slc26a3) and B cell leukemia/lymphoma 2-like 11 (Bcl2l11/Bim), and the

increases of Cxcl2 and Cxcl5 expression in mouse colon. As a consequence, there is an

electrolyte imbalance, hyperproliferation, and neutrophil infiltration in the colonic mucosa

during infection [34, 35]. In addition, gene-specific approaches identified that genes endothelial

PAS domain protein 1 (Epas1) and solute carrier family 15 member 1 (Slc15a1) involved in

intestinal barrier function were deregulated [324-326]. However, post-transcriptional regulation

of gene expression has not been thoroughly investigated. MiRNAs are non-coding RNA

molecules working in a cooperative manner to fine-tune gene expression. To date, 2,588

miRNAs have been identified in humans and 1,915 in mice (miRBase release 21, June 2014)

[36] and they are believed to regulate up to 60% of the transcriptome [243]. Recent studies have

suggested that various pathogenic bacteria can influence host miRNA expression in the GI tract.

For instance, H. pylori-positive patients exhibit a different miRNA signature in gastric mucosa

compared to controls [262]. The expression of the miR-200 family is downregulated in Listeria

infected mouse ileal tissue [38, 264], while miR-146 and miR-155 were upregulated in Listeria-

infected human intestinal epithelial caco-2 cells [251] and in Salmonella-infected mouse

phagocytes [39]. Intriguingly, miR-155-deficient mice have been shown to be more susceptible

to C. rodentium infection than wild-type mice [268], suggesting that miRNAs may also play a

role in the colonic infection by C. rodentium. However, a comprehensive analysis of the host

response to this pathogen at the miRNA level is lacking. Thus, the aim of this study was to

examine if C. rodentium infection alters miRNA signature of the mouse colon and if the

alterations are involved in the host response to the infection.

51

4.3 Materials and Methods

Mice

Animal study design and procedures were approved by the animal ethics committee at the

University of Toronto (Animal Use Protocol Number: 20010228) and were in accordance with

the Regulations of the Animals for Research Act in Ontario and the Guidelines of the Canadian

Council on Animal Care. Forty conventional C57Bl6/J male mice, six weeks of age, were

obtained from Jackson Laboratories (Bar Harbor, ME), and housed in filtered cages with sterile

bedding, and sterile chow diet and water ad libitum. Mice were randomized into two groups

(n=20/group), sham infected and C. rodentium infected. Sample size was calculated based on a

priori sample size calculation performed in G*Power 3.1.9.2 [327] (n=9 based on outcomes of

intestinal permeability and crypt hyperplasia in a published paper [323]). Infection was

performed by intra-gastric gavage of 100 µl Luria-Bertani (LB)-cultured Citrobacter rodentium

(16 hours overnight culture, 109

colony forming units (CFU)/ml) or an equal volume of sterile

LB (sham) as previously described by Bhinder et al [328]. Body weights were measured and

freshly passed fecal pellets were collected every other day and just before sacrifice. All mice

were sacrificed on day 10 post-infection (p.i.), which is the peak of infection as discussed in

chapter 2, by cervical dislocation after a brief exposure to carbon dioxide (Figure 4.1); distal

colon (the major site of infection on day 10 p.i. and defined as the distal 3.5 cm of the colon after

excision of the rectum), kidneys, spleen, liver, and adipose tissue were dissected on ice and fixed

in 10% formalin or snap-frozen in liquid nitrogen and stored at -80°C.

52

Figure 4.1 Study Design

Forty male C57BL/6J mice were randomized into a sham infected and a C. rodentium infected group. Infection was

performed by intra-gastric gavage on day 0. Body weight and fecal C. rodentium load were monitored every other

day. All mice were sacrificed at 10 days post infection where their intestinal integrity and intestinal miRNA

expression were examined.

53

C. rodentium culturing and quantification

For gavage, C. rodentium DBS100 (kindly provided by Dr. Philpott, Department of

Immunology, University of Toronto) was grown in LB broth aerobically at 37°C for 16 hours, as

previously described [328]. Viable counts of C. rodentium in liquid culture, fecal and liver

samples were determined by plating on MacConkey Agar (BioShop) after aerobic incubation for

24 hours at 37°C. Colonies were identified based on morphology (round shape with red color at

the centre and white color at the edges) [152].

Histological analysis

Paraffin blocks of the distal colon (the distal 1 cm of the colon after excision of the rectum) were

used for hematoxylin and eosin (H&E) staining. Five μm sections were assessed microscopically

for scoring the severity of mucosal damages using a scale 0-4 (0, no signs of inflammation; 1,

minimal evidence of inflammatory cell infiltration; 2, significant evidence of inflammatory cell

infiltration; 3, significant evidence of inflammation with goblet cell depletion; 4, sever

inflammation characterized by widespread inflammatory cells infiltration, goblet cell depletion

and destruction of the mucosal architecture) as previously described [329]. The depths of at least

5 well-oriented far-separated crypts per section were measured to determine hyperplasia. A

minimum of 1 transverse and 2 longitudinal sections per distal colon and 5 fields per section

(total 15 measurements) were assessed to determine the mean mucosal damage score and crypt

hyperplasia for each mouse. To reduce bias, samples were coded such that the examiner was not

aware of which group a sample belongs to.

RNA extraction

Total RNA was isolated from one-half longitudinal segment of the distal colon tissues using the

mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA), as per the manufacturer’s

instructions, eluted in 50 μl of RNAse-free water and stored at -80°C. Recovered total RNA

concentration and purity were assessed using Thermoscientific’s Nanodrop 1000

Spectrophotometer.

54

NanoString nCounter miRNA profiling and data analysis

The expression of 578 miRNAs was assessed using 100 ng of total RNA (n=6/group) and the

nCounter Mouse miRNA Assay (NanoString Technologies, Seattle, WA) (miRbase built version

15) at the Princess Margaret Genomics Centre, Toronto, Canada. Data were processed with the

nSolver Analysis Software v1.1 (NanoString). Code counts were normalized to the geometric

mean of the top 100 miRNAs and background (Mean negative controls + 2 SD) was subtracted; only

probes that were above the background in 80% of the samples of any of the two groups were

retained. For the purpose of this study, probes detecting more than one miRNAs simultaneously

are counted as one miRNA. The final dataset was log2-transformed before statistical analysis.

The relative expression between groups was expressed as Infected/Sham. Student’s t-test was

used to calculate statistical significance (p<0.05). Unsupervised hierarchical clustering was

performed using MultiExperiment Viewer software (MeV 4.9; Institute of Genomic Research,

Rockville, MD) operating under the R statistical computing environment.

MiRNAs target prediction and pathway analysis

Experimentally validated target genes of the differentially expressed miRNAs were identified in

silico using the miRWalk database [330] with a complementary manually curated search. These

gene targets were used for pathway analysis using the PANTHER Classification System version

10.0 [331]. Regulatory networks of miRNA-mRNA targets were visualized using Cytoscape

3.2.1 [332].

Real-Time quantitative PCR (qPCR)

Total RNA (10ng) was reverse transcribed with the Taqman® MicroRNA Reverse Transcription

Kit and primers specific for miR-148a (Assay ID: 000470), miR-200a (Assay ID: 000502), miR-

200b (Assay ID: 002251), and the endogenous control snoRNA135 (Assay ID: 001230) (Applied

Biosystems, Foster City, CA). Real-time PCR was then conducted using undiluted cDNA, and

the TaqMan 2X Universal PCR Master Mix, No AmpEraseUNG (Applied Biosystems) in a 10 μl

PCR reaction. Each reaction was run in triplicates in a 384-well optical plate in Applied

Biosystems’ 7900 HT thermocycler using the 9600 emulation mode with an initial hold at 95°C

for 10 minutes followed by 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds.

55

For gene expression analysis, 2µg of purified total RNA were reverse transcribed with the High

Capacity cDNA RT kit (Applied Biosystems) according to the manufacturer’s instructions. Real-

time PCR was carried out using 100ng of cDNA product in 10µl PCR reaction with TaqMan

Gene Expression Assay (Hprt assay ID: Mm01545399_m1, Zeb1 assay ID: Mm00495564_m1,

Bcl2l11 assay ID: Mm00437796_m1, Ctnnb1 assay ID: Mm00483039_m1, Epas1 assay ID:

Mm01236112_m1, Slc15a1 assay ID: Mm04209483_m1) and TaqMan Gene Expression Master

Mix (Applied Biosystems). Each reaction was run in triplicates in a 384-well optical plate in

Applied Biosystems’ 7900 HT Real-Time PCR machine with default thermocycling conditions.

The 2−ΔΔ

CT method was used to calculate relative expression levels [333] for both miRNA and

mRNA using sno-135 and Hprt as normalizers, respectively. Significance was determined using

QIAGEN 2009 Relative Expression Software Tool (REST) [334].

Fluorescein Isothiocyanate–Dextran in vivo Permeability Assay

On day 10 p.i. overnight fasted mice (10/group) were gavaged with 4 kDa isothiocyanate

(FITC)-dextran (Sigma – Aldrich) at 88 mg/mL in 100 µL of sterile PBS. Four hours after

gavage, blood was collected by cardiac puncture and serum FITC-dextran concentration was

quantified by fluorometry (FusionTM

, PerkinElmer) with excitation wavelength of 485 nm and

emission wavelength of 535 nm as previously described [328].

Statistical Analysis

Student’s t-test was use to determine statistically significant differences in fecal C. rodentium

load and body and organ weights, crypt hyperplasia, colon damage scores, intestinal

permeability, and bacterial translocation between the infected and the sham group. Bacterial

counts were expressed as log10/g of feces. A p-value of p<0.05 was considered as statistically

significant, with an exception in PANTHER pathway analysis where p<0.0001 was considered

as statistically significant. All statistical analyses were performed in GraphPad Prism 6 software

(La Jolla, CA, USA). Analysis of NanoString and qPRC miRNA expression is described above.

56

4.4 Results

C. rodentium infection kinetics and characteristics

C. rodentium was detectable in the feces two days after infection. Viable count continued to

increase until reaching a plateau (109 CFU/g) at day 8-10 p.i. (Figure 4.2a). Infection did not

result in significant body weight change (Figure 4.2b). Spleen weights of the infected mice were

significantly higher than the sham (0.53 ± 0.007 vs. 0.29 ± 0.071 % body weight; p<0.01); there

was no significant difference in kidney, liver and adipose tissue weights between the two groups

(Figure 4.2c).

Intestinal histology and barrier integrity

C. rodentium infected mice exhibited a significant increase in distal colon crypt lengths

compared to the sham (262.3 ± 11.5 vs.186.8 ± 5.2 μm; p<0.0001) (Figure 4.3a). The infection

resulted in significant histological damages to the distal colon (damage score 3.0 ± 0.17 vs. 0.4 ±

0.04; p<0.0001) (Figure 4.3b), as evidenced by significant signs of lymphocyte infiltration,

goblet cell depletion, and even loss of mucosal architecture (Figure 4.3c,d,e,f). C. rodentium

infected mice also experienced loss of intestinal barrier integrity. Intestinal permeability was

significantly higher in the infected mice compared to the sham mice (1042.5 ± 233.4 vs. 443 ±

71.6 ng/ml; p<0.05) on day 10 p.i. (Figure 4.4a). Moreover, C. rodentium was detectable in the

liver of infected but not control mice (102 CFU/g, Figure 4.4b).

Distal colon microRNA signature

One hundred and forty-six probes were detectable in the distal colon. Of these, 91 were

differentially expressed between C. rodentium-infected and sham-infected mice (p<0.05), where

42 were downregulated and 49 were upregulated in response to C. rodentium (0.2-8.6 fold)

(Table 4.1). Some probes can detect more than one miRNAs (eg. mmu-miR-20a+mmu-miR-

20b); one probe is counted as one miRNA in this project. Unsupervised hierarchical clustering

analysis revealed that infected samples clearly clustered together and separately from the

controls based on the expression profiles of these 91 miRNAs (Figure 4.5a). The expression of 3

selected miRNAs (miR-148a, miR-200a, and miR-200b), which were statistically significant

down-regulated by C. rodentium, was validated by qPCR (Figure 4.5b).

57

a.

b.

c.

Figure 4.2 C. rodentium infection kinetics and effect on mouse body and organ weights.

(a) Viable C. rodentium counts in infected mice feces collected every other day post-infection (p.i.); (b) body

weights of sham and infected mice p.i., n=20/group; (c) percent of organ weight per gram of body weight between

sham and infected mice on day 10 p.i., n=10/group. Statistical significance was determined by Student’s t-test, **P

< 0.01.

58

a.

b.

c.

d.

e.

f.

Figure 4.3 C. rodentium induced intestinal lesions, crypt hyperplasia and barrier

dysfunction.

(a) Tissue damage score and (b) crypt length of the distal colon were assessed blindly (n=10/group). Representative

hematoxylin and eosin stained histological slides were taken on day 10 p.i. (c) score 0: no inflammation with normal

crypt and goblet cells (black arrows), (d) Score 1: minimal evidence of inflammatory infiltrate (black circle), (e)

Score 3: significant evidence of inflammatory infiltrate (black circle) with goblet cell depletion, (f) Score 4: severe

inflammation with widespread lymphocyte infiltration, goblet cell depletion, and destruction of architecture (red

arrow). m: mucosa; s: submucosa; ms: muscularis. 400X. Results are expressed as mean ± SE. Statistical

significance was determined by Student’s t-test, ***P < 0.001.

s

m

ms

m s ms

m

s

m

s

59

a.

b.

Figure 4.4 Loss of barrier integrity of C. rodentium infected mice on day 10 p.i.

(a) Infected mice had an increased serum concentration of the orally administered macromolecule FITC-dextran (4-

kDa), n=8-10. Barrier intergrity was also assessed based (b) translocation of C. rodentium to the liver, n=10/group.

Results are expressed as mean ± SE. Statistical significance was determined by Student’s t-test, *P < 0.05. N.D, not

detectable.

60

Table 4.1 Differentially Expressed miRNAs

61

a.

b.

Figure 4.5 C. rodentium infected mice exhibit distinct miRNA signature.

(a) Heat map shows unsupervised hierarchical clustering of the 91 differentially expressed miRNAs between

infected and sham samples, n=6/group; statistical significance was determined by Student’s t-test. The red color

denotes log2 expression level above the mean, and green color denotes log2 expression level below the mean. (b)

qPCR validation of expression levels of selected miRNAs, n=8-9/group (for 2 of the samples in the Sham group and

1 in the CR group the amount of RNA was not sufficient for qPCR). Results are expressed as mean ± SE. The three

miRNAs were significantly decreased in the CR group (*P<0.05, **P < 0.01, ***P < 0.001 based on Student’s t-

test), and there are no statistical significant differences between the qPCR and Nanostring techniques.

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Identification of gene targets of differentially expressed miRNAs and pathway analysis

Fifty-eight differentially expressed miRNAs with fold change > 1.5 or < 0.67 were carried

forward for gene target analysis. In silico analysis identified 11579 putative and 865

experimentally validated gene targets of these 58 miRNAs. Of the 865 genes, 824 were mapped

to the Gene ontology PANTHER classification system. Gene ontology classification analysis

revealed that several categories were significantly enriched (p<0.0001) among these validated

targets: (1) biological processes, including 55, 53, and 36% of the annotated genes classified into

the metabolic process, cellular process (eg. cell cycle and communication), and biological

regulation categories, respectively (Table 4.2); (2) molecular functions, including 48, 31, and

18% of the annotated genes classified as protein and nucleic acid binding, catalytic activity, and

transcription factor activity genes, respectively (Table 4.3); (3) cellular component, with 37 and

13% of the annotated genes classified to the cell part, and organelle (eg. cytoskeleton) categories

(Table 4.4). The majority of the genes were annotated to distinct pathways (Table 4.5), and

PANTHER Pathway Overrepresentation test revealed that many of the pathways involved in

inflammatory response and cell cycle regulation such as the Gastrin and cholecystokinin receptor

(CCKR), Tlr, TGF-β, apoptosis, and Wnt signalling pathways, are significantly enriched

(p<0.0001) (Figure 4.6).

63

Table 4.2 GO Biological Process (total number of genes: 824; total number process hits: 2158).

Category name (Accession)

# of

genes

Percent of gene hit

against total # genes

Percent of gene hit against

total # Process hits

cellular component organization or

biogenesis (GO:0071840) 74 9.00% 3.40%

cellular process (GO:0009987) 434 52.70% 20.10%

localization (GO:0051179) 116 14.10% 5.40%

apoptotic process (GO:0006915) 59 7.20% 2.70%

reproduction (GO:0000003) 34 4.10% 1.60%

biological regulation (GO:0065007) 300 36.40% 13.90%

response to stimulus (GO:0050896) 177 21.50% 8.20%

developmental process (GO:0032502) 232 28.20% 10.80%

multicellular organismal process

(GO:0032501) 115 14.00% 5.30%

locomotion (GO:0040011) 11 1.30% 0.50%

biological adhesion (GO:0022610) 39 4.70% 1.80%

metabolic process (GO:0008152) 451 54.70% 20.90%

growth (GO:0040007) 1 0.10% 0.00%

immune system process (GO:0002376) 115 14.00% 5.30%

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Table 4.3 GO Molecular Function (total number of genes: 824; total number process hits:

1062).

Category name (Accession)

# of

genes

Percent of gene hit

against total # genes

Percent of gene hit against

total # Function hits

transporter activity (GO:0005215) 41 5.00% 3.90%

translation regulator activity

(GO:0045182) 6 0.70% 0.60%

protein binding transcription factor activity

(GO:0000988) 8 1.00% 0.80%

enzyme regulator activity (GO:0030234) 50 6.10% 4.70%

catalytic activity (GO:0003824) 256 31.10% 24.10%

receptor activity (GO:0004872) 101 12.30% 9.50%

nucleic acid binding transcription factor

activity (GO:0001071) 145 17.60% 13.70%

antioxidant activity (GO:0016209) 5 0.60% 0.50%

structural molecule activity (GO:0005198) 55 6.70% 5.20%

binding (GO:0005488) 395 47.90% 37.20%

65

Table 4.4 GO Cellular Component (total number of genes: 824; total number process hits:

487).

Category name (Accession)

# of

genes

Percent of gene hit

against total # genes

Percent of gene hit against

total # Component hits

synapse (GO:0045202) 5 0.60% 1.00%

cell junction (GO:0030054) 4 0.50% 0.80%

membrane (GO:0016020) 62 7.50% 12.70%

macromolecular complex (GO:0032991) 40 4.90% 8.20%

extracellular matrix (GO:0031012) 23 2.80% 4.70%

cell part (GO:0044464) 180 21.80% 37.00%

organelle (GO:0043226) 110 13.30% 22.60%

extracellular region (GO:0005576) 63 7.60% 12.90%

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Table 4.5 Panther Pathways (total number of genes: 824; total number process hits: 895).

Category name (Accession)

# of

genes

Percent of gene hit

against total # genes

Percent of gene hit against

total # Pathway hits

Toll_pathway_drosophila (P06217) 1 0.10% 0.10%

SCW_signaling_pathway (P06216) 1 0.10% 0.10%

DPP_signaling_pathway (P06213) 1 0.10% 0.10%

DPP-SCW_signaling_pathway (P06212) 1 0.10% 0.10%

BMP_signaling_pathway-drosophila

(P06211) 1 0.10% 0.10%

Axon guidance mediated by netrin

(P00009) 1 0.10% 0.10%

Axon guidance mediated by Slit/Robo

(P00008) 4 0.50% 0.40%

Axon guidance mediated by semaphorins

(P00007) 1 0.10% 0.10%

Apoptosis signaling pathway (P00006) 31 3.80% 3.50%

Gonadotropin releasing hormone receptor

pathway (P06664) 63 7.60% 7.00%

Angiogenesis (P00005) 37 4.50% 4.10%

Ornithine degradation (P02758) 1 0.10% 0.10%

Alzheimer disease-presenilin pathway

(P00004) 22 2.70% 2.50%

Alzheimer disease-amyloid secretase

pathway (P00003) 8 1.00% 0.90%

Alpha adrenergic receptor signaling

pathway (P00002) 2 0.20% 0.20%

67

Methylmalonyl pathway (P02755) 1 0.10% 0.10%

Adrenaline and noradrenaline biosynthesis

(P00001) 1 0.10% 0.10%

CCKR signaling map (P06959) 51 6.20% 5.70%

Lysine biosynthesis (P02751) 1 0.10% 0.10%

Ubiquitin proteasome pathway (P00060) 3 0.40% 0.30%

p53 pathway (P00059) 14 1.70% 1.60%

Wnt signaling pathway (P00057) 36 4.40% 4.00%

Heme biosynthesis (P02746) 1 0.10% 0.10%

VEGF signaling pathway (P00056) 14 1.70% 1.60%

Transcription regulation by bZIP

transcription factor (P00055) 2 0.20% 0.20%

Toll receptor signaling pathway (P00054) 18 2.20% 2.00%

Formyltetrahydroformate biosynthesis

(P02743) 2 0.20% 0.20%

T cell activation (P00053) 17 2.10% 1.90%

Folate biosynthesis (P02742) 1 0.10% 0.10%

TGF-beta signaling pathway (P00052) 28 3.40% 3.10%

Plasminogen activating cascade (P00050) 5 0.60% 0.60%

De novo pyrimidine deoxyribonucleotide

biosynthesis (P02739) 2 0.20% 0.20%

Parkinson disease (P00049) 13 1.60% 1.50%

De novo purine biosynthesis (P02738) 3 0.40% 0.30%

PI3 kinase pathway (P00048) 14 1.70% 1.60%

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PDGF signaling pathway (P00047) 21 2.50% 2.30%

Oxidative stress response (P00046) 9 1.10% 1.00%

Notch signaling pathway (P00045) 10 1.20% 1.10%

Nicotinic acetylcholine receptor signaling

pathway (P00044) 10 1.20% 1.10%

Muscarinic acetylcholine receptor 2 and 4

signaling pathway (P00043) 5 0.60% 0.60%

Muscarinic acetylcholine receptor 1 and 3

signaling pathway (P00042) 7 0.80% 0.80%

Metabotropic glutamate receptor group I

pathway (P00041) 3 0.40% 0.30%

Asparagine and aspartate biosynthesis

(P02730) 1 0.10% 0.10%

Metabotropic glutamate receptor group II

pathway (P00040) 4 0.50% 0.40%

Synaptic_vesicle_trafficking (P05734) 1 0.10% 0.10%

GABA-B_receptor_II_signaling (P05731) 5 0.60% 0.60%

Endogenous_cannabinoid_signaling

(P05730) 2 0.20% 0.20%

Ascorbate degradation (P02729) 1 0.10% 0.10%

Arginine biosynthesis (P02728) 1 0.10% 0.10%

Metabotropic glutamate receptor group III

pathway (P00039) 6 0.70% 0.70%

JAK/STAT signaling pathway (P00038) 6 0.70% 0.70%

Ionotropic glutamate receptor pathway

(P00037) 5 0.60% 0.60%

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Interleukin signaling pathway (P00036) 24 2.90% 2.70%

Interferon-gamma signaling pathway

(P00035) 7 0.80% 0.80%

Xanthine and guanine salvage pathway

(P02788) 1 0.10% 0.10%

Adenine and hypoxanthine salvage

pathway (P02723) 1 0.10% 0.10%

Integrin signalling pathway (P00034) 23 2.80% 2.60%

Insulin/IGF pathway-protein kinase B

signaling cascade (P00033) 10 1.20% 1.10%

Insulin/IGF pathway-mitogen activated

protein kinase kinase/MAP kinase cascade

(P00032) 14 1.70% 1.60%

p53 pathway feedback loops 2 (P04398) 10 1.20% 1.10%

Inflammation mediated by chemokine and

cytokine signaling pathway (P00031) 40 4.90% 4.50%

p53 pathway by glucose deprivation

(P04397) 4 0.50% 0.40%

Hypoxia response via HIF activation

(P00030) 3 0.40% 0.30%

Vitamin D metabolism and pathway

(P04396) 2 0.20% 0.20%

Thyrotropin-releasing hormone receptor

signaling pathway (P04394) 4 0.50% 0.40%

Thiamine metabolism (P02780) 1 0.10% 0.10%

Ras Pathway (P04393) 17 2.10% 1.90%

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Oxytocin receptor mediated signaling

pathway (P04391) 3 0.40% 0.30%

Huntington disease (P00029) 19 2.30% 2.10%

Heterotrimeric G-protein signaling

pathway-rod outer segment

phototransduction (P00028) 3 0.40% 0.30%

Heterotrimeric G-protein signaling

pathway-Gq alpha and Go alpha mediated

pathway (P00027) 10 1.20% 1.10%

p38 MAPK pathway (P05918) 14 1.70% 1.60%

Heterotrimeric G-protein signaling

pathway-Gi alpha and Gs alpha mediated

pathway (P00026) 10 1.20% 1.10%

Opioid proopiomelanocortin pathway

(P05917) 4 0.50% 0.40%

Hedgehog signaling pathway (P00025) 4 0.50% 0.40%

Opioid prodynorphin pathway (P05916) 2 0.20% 0.20%

Glycolysis (P00024) 1 0.10% 0.10%

Opioid proenkephalin pathway (P05915) 2 0.20% 0.20%

General transcription regulation (P00023) 1 0.10% 0.10%

Nicotine pharmacodynamics pathway

(P06587) 3 0.40% 0.30%

Enkephalin release (P05913) 5 0.60% 0.60%

FGF signaling pathway (P00021) 21 2.50% 2.30%

Dopamine receptor mediated signaling

pathway (P05912) 5 0.60% 0.60%

71

FAS signaling pathway (P00020) 7 0.80% 0.80%

Angiotensin II-stimulated signaling

through G proteins and beta-arrestin

(P05911) 8 1.00% 0.90%

Histamine H2 receptor mediated signaling

pathway (P04386) 1 0.10% 0.10%

Pyruvate metabolism (P02772) 1 0.10% 0.10%

Histamine H1 receptor mediated signaling

pathway (P04385) 3 0.40% 0.30%

Gamma-aminobutyric acid synthesis

(P04384) 1 0.10% 0.10%

Cortocotropin releasing factor receptor

signaling pathway (P04380) 2 0.20% 0.20%

Endothelin signaling pathway (P00019) 15 1.80% 1.70%

EGF receptor signaling pathway (P00018) 21 2.50% 2.30%

DNA replication (P00017) 2 0.20% 0.20%

Cytoskeletal regulation by Rho GTPase

(P00016) 11 1.30% 1.20%

Purine metabolism (P02769) 1 0.10% 0.10%

Circadian clock system (P00015) 3 0.40% 0.30%

Cholesterol biosynthesis (P00014) 1 0.10% 0.10%

Cell cycle (P00013) 4 0.50% 0.40%

Cadherin signaling pathway (P00012) 15 1.80% 1.70%

Blood coagulation (P00011) 6 0.70% 0.70%

Beta2 adrenergic receptor signaling 1 0.10% 0.10%

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pathway (P04378)

B cell activation (P00010) 12 1.50% 1.30%

Beta1 adrenergic receptor signaling

pathway (P04377) 1 0.10% 0.10%

5HT4 type receptor mediated signaling

pathway (P04376) 1 0.10% 0.10%

5HT3 type receptor mediated signaling

pathway (P04375) 1 0.10% 0.10%

5HT2 type receptor mediated signaling

pathway (P04374) 5 0.60% 0.60%

5HT1 type receptor mediated signaling

pathway (P04373) 4 0.50% 0.40%

5-Hydroxytryptamine degredation

(P04372) 1 0.10% 0.10%

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Figure 4.6 Enriched signaling pathways among miRNA-regulated gene targets.

In silico analysis identified 865 genes as experimentally validated targets of the top differentially expressed miRNA

(fold-change > 1.5 or < 0.67). Listed pathways were significantly (p<0.0001) enriched among these genes based on

the PANTHER Pathways Overrepresentation Test version 10.0. s.p., signalling pathway.

74

Regulatory networks of miRNA-gene targets

A regulatory network of 6 miRNAs of interest and their experimentally verified targets involved

in the Tlr and Wnt signalling pathways was constructed (Figure 4.7). These selected miRNAs are

highly expressed with large fold-changes based on NanoString data, and have been previously

implicated to play a role in host-microbial interaction, inflammatory response and cell cycle

regulation based on the manual curated search and as discussed in Chapter 2.4. Each connection

in the network represents a putative regulatory relationship between miRNA and mRNA during

C. rodentium infection in mouse colon. The network suggests that the selected miRNAs

cooperate to impact the two important pathways in C. rodentium infection. In particular, miR-

146a targets largely Tlr genes, whereas miR-21 and miR-200s predominately target genes of the

Wnt pathway. Four selected miRNA:mRNA pairs (miR-200a/b:Zeb1; miR-148a/152:Ctnnb1,

200a/b:Ctnnb1; miR-146a:Tlr4) did not show inverse correlation, since the gene targets were

downregulated (Zeb1: 0.37 fold, Ctnnb1: 0.58 fold) or unchanged (Tlr4) in infected mice (Figure

4.8). Because the currently available databases are incomplete, additional pairs of interest were

identified by manually curated search. These include: miR-148a:Epas1 and miR193:Slc15a1

(fold-change 0.30 and 0.07) (Figure 4.8) but these pairs again did not show inverse correlation.

75

Figure 4.7 Putative regulatory network of selected miRNAs.

A regulatory network linking selected miRNAs to their experimentally verified gene targets. Purple ovals are genes

associated with the Wnt pathway, and yellow circles are genes associated with the TLR pathway. Red and green

rectangles are upregulated and downregulated miRNAs by C. rodentium, respectively.

76

Figure 4.8 Expression of selected genes in distal colon of sham and infected mice.

Gene expression was evaluated by qPCR (n=7-9/group, for 3 of the samples in the Sham group and 1 in the CR

group the amount of RNA was not sufficient for qPCR). Data are presented as relative fold change in infected mice

versus sham mice. Delta CTs were normalized to Hprt1 (except for Tlr4 that was normalized to β-actin). The tested

genes were all significantly downregulated in C. rodentium infected mice based on the 2−ΔΔ

CT method. Statistical

significance was determined by REST, ***P < 0.001.

77

Finally, crossing the 865 experimentally validated gene targets of the deregulated miRNAs in the

present study with published DNA microarray data of differentially expressed genes in C.

rodentium-infected mouse colon from two independent studies [34, 35] revealed 103 overlapping

genes. Of these genes, Bim was of interest for its expression correlated inversely with a number

of highly upregulated miRNAs targeting it. More importantly, Bim is annotated to apoptosis

process under the biological process category; although it is not currently annotated into any

signaling pathways on PANTHER, Bim is a potential downstream effector of the enriched CCK

[335-337] and the apoptosis [338] signaling pathways, based on literature search. The

downregulation of Bim (0.49 fold) was validated in the present study by qPCR. Based on

expression levels of miRNAs targeting Bim a regulatory network was defined (Figure 4.9). The

network illustrates miRNA regulation of Bim expression and reveals the simultaneous impact of

the differentially expressed miRNAs on the expression of this gene with a net suppressive effect

on Bim expression during C. rodentium infection. Specifically, miR-92a (8.6 fold), miR-106/17

(3.0 fold), miR-19a (2.9 fold), miR-20a/b (2.9 fold), miR-93 (2.0 fold), miR-19b (1.5 fold), miR-

130b (1.5 fold), miR-32 (1.5 fold), miR-25 (1.3 fold) were upregulated and miR-148a (0.3 fold)

and miR-181a (0.6 fold) were downregulated by C. rodentium, suggesting that Bim is a key

candidate gene target of miRNA-mediated response of the host to C. rodentium infection.

Notably, most of these overexpressed miRNAs belong to the miR-17-92 cluster (miR-17, -18a, -

19a, -20a, -19b-1, -92a-1), and its two paralogues miR-106a-363 cluster (miR-106a, -18b, -20b, -

19b-2, -92a-2, -363) and miR-106b-25 cluster (miR-106b, -93, -25), which together will be

referred to as miR-17-92-related clusters from this point on.

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Figure 4.9 Action of 11 differentially expressed miRNAs on Bim.

Differentially expressed miRNAs that have been experimentally verified to directly target Bim are shown in

rectangles. The color intensity represents miRNA fold-change in infected mice versus sham mice, where red and

green denote over- and under-expression, respectively, in C. rodentium infected mice.

79

4.5 Discussion

Alteration of the transcriptome underlies the response of the host to C. rodentium infection. This

study explored the impact of this pathogen at the post-transcriptional level. It was found that C.

rodentium infection alters the colonic miRNA signature with implications for several pathways

including Wnt, Tlr and apoptosis.

There are only a limited number of studies investigating miRNA expression in response to an

enteric pathogen infection. These include Salmonella [39], Listeria [251] and Campylobacter

infection [40], which, unlike C. rodentium, are pathogens of the small and but not large intestine.

Though, comparing results from the present study with these studies, miR-146 and miR-16 (all

up-regulated), and miR-200a/b and miR-148a (all down-regulated) are also been found to be

differentially expressed in a consistent manner in at least one other enteric pathogen infection. In

particular, miR-146 and miR-16 are upregulated in H. pylori-infected gastric epithelial cells

[263, 273] and Listeria-infected intestinal epithelial Caco-2 cells [251]; miR-200a/b are

downregulated in H. pyori-infected patients’ gastric mucosa [262] and Listeria-infected mouse

ileal tissue [38, 264]; miR-148a is downregulated in Listeria-infected mouse ileal tissue[38].

Thus, there might be a consensus of pathogen-induced miRNA alterations at different regions of

the GI tract. However, this requires further examination, since most of the current published

studies did not examine miRNA profile in a comprehensive manner and many of our

differentially expressed miRNAs were not included in those analyses. Indeed, it might not be

surprising that miR-146 is consistently up-regulated in response to all of these pathogens, as it is

a well-established negative regulator of innate immune response and its expression is induced

upon Tlr signal activation. The increase of miR-146 is associated with improved immune

tolerance by suppressing immune-related genes like interleukin 1 receptor-associated kinase

(Irak1) and Tumor necrosis factor receptor-associated factor 6 (Traf6) [254, 256, 257, 339, 340].

Although in the present study the increase of miR-146a did not significantly suppress Tlr4

expression, it does not exclude that this gene is regulated by the miRNA. It is known that C.

rodentium activates host innate immune response via Tlr4 signaling cascade [120, 122, 123], and

Tlr4 expression has protective effect against Gram-negative pathogens such as Salmonella and

uropahogenic E. coli [341, 342]. However, C. rodentium infection did not induce Tlr4 expression

changes in the present study. This is in line with Khan et al. who previously suggested that Tlr4

expression in C. rodentium was a maladaptive response that is dispensable for the host defense

80

against the pathogen [122]. This was based on the finding that Tlr4-deficient mice have

attenuated disease severity including C. rodentium colonization, hyperplasia, and weight loss

compared to the wild-type. They also observed that Tlr4 mRNA was expressed at comparable

levels on day 6 and 10 p.i. [122]. This is in line with our results, indicating that Tlr4 expression

might be tightly regulated as its expression does not seem to confer benefit to the host during

infection. Hence, it can be postulated that miR-146a is perhaps involved in this tight regulation

of Tlr4 level. Furthermore, some of these miRNA alterations might be microbiota-dependent, as

it was found that the decreases of miR-148a and miR-200b expression were only observed in

conventional, but nor germ-free, mice upon L. monocytogenes infection [38]. Similarly, it is

known that the expression of miR-148a in the intestine depends on the presence of the

microbiota [37]. A few studies showed that C. rodentium infection can be mitigated by probiotic

administration [152, 319-323] and one of the hallmarks of probiotic action is their effect on

microbiota composition. Thus, miRNA regulation may be one of the mechanisms underlying

beneficial effects of probiotics in C. rodentium infection. However, it is important for future

studies to consider the incorporation of control groups with chemically induced inflammation,

such as DSS-induced colitis, to further isolate miRNA response specifically due to C. rodentium

infection.

C. rodentium causes intestinal inflammation and hyperplasia that resemble IBD and colon

tumorigenesis [343]. Interestingly, among the 58 top-regulated miRNAs, 8 (miR-200b, miR-192,

miR-16, miR-21, miR-146a, miR-93, miR-132 and mir-106a+miR-17) [344, 345] and 16

miRNAs (miR-26a, miR-200a, miR-200b, miR-148a, miR-152, miR-192, miR-194, miR-150,

miR-21, miR-146a, miR-223, miR-92a, miR-106a+miR-17, miR-20a+miR-20b, miR-19a, miR-

93) [346-352] are consistently modulated in these two pathologies, respectively. Among the

above, miR-21 is an oncomir associated with IBD. Overexpression of miR-21 was seen in IL-10

knockout mice[287], DSS-treated mice[284] and mucosal biopsies of UC patients[285]. Shi et al.

showed that knocking out miR-21 in DSS-treated mice improves survival rate and attenuates

inflammation [284]. Intestinal barrier dysfunction is a marked characteristic of IBD as well as C.

rodentium infection. Yang et al. found in Caco-2 cells that increased expression of miR-21 can

impair the tight junction gene, RhoB, leading to increased intestinal permeability [285]. In this

study, paracellular permeability was increased in response to C. rodentium, suggesting that miR-

21 may play a role in this phenotypic outcome. MiR-93 and miR-106a, together with miR-17,

81

miR-19a, miR-20a/b, miR-92a and miR-93 belong to the miR-17-92-related clusters, which are

highly up-regulated clusters by C. rodentium. Overexpression of miR-93 and miR-106a are

believed to disrupt autophagy events and pathogen clearance, resulting in chronic inflammatory

state seen in IBD [353, 354]. The miR-17-92 cluster is also known as oncomir-1 [355] and its

overexpression promotes proliferation and reduces apoptosis of epithelial cells in CRC [356-

358]. Thus, the increased expression of these clusters may have implications in inflammatory

response as well as crypt hyperplasia during C. rodentium infection. Moreover, miR-200 family

is one of the top downregulated miRNAs in our study. This family inhibits epithelial-

mesenchymal-transition (EMT) in CRC by directly targeting the zinc finger E-box-binding

homeobox (Zeb) to maintain expression of the epithelial marker E-cadherin [359]. The

downregulation of miR-200 family will result in loss of inhibition on Zeb, followed by loss of

epithelial phenotype and cell-cell adhesion, contributing to tumorigenesis and metastasis [305,

360, 361]. Hence, reduced levels of miR-200a/b in C. rodentium infection may also contribute to

the hyperproliferation of TA cells, which do not possess epithelial phenotype and can proliferate

without being restricted by cell-cell contact [15]. Generally speaking, the fact that some miRNA

alterations induced by C. rodentium are commonly seen in both IBD and CRC may reflect the

fundamental pathological similarities between these conditions and may infer a regulatory role of

miRNAs in the pathogenesis of these conditions.

Pathway analysis of the miRNA targets revealed a number of enriched pathways, with apoptosis,

Tlr, and Wnt signaling pathways being of particular interest in the context of C. rodentium

pathology. Based on the findings discussed above, miR-21, miR-146a, miR-148a, miR-200a/b,

and miR-17-92 cluster were identified as candidate miRNAs involved in regulating these

pathways. Potential regulatory networks of the selected miRNAs were generated for the three

enriched pathways based on bioinformatics in order to identify differentially expressed genes

corresponding to miRNA alterations. In these networks, genes, including Bim, Bcl2, Zeb1/2,

Ctnnb1, and Apc, have been previously shown to be deregulated in tumorigenesis. It is worth

noticing that pathway analysis based on the available databases is often limited due to the

inherent bias in the amount of data available for different biological pathways. Some pathways,

for example cancer-related, are better studied than others; thus, genes in those pathways might be

more represented in the current databases. In addition, since the current databases are still being

optimized, genes can often be misclassified. For example, Bim belongs to the Bcl2 family of

82

apoptosis-related genes and functions as an apoptosis facilitator [338], but it has not yet been

categorized into the apoptosis regulator pathway on PANTHER Classification System.

Therefore, one limitation of using bioinformatics analysis in this study is that confounding

results can be produced while important information can be lost. With that said, the current study

incorporates an extensive manual literature search to complement these disadvantages. Several

miRNA:mRNA pairs are of interest in the context of C. rodentium infection. These include miR-

200a/b:Zeb1; miR-148a/152:Ctnnb1, 200a/b:Ctnnb1; miR-146a:Tlr4; miR-148a:Epas1 and miR-

193:Slc15a1; miR-17-92-related clusters:Bim. While Zeb1, Ctnnb1, EPAS1 and Slc15a1 are

experimentally validated targets of their corresponding miRNA, they were not inversely

regulated upon C. rodentium infection.

C. rodentium infection can also induce hypoxia response. One study by Xue et al. showed that

expression of endothelial PAS domain protein 1(Epas1), a hypoxia-inducible factor, is increased

upon C. rodentium infection, and genetic deletion of Epas1 attenuates inflammation in mouse

colon [324]. On the contrary, the present study revealed that Epas1 was significantly down-

regulated in response to C. rodentium infection. This could be due to fundamental differences

between the two studies. In the study of Xue et al. Epas1 expression was examined 7 days p.i. at

protein level compared to 10 days p.i. at mRNA level in the present study. In the microarray

study by Borenshtein et al. many genes significantly increased on day 4 post-C. rodentium

infection were significantly reduced on day 9 p.i.[34]. Although it requires further validation, it

is possible that Epas1 expression is time-dependent as infection progresses, where by day 10 p.i.

the expression of Epas1 at mRNA level might have been reduced after the initial increase

responsible for the elevation of protein expression observed on day 7 p.i.. It has been shown that

miR-148a and miR-20b directly regulate Epas1 expression in human embryonic stem cells; and

underexpression of both miR-148a and miR-20b increases Epas1 resulting in mesenchymal stem

cell phenotype [362]. Notably, as opposed to miR-148a, miR-20b (a member of miR-106a-363

cluster) is highly up-regulated in the current study. As a matter of fact, Epas1 is a predicted

target of the miR-17-92, miR-106a-363 and miR-106b-25, which are all highly up-regulated.

Hence, miRNA regulation may account for the low expression of Epas1 at mRNA level 10 days

after C. rodentium infection.

Another gene of interest is Slc15a1 (solute carrier family 15 member 1), which functions to

transport bacterial peptide products into epithelial cells to induce inflammation [325]. It has been

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shown that being a target of miR-193a, Slc15a1 is induced at mRNA and protein levels in mouse

colon 6 hours and 7 days after C. rodentium infection [325, 326]. However, there is a robust

reduction of Slc15a1 mRNA on day 10 p.i. with a decrease of miR-193a expression in the

current study. This could be a consequence of the activation of inflammation resolving

mechanisms at the peak of infection (day 10 p.i.) to prevent further Slc15a1 protein elevation. In

fact, the discrepancy between Slc15a1 mRNA and protein levels has been observed at later

stages of inflammation. Dai et al. showed that expression of miR-193a correlated inversely with

Slc15a1 protein but not with mRNA in both UC tissues and 12 weeks after DSS-induced colitis

in mice [326]. Therefore, future investigation can examine Slc15a1 expression at both mRNA

and protein levels and at different time points as inflammation progresses. This is a future

direction for other genes, including Zeb1, and Ctnnb1, whose mRNA levels did not show an

inverse relationship with miRNA expression, to elucidate the timing effects of miRNA

regulation on gene expression.

On the contrary the miR-17-92-related clusters:Bim pair displayed an inverse correlation (Figure

4.9). Bim is a potent downstream effector of the TGF-β pathway that promotes physiological

apoptosis in various tissues. Down-regulation of Bim has been associated with resistance to TGF-

β-induced apoptosis and thereby tumorigenesis [363-365]. Sinicrope et al. showed that loss of

Bim expression is linked to poor overall survival rates in patients with colon carcinomas [366].

Low expression of Bim at mRNA and protein levels has also been implicated in skin and renal

carcinoma; therefore, Bim is recognized as a tumor suppressor gene [367, 368]. Here, Bim is

significantly down-regulated. This is in line with published genome-wide microarray data, which

revealed that Bim is one of the significantly down-regulated genes in mouse colon 9-day post C.

rodentium infection [34]. The mechanisms underlying Bim suppression in cancers and C.

rodentium infection are unclear. Several studies have verified that Bim is a direct target of

members in the miR-17-92, miR-106a-363 and miR-106b-25 clusters, as well as miR-130b and

miR-32 [369-371], In particular, Tsuchida et al. determined that high expression of miR-92a

promotes malignant transformation in CRC development by directly suppressing Bim expression

[300]; other studies showed that over-expressing miR-25 or miR-130b in gastric cancer cell lines

impairs TGF-β-induced apoptosis via targeting Bim [252, 370]. Remarkably, all of these

miRNAs targeting Bim were up-regulated in C. rodentium infection. Moreover, Bim has also

been shown to regulate immune responses, where Bim-deficient mice were not able to terminate

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T-cell-mediated responses after viral infection [372, 373]. A recent study found that high

expression of miR-148a in T-lymphocytes upon viral infection reduces Bim expression and

contributes to persistent immune responses in chronic inflammation [374]. In contrast to this

study, miR-148a was down-regulated in response to C. rodentium infection. Yet, C. rodentium

infection does not provoke chronic inflammation and it is unknown whether miR-148a also

regulates Bim in colonic epithelial cells. As Figure 4.9 demonstrates, the effect of the up-

regulated miRNAs is likely to overrule that of the down-regulated miRNAs based on the number

and fold-change of the up- and down-regulated miRNAs targeting Bim, resulting in an overall

down-regulation of Bim in C. rodentium infection. Therefore, these findings suggest that the loss

of Bim-induced apoptosis may contribute to hyperproliferation of the colonic cells in response to

C. rodentium infection and this process is regulated post-transcriptionally via the coordination of

multiple miRNAs.

Taken together, this study shows that C. rodentium–infected mice displayed an altered miRNA

signature. The miRNA alterations are involved the host response to the infection; particularly,

physiological apoptosis is disrupted as a result of miRNA post-transcriptional regulation of the

apoptosis facilitator Bim.

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Chapter 5

Study 2- Effects of Bifidobacterium bifidum on Citrobacter

rodentium Infection via microRNA Modulation

Study Contributions:

The quantitative data for fecal C. rodentium load in Figure 5.2a titled “C. rodentium infection

kinetics and effect on mouse body and organ weights”, and the data for C. rodentium

translocation to liver and spleen in Figure 5.5b titled “B. bifidum effects on intestinal barrier at

day 10 p.i.” were provided by a fourth-year summer research project student, Sofia Sagaidak.

The quantitative data for mouse body weights and organ weights in Figure 5.2b,c titled “C.

rodentium infection kinetics and effect on mouse body and organ weights”, and the data for

caecal crypt lengths and damage score in Figure 5.4a,c titled “B. bifidum effects on intestinal

crypt hyperplasia and tissue damage at day 10 p.i.” were partially contributed by a fourth-year

summer research project student, Alex Lee.

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5.1 Abstract

B. bifidum is a common probiotic. Various strains of B. bifidum have been shown to confer

beneficial effects against pathogen infection and inflammation in animal studies. Administration

of B. bifidum MIMBb75, a highly adhesive strain, was found to be able to influence miRNA

expression in mouse caecum. This study aimed to examine if administration of B. bifidum

MIMBb75 attenuates pathology and miRNA alterations associated with C. rodentium infection.

C57Bl6/J male mice were randomized into four groups, sham, C. rodentium infection, C.

rodentium infection with daily B. bifidum administration initiated on the same days as infection,

and C. rodentium infection with daily B. bifidum administration initiated 7 days before infection.

Mice were sacrificed at day 10 post-infection. Fecal C. rodentium and B. bifidum fecal load were

monitored throughout the study. Crypt hyperplasia and intestinal inflammation were examined

by histology and in vivo intestinal permeability test. Expressions of selected miRNAs were

quantified by real-time PCR in the proximal colon. Presence of B. bifidum did not prevent or

reduce C. rodentium colonization. Intestinal permeability was not different among the groups. B.

bifidum treatment did not attenuate pathology nor normalize miRNA alterations associated with

C. rodentium infection.

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5.2 Introduction

B. bifidum is a Gram-positive anaerobe belonging to the Actinobacteria phylum. B. bifidum is

one of the first colonizers of the infant gut [177] and members of this species have many health-

promoting effects, including microbiota composition improvement [375], immunomodulation

[376], bacteriocin production [377] and pathogen exclusion [211]. These beneficial effects may

have implications in enteric infection and inflammation. For instance, Bayoumi et al. recently

showed that B. bifidum ATCC 29521 interferes with EHEC attachment and colonization in vitro

[31]. López et al. revealed that HT29 cell cultures treated with B. bifidum LMG13195 display

improved monolayer integrity [27]. It was also reported that oral administration of B. bifidum

BGN4 alleviates lymphocytes infiltration and Th1-type cytokines production in the naive T-cell

transfer mouse model of IBD [212]. B. bifidum MIMBb75 exhibits strong adhesive ability to

intestinal epithelial cells [29, 179] and it was found to have beneficial effects in mitigating

intestinal discomfort of IBS patients [23]. The molecular basis for these beneficial effects

attributed to B. bifidum is largely unknown. It is known that microorganisms influence gut

physiology through host gene expression modulation [33]. For instance, global transcriptome

analysis of mouse intestinal cells revealed that B. bifidum PRL2010 colonization was able to

modulate host innate immune response with enhanced production of IL-6 and IL-8 cytokines

[30]. Recent evidence suggests that some probiotic bacteria, such as Lactobacillus casei [264]

and E. coli Nissle 1907 [271], can influence the expression of host epithelial miRNAs, which are

important posttranscriptional regulators of gene expression. Our research group previously found

that mice supplemented with B. bifidum MIMBb75 exhibit a different miRNA signature in the

caecum, where miR-148a was up-regulated in treated mice [272]. Intriguingly, study 1 in the

present project has demonstrated that C. rodentium infection, a model for IBD and colonic

tumorigenesis, substantially altered miRNA signature in mouse colon, with miR-148a being one

of the top down-regulated miRNAs among other differentially expressed miRNAs such as miR-

200a/b, miR-146a, miR-21 and miR-17-92 cluster. However, whether miRNA-mediated

posttranscriptional regulation play a part in the amelioration effects of B. bifidum MIMBb75 has

not been investigated, especially in the context of enteric inflammation induced by C. rodentium

infection. The aim of study 2 was to explore if daily B. bifidum MIMBb75 administration in mice

before and after C. rodentium infection would alleviate C. rodentium colonization and intestinal

barrier dysfunction and inflammation via modulating host miRNA expression.

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5.3 Materials and Methods

Mice

Animal study design and procedures were approved by the animal ethics committee at the

University of Toronto (Animal Use Protocol Number: 20010228) and were in accordance with

the Regulations of the Animals for Research Act in Ontario and the Guidelines of the Canadian

Council on Animal Care. Eighty C57Bl6/J male mice, six weeks of age, were obtained as two

staggered batches (n=40/batch) from Jackson Laboratories (Bar Harbor, ME), and housed in

filtered cages with sterile bedding, and sterile chow diet and water ad libitum. Mice in each batch

were randomized into four groups (total n=20/group): (1) sham infected, (2) C. rodentium

infected (CR), (3) C. rodentium infected with daily administration of B. bifidum initiated on the

same day as, but temporally after, the infection (BB post-CR), (4) C. rodentium infected with

daily B. bifidum administration initiated one week (7 days) before the infection (BB pre-post-

CR) (Figure 5.1). Infection was performed by intra-gastric gavage of 100 µl LB-cultured C.

rodentium (16 hours overnight culture, 109

CFU/ml) or an equal volume of sterile LB (Sham) as

previously described [328]. Daily B. bifidum treatment was performed by intra-gastric gavage of

200 µl B. bifidum suspension (109

CFU/ml) or an equal volume of sterile PBS. C. rodentium

infection and sham infection were performed at 9 until 11 AM on the day of infection; gavage of

B. bifidum and PBS (control) were performed at 2 until 4 PM everyday throughout the study.

Body weights were measured and freshly passed fecal pellets were collected on p.i. days 2, 4, 6,

8, 9 and just before sacrifice. A subset of mice (n=16/group) were sacrificed on day 10 p.i.,

which is the peak of infection as previously discussed, by cervical dislocation after a brief

exposure to carbon dioxide; caecum (the initial site of infection about 2-3 days p.i.) and distal

colon (the major site of infection at day 10 p.i. and defined as the distal 3.5 cm of the colon after

excision of the rectum), kidneys, spleen and liver were dissected on ice and fixed in 10%

formalin or snap-frozen in liquid nitrogen and stored at -80°C. To collect preliminary evidence,

four mice from each group were retained to monitor C. rodentium clearance after day 10 p.i. and

thereafter, freshly passed fecal pellets were collected every other day up to day 28 p.i. based on

the infection cycle of C. rodentium previously described where clearance generally takes place

within 2-3 weeks p.i. [104, 105] . Mice were sacrificed when C. rodentium was completely

cleared (C. rodentium load under the detection limit of 3x103 CFU/ml for two consecutive days).

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Figure 5.1 Study Design.

Eighty male C57BL/6J mice were randomized into four groups after one week of acclimatization. (1) sham infected

(Sham), (2) C. rodentium infected (CR), (3) C. rodentium infected with B. bifidum daily administration initiated on

the same day after the infection (BB post-CR), (4) C. rodentium infected with daily B. bifidum administration

initiated one week (day -7 p.i.) before the infection (BB pre-post-CR). B. bifidum (yellow bars) was administered

from 2 to 4 PM every day by intra-gastric and sterile PBS (blue bars) was used to control for gavage. C. rodentium

(arrows) infection was performed from 9 to 11 AM on day 0 p.i. by intra-gastric gavage. On day 10 p.i., 16

mice/group were sacrificed with a subset (n=8/group) used to perform FITC-dextran intestinal permeability test, and

the rest were used for histology and gene expression analyses. The remaining mice (n=4/group) were kept alive and

used to monitor C. rodentium clearance. Body weights were recorded and fecal samples were collected every other

day throughout the study.

90

Bacteria culturing and quantification

For gavage, C. rodentium DBS100 (kindly provided by Dr. Philpott, Department of

Immunology, University of Toronto) was grown in LB broth aerobically at 37°C for 16 hours, as

previously described [328]. Viable counts of C. rodentium in liquid culture (gavage), fecal and

liver samples were determined by classical culturing on MacConkey Agar (BioShop). After

aerobic incubation for 24 hours at 37°C, C. rodentium colonies were identified based on

morphology (round shape with red color at the centre and white color at the edges) [152].

B. bifidum MIMBb75 was grown anaerobically at 37°C in Man Rogosa Sharpe broth

supplemented with 0.05% L-cysteine hydrochloride (cMRS) for 24 hours. Culture was washed

and re-suspended in sterile pre-reduced PBS (200 µl) immediately before gavage. Viable counts

of B. bifidum in gavage culture were enumerated microscopically and by classical culturing on

L-cysteine supplemented MRS agar. To assess B. bifidum colonization in mice, fecal pellets were

collected from uninfected mice 2 days post-B. bifidum initiation (i.e. BB pre-post-CR) and all

groups on day 9 p.i.. DNA was extracted from fecal pellets using the Omega E.Z.N.A.TM Stool

DNA Isolation Kit as per the manufacturer’s instructions. Quantitative Real-Time PCR was

performed with 50 ng of DNA using the SYBR Green Master Mix (Applied Biosystems,

Carlsbad, CA) with specific primers targeting the BopA gene, which is specific to the B. bifidum

species[29].(Forward: 5’ACCGAATTCGCCTGTCACTT3’; Reverse:

5’ACGGCGCGGATTCGT3’) at optimized concentrations (F-R: 100-100 nM/reaction). To

determine absolute B. bifidum counts, each reaction (10 μl) was run in triplicates in a 384-well

optical plate using a 7900 HT Real-Time PCR machine (Applied Biosystems) with default

conditions. Bacterial counts were determined using pre-constructed standard curves and data

were expressed as log10 CFU per gram of wet feces.

Fluorescein Isothiocyanate–Dextran in vivo Permeability Assay

This assay was conducted as described in Study 1.

Histological analysis

These analyses were conducted as described in Study 1.

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RNA extraction

Total RNA was isolated from one-half longitudinal segment of the distal colon tissues using the

mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA), as per the manufacturer’s

instructions, eluted in 50 μl of RNAse-free water and stored at -80°C. Recovered total RNA

concentration and purity were assessed using Thermoscientific’s Nanodrop 1000

Spectrophotometer.

Real-Time quantitative PCR (qPCR)

Total RNA (10ng) was reverse transcribed with the Taqman® MicroRNA Reverse Transcription

Kit and primers specific for miR-146a (Assay ID: 000468), miR-148a (Assay ID: 000470), miR-

19a(Assay ID: 000395), miR-200a (Assay ID: 000502), miR-200b (Assay ID: 002251), miR-21

(Assay ID: 000397), and the endogenous control snoRNA135 (Assay ID: 001230) (Applied

Biosystems, Foster City, CA). Real-time PCR was then conducted using undiluted cDNA, and

the TaqMan 2X Universal PCR Master Mix, No AmpEraseUNG (Applied Biosystems) in a 10 μl

PCR reaction. Each reaction was run in triplicates in a 384-well optical plate in Applied

Biosystems’ 7900 HT thermocycler using the 9600 emulation mode with an initial hold at 95°C

for 10 minutes followed by 40 cycles of 95°C for 15 seconds, and 60°C for 60 seconds. The

2−ΔΔ

CT method was used to calculate relative expression levels [333] using sno-135 as a

reference gene. Significance was determined based on ΔCT values using one-way ANOVA,

followed by Bonferroni’s post-hoc test.

Statistical Analysis

One-way ANOVA followed by the Bonferroni’s post-hoc test was used to determine statistically

significant differences in fecal C. rodentium load and body and organ weights, crypt hyperplasia,

colon damage scores, intestinal permeability, and bacterial translocation. Difference of fecal B.

bifidum counts before and after infection, between B. bifidum-treated groups, and between

batches were assessed by Student’s t-test. Bacterial counts were expressed as log10/g of feces.

MiRNA expression among groups was assessed based on ΔCt values using one-way ANOVA,

followed by Bonferroni’s post-hoc test. Outliers were determined by the Grubbs’ Outlier Test. A

p-value of p<0.05 was considered as statistically significant. All statistical analysis were

performed in GraphPad Prism 6 software (La Jolla, CA, USA).

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5.4 Results

C. rodentium infection kinetics and characteristics

C. rodentium was detectable in the feces two days after infection in all three C. rodentium

infected groups. In all infected groups viable count continued to increase until reaching a plateau

(1010

CFU/g) at day 10 p.i. (Figure 5.2a). Four mice from each group were retained for

monitoring C. rodentium clearance. C. rodentium counts decreased rapidly after the peak and

were below detection limit (103 CFU/g) after day 24 p.i. in most mice, although in a few mice

fecal C. rodentium counts fluctuated after 28 days of infection. However, there was no

significant difference on daily C. rodentium count or bacterial clearance rate among the groups.

Infection did not result in significant body weight change among the groups; growth rate was

slowed down from day 2 to day 8 p.i. in the infected groups compared to the sham, though not

significant (Figure 5.2b). Spleen weights were significantly higher in the infected groups

compared to the sham but did not differ among the infected groups (CR: 0.54 ± 0.075, BB post-

CR: 0.61 ± 0.06, BB pre-post-CR: 0.58 ± 0.07, Sham: 0.28 ± 0.02 % body weight; p<0.01); there

was no significant difference in kidney and liver weights among all groups (Figure 5.2c).

B. bifidum colonization

Before infection, B. bifidum load in feces of the BB pre-post-CR mice after 2 day of

administration was about 107cells/g and there was no batch difference (batch 1: 7.4 ± 0.20, batch

2: 6.9 ± 0.15 log cell/g) (Figure 5.3a). On day 9 p.i., B. bifidum remained detectable in the B.

bifidum-treated groups and there was no significant difference in B. bifidum load between the

two groups (BB post-CR: 7.0 ± 0.4, BB pre-post-CR: 6.7 ± 0.4 log cell/g) (Figure 5.3b). There

was no significant difference of B. bifidum load in group BB pre-post-CR before (-5 p.i.) and

after infection (9 p.i.) (Figure 5.3b). B. bifidum was not detected in CR and Sham, which did not

receive B. bifidum treatment.

93

a.

b.

c.

Figure 5.2 C. rodentium infection kinetics and effect on mouse body and organ weights.

(a) Viable C. rodentium counts in infected mice feces were quantified every other day p.i., n=9-13/group (day 2-10

p.i.), and n=1-4/group (day 12-28 p.i.); (b) body weights among sham and infected groups, including C. rodentium-

infected only (CR), C. rodentium-infected with B. bifidum treatment 10 days post-infection (BB post-CR), and B.

bifidum treatment one week prior to C. rodentium infection and 10 days after infection (BB pre-post-CR),

n=20/group (except for day 10 p.i., only non-fasted mice (n=8/group) used for sacrifice were measured); (c) percent

of organ weight per gram of body weight among sham and infected groups on day 10 p.i., n=8/group. Data are

presented as mean ± SE. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s

post-hoc test. Different superscripts (a, b) indicate statistic significance among groups, p<0.05.

94

a.

b.

Figure 5.3 Fecal B. bifidum load before and after infection.

(a) QPCR quantification of fecal B. bifidum load two days after B. bifidum initiation in the group receiving B.

bifidum prior infection, n=10/batch. (b) qPCR quantification of fecal B. bifidum in all groups of mice after C.

rodentium (CR) infection (n=9/group), and in BB pre-post-CR group before CR infection (n=20/group). Statistical

significance was determined by Student’s t-test, data presented as mean ± SE. N.D, not detectable.

95

Intestinal histology and barrier integrity

C. rodentium infected mice exhibited a significant increase of caecal crypt length compared to

the sham (CR: 117 ± 5.57, BB post-CR: 120 ± 2.90, BB pre-post-CR: 109.7 ± 4.39, vs. Sham:

79.9 ± 2.28 μm; p<0.01) (Figure 5.4a). This is also the case in the distal colon (CR: 232.9 ±

14.61, BB post-CR: 242.2 ± 7.19, BB pre-post-CR: 224.4 ± 14.48, vs. Sham: 160.6 ± 6.59 μm;

p<0.0001) (Figure 5.4b). B. bifidum-treatment did not yield significant difference in caecal or

distal colon crypt lengths compared to the infected CR group without the treatment. Histological

analysis of the ceacum and distal colon tissue revealed significant difference in tissue damage

scores between infected mice and sham mice, but no difference between B. bifidum-treated and

infection-only mice (caecum damage score CR: 1.5 ± 0.2, BB post-CR: 1.3 ± 0.2, BB pre-post-

CR: 1.2 ± 0.2, vs. Sham: 0.3 ± 0.1 μm; distal colon damage score CR: 2.6 ± 0.4, BB post-CR: 3.3

± 0.2, BB pre-post-CR: 3.0 ± 0.3, vs. Sham: 0.6 ± 0.2, p<0.0001) (Figure 5.4c,d). There was no

difference in intestinal permeability measured by the FITC-dextran assay among the four groups

on day 10 p.i. (Figure 5.5a). C. rodentium was detectable in the liver and spleen of infected but

not control mice; but there was no difference in the degree of bacterial translocation with or

without B. bifidum treatment (Figure 5.5b).

Distal colon microRNA expression

Based on results in Study 1, the expression of 6 relevant miRNAs (miR-148a, miR-200a, miR-

200b, miR-19a, miR-21, and miR-146a) was examined (Figure 5.6). MiR-148a, miR-19a were

numerically, but not significantly, reduced and increased, respectively, in the C. rodentium

infected groups. MiR-21 was differentially expressed among the four groups (one-way ANOVA:

p<0.05). Post-hoc Bonferroni’s test revealed that the expression of miR-21 was significantly

elevated in groups BB post-CR (fold=3.4 ± 0.1, p<0.05) and BB pre-post-CR (fold=3.1 ± 0.2,

p<0.05), but not in the CR (fold=2.1 ± 0.2).

96

a.

b.

c.

d.

Figure 5.4 B. bifidum effects on intestinal crypt hyperplasia and tissue damage at day 10

p.i..

(a) Distal colon and (b) ceacal crypt lengths were measured with the examiner unaware of the group of the samples

(n=10/group; 2 additional samples collected from mice used for intestinal permeability test were included); infected

groups showed significantly greater crypt lengths than the uninfected group (p<0.001); B. bifidum treatment did not

influence crypt hyperplasia induced by C. rodentium. (c) Distal colon and (d) ceacal damage score were evaluated in

the same manner as for distal colon (n=10/group; 2 additional samples collected from mice used for intestinal

permeability test were included); infected groups had significantly higher tissue damage scores compared to the

uninfected control (p<0.001); B. bifidum treatment did not influence intestinal colitis induced by C. rodentium.

Results are expressed as mean ± SE. Statistical significance was determined by one-way ANOVA, followed by the

Bonferroni’s post-hoc test, p<0.05. Different superscripts (a, b) indicate statistic significance between groups.

97

a.

b.

Figure 5.5 B. bifidum effects on intestinal barrier at day 10 p.i.

(a) No statistical significance was observed in intestinal permeability among the groups, despite of C. rodentium

infection or B. bifidum treatment, n=8/group. (b) Barrier integrity was also assessed based on C. rodentium

translocation to secondary organs (n=8/group); no difference in C. rodentium load was observed in spleen and liver

among the infected groups, regardless of B. bifidum treatment. Results are expressed as mean ± SE. Statistical

significance was determined by one-way ANOVA. N.D, not detectable.

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Figure 5.6 Distal colon miRNA expression at day 10 p.i..

Real-time PCR quantification of 6 selected miRNA expression; data were normalized to snoRNA135, n=7-8/group

(one statistical outlier was taken out from the Sham group, with a Ct value comparable to the negative control).

Results are expressed as mean ± SE. Statistical significance was determined based on the ΔCt values using one-way

ANOVA, followed by Bonferroni’s post-hoc test. Different superscripts (a, b) indicate statistical significance

between groups.

99

5.5 Discussion

The current study investigated the impact of B. bifidum MIMBb75 on C. rodentium infection in

the framework of miRNA modulation. It was found that administration of B. bifidum MIMBb75

before and after C. rodentium infection did not improve intestinal pathology (C. rodentium

colonization, crypt hyperplasia, inflammation, and barrier dysfunction) or miRNA alterations

associated with the infection.

Several probiotic strains belonging to the Lactobacillus and Bifidobacterium species and their

fermented products have been implicated in improving C. rodentium induced colitis in vitro and

in vivo [152, 319-323]. Some studies found that the use of probiotic bacteria resulted in a

reduction in bacterial load during C. rodentium infection [152, 319, 322]. For example, treatment

of L. rhamnosus R0011 and L. acidophilus R0052 reduced C. rodentium attachment to T84

epithelial cells in vitro, and reduced C. rodentium load in colonic luminal contents on day 9 p.i.

in C57Bl/6 mice [152]. A 3-day pretreatment of B. breve UCC2003 in BALB/c mice reduced

fecal C. rodentium load from day 3-14 p.i.[319]. On the contrary, other studies revealed that

probiotics may prevent C. rodentium-induced pathology independently from colonization

resistant effects. Particularly, Collins et al. showed that treatment of B. breve UCC2003 for 3

days prior to infection attenuated crypt hyperplasia without affecting C. rodentium colonization

or A/E lesion formation in C57Bl/6 mice [320]. A more recent study conducted by this group

also found that administration of fermented product from Lactobacillus species to C57Bl/6 mice

improved infection but did not alter C. rodentium colonization kinetics [321]. In the present

study, B. bifidum MIMBb75 administration did not suppress C. rodentium colonization nor

improved clearance. The discrepancies in colonization resistance conferred by different

probiotics could be attributed to strain-specific effects of probiotics and variations in study

conditions such as treatment duration and animal strains used. Also, it was of interest to examine

if C. rodentium would in turn compete with B. bifidum MIMBb75 intestinal counts in particular

taking into consideration that B. bifidum MIMBb75 is a human-restricted strain while C

rodentium is only pathogenic in murine. It was found that fecal B. bifidum counts in BB pre-post-

CR group before and after infection (Figure 5.3) were not significantly different and the number

was comparable to data previously obtained in our group where fecal B. bifidum load 24 hours

post-gavage was about 6.8 ± 0.3 log cell/g [378]. This indicates that infection did not interfere

with B. bifidum fecal load. Knowing that C. rodentium can replace almost 90% of the resident

100

microbiota, the fact that B. bifidum MIMBb75 was not displaced indicates that this

bifidobacterium strain is a good colonizer [14]. A previous study from our group also revealed

that B. bifidum predominately resides in the ceacum instead of the distal colon [378], where C.

rodentium preferentially localizes. Hence, it might be of interest for future studies to examine

colonization competition between the two bacteria in a region-specific manner.

The use of selected probiotic bacteria, especially when given before infection, has been shown to

alleviate TMCH and colitis caused by C. rodentium in mice [152, 160, 320-323]. For instance, in

the study with B. breve UCC2003, Collins et al. found a significant reduction of colonic crypt

hyperplasia and mucosal infiltration of immune cells in mice treated with the probiotic strain 3

days before and 8 days after infection [320]. Rodrigues et al. demonstrated that C. rodentium-

induced Th17 and Th1 response and crypt hyperplasia were mitigated when probiotic treatment

(L. rhamnosus R0011 and L. helveticus R0052) was initiated one week before or concurrently

with the infection but not after the infection[323]. In the present study, TMCH and histological

damage of the ceacum and distal colon were not attenuated in mice treated with B. bifidum

MIMBb75 whether before or after C. rodentium challenge. This probiotic strain was chosen

based on its adhesion properties and ability to modify intestinal miRNAs such as miR-148a. It is

possible that this particular strain, B. bifidum MIMBb75, is ineffective in preventing or treating

infectious colitis, or the conditions in the study are not optimal for detecting benefits. Indeed,

even the same strain of probiotic may impose different influence on the host under different

conditions. In the study by Rodrigues et al. the same probiotic intervention prevented mortality

but did not reduce TMCH in neonatal mice as opposed to adult mice [323]. Interestingly, an

earlier study by the group, however, observed crypt reduction in probiotic-treated neonatal mice

[160]. These findings imply that experimental conditions may be an indispensable factor

determining the efficacy of a probiotic; namely, age of animals, dosage of C. rodentium

challenges, time and duration of intervention may all potentially mask or interfere with probiotic

effects.

One unexpected observation in the present study was the general increase of intestinal

permeability in all mice including the uninfected ones. It is known that C. rodentium infection

results in intestinal barrier dysfunction and therefore, increased paracellular translocation of

macromolecular tracer FITC-dextran, as seen in Study 1. Uninfected control mice were expected

to have marginal permeability based on Study 1 and the current literature [152, 160, 323, 328].

101

For example, using the in vivo intestinal permeability assay Rodrigues et al. showed that on day

10 p.i. serum FITC-dextran level of the infected adult mice was two-times higher than that of the

sham mice, and this high level was normalized to be comparable to the sham when treated with

probiotics [323]. Surprisingly, serum FITC-dextran concentration of the sham mice in the present

study was as high as the infected groups on day 10 p.i., suggesting impaired barrier integrity in

healthy uninfected mice. This could be a consequence of the daily intra-gastric gavage treatment

on the mice for a prolonged period of time. Although the employment of intra-gastric gavage

allows for a better control on the amount of probiotic administered to each mouse, it imposes

physical and psychological stress to the animals. It was suggested that chronic psychological

stress can trigger stress glucocorticoid hormone release leading to mucosal barrier dysfunction,

thereby increased intestinal permeability and host defense mechanism impairments [379]. As a

matter of fact, in the studies that showed positive effects of probiotic treatment with normal

ranges of intestinal permeability in the sham controls, probiotics were administered daily in

drinking water instead of intra-gastric gavage [152, 160, 323]. Other studies, such as with B.

breve UCC2003, used prolonged daily gavage of probiotics, but did not measure permeability

[320]. The present study is actually the first study to examine intestinal permeability under the

condition of daily gavage. Therefore, barrier integrity in all groups may have been disrupted due

to the daily gavage event. In line with this, addition, C. rodentium translocation to secondary

organs, spleen and liver, was similar among the infected groups, indicating similar alteration of

the barrier integrity. Although it is still possible for a probiotic to confer other health benefits

even when given via daily gavage, based on our data this administration method may increase

variability within experimental groups and thereby, a larger sample size may be needed to attain

statistical power for detecting any potential significant effects. Future studies should consider

exploiting other administration methods such as by drinking water or perorally behind the mouse

incisors, to minimize perturbation caused to animals. Importantly, data generated from studies

where probiotics were administered by daily gavage should be interpreted with caution in the

future, considering that the data may be affected by the altered barrier function.

The field of bacteria-host interaction at the level of miRNAs is still in its infancy. To date, there

is only one study that investigated the impact of probiotic Lactobacillus species on shaping

pathogenic Listeria-induced miRNA aberrancy [264]. The present study examined the

expression of six selected miRNAs (miR-148a, miR-200a, miR-200b, miR-19a, miR-21, and

102

miR-146a) that were deregulated during C. rodentium infection based on findings from Study 1,

and found that B. bifidum MIMBb75 treatment did not result in normalization of these miRNAs.

Importantly, there are inconsistencies between expression data of Study 1 and Study 2. Unlike

Study 1, which was based on NanoString and qPCR, miR-148a and miR-200a/b were not

significantly down-regulated upon infection and miR-19a, miR-21 and miR-146a were not

significantly up-regulated, even though a numerical increase was observed. There seems to be a

trend that the expression of miR-19a, miR-21 and miR-146a were induced upon C. rodentium

infection, which are in line with those in Study 1, and the increase of miR-21 was significant in

the probiotic-treated groups The statistical insignificance observed in Study 2 might be attributed

to a relatively small sample size (n=8 vs n=10 in study 1) and high variability. Furthermore, one

of the limitations of this study was the lack of an uninfected healthy group receiving daily B.

bifidum treatment. Therefore, it is unclear what would be the impact of B. bifidum alone on

shaping the expression of these miRNA. A previous study in our group found that miR-148a is

up-regulated in healthy mice treated with B. bifidum MIMBb75 for two days [272]. In the current

study, expression of miR-148 was decreased in the CR group, and miR-148a expression levels

were slightly elevated in the probiotic-treated groups. Although none of these attained

statistically significance, most likely because of the high variability, future studies may further

focus on this specific miRNA. Finally, even though B. bifidum MIMBb75 treatment did not

normalize the selected miRNA alterations associated with C. rodentium, it will be important to

analyze and compare genome-wide miRNA expression profiles between the experimental groups

in order to decipher potential miRNA modulations mediated by B. bifidum MIMBb75.

In summary, this is the first study looking at effects of B. bifidumMIMBb75 on infectious colitis

and host miRNA responses. It was found that administration of this strain did not attenuate C.

rodentium colonization, intestinal inflammation and crypt hyperplasia, barrier dysfunction, or

miRNA alterations associated with C. rodentium infection. Although the expression of the

selected miRNAs was not influenced by B. bifidum treatment, follow-up studies are needed to

examine genome-wide miRNA signature and transcriptome among the experimental groups in

the distal colon as well as the caecum. Findings from this study may provide insights for future

studies with respect to the administration method of probiotics as well as probiotics of choice for

mitigating intestinal inflammation.

103

Chapter 6

General Discussion

This research project includes two studies that investigated host-microbial crosstalk at the level

of posttranscriptional regulation of host gene expression. The first study (Chapter 4) showed that

C. rodentium infection alters the murine colonic miRNA signature, which might play a role in

the deregulation of apoptosis pathways upon infection as shown by bioinformatic and gene

expression analyses. In the second study (Chapter 5), administration of the probiotic strain B.

bifidum MIMBb75 before and after infection did not attenuate C. rodentium-induced pathology.

Moreover, based on the data of six selected miRNAs (miR-148a, miR-200a/b, miR-146a, miR-

21 and miR-19a of the miR-17-92 cluster), the presence of B. bifidum MIMBb75 did not have

significant impact on colonic miRNA alterations in response to C. rodentium infection.

To date, only a limited number of studies have explored miRNA expression in response to an

intestinal pathogen infection. Most of these studies focused on pathogens of the stomach

(Helicobacter) [252, 253], and small intestine (Listeria [251] and Salmonella [39]), and only

examined selected miRNAs. The present work reports for the first time a comprehensive analysis

of miRNA signature in the context of a colon-specific pathogen infection. Although there is a

lack of comprehensive data available in the literature, some of the differentially expressed

miRNAs in Study 1, including miR-146a/b, miR-21, miR-16, miR-200a/b and miR-148a, were

identified to be deregulated in a consistent manner in at least one other pathogen infection. This

is indicative of a plausible consensus of pathogen responsive miRNAs along the gastrointestinal

tract. The fact that these miRNAs have been implicated in regulating immune responses [254,

266] and cell cycle [267, 295, 380] highlights the critical role of miRNA in the host response to

pathogen infections. Meanwhile, some other miRNA could be deregulated in a pathogen-specific

manner. Exploiting bioinformatic approaches with gene expression analysis, this study proposes

the existence of a miRNA regulatory network, involving 9 upregulated and 2 downregulated

miRNAs, repressing the expression of the apoptosis factor Bim during infection. A low level of

Bim expression is associated with colon carcinomas [300, 366] and thereby, may contribute to

the development of crypt hyperplasia, which is a hallmark of C. rodentium pathogenicity.

Recent evidence has also revealed that miRNA expression patterns can be influenced by the

presence of gut microbiota [37, 38, 250] or probiotic bacteria [264, 269, 271, 272]. The probiotic

104

strain B. bifidum MIMBb75 has been previously demonstrated in our group by Singh et al. to be

able to modulate ceacal miRNA signature when administered to conventional mice for two days

[272]. It was found that this strain of B. bifidum induced expression of miR-148a, which was

downregulated in C. rodentium-infected colon based on Study 1. In light of this, Study 2 in this

project attempted to explore colonic miRNA responses in the interplay between the host, a

pathogen, and a probiotic bacterium. Six C. rodentium responsive miRNAs in Study 1 were

further investigated in Study 2. They are miR-148a, miR-200a/b, miR-146a, miR-21 and miR-

19a of the miR-17-92 cluster. Interestingly, in Study 2, none of the selected miRNAs were

significantly deregulated in mice infected with C. rodentium without probiotic treatment. These

findings are inconsistent with those in Study 1. Though, it should be noted that the expression of

miR-21, miR-19a and miR-146a were numerically increased in the all C. rodentium infected

groups, and miR-21 was significantly increased in the probiotic-treated infected groups. The lack

of significant differences of the C. rodentium group could be attributed to the relatively small

sample size with a high sample variability that lowered the statistical power for detecting

significance. Administration of B. bifidum MIMBb75 did not have a significant impact on these

C. rodentium-induced miRNA alterations. It is possible that the influence of a pathogen on the

host may outcompete that of a probiotic or that the host exhibits a time-dependent response to the

two bacteria. Indeed, in the study of Singh et al. mir-148a was only differentially expressed after

B. bifidum MIMBb75 was given for 2 days but not 14 days [272]. This may explain the lack of

influence of probiotic administration in Study 2, where B. bifidum was given for 17 or 10 days.

The postulation that host may respond early to probiotic colonization was also shown in Listeria

infection. Infection-induced miRNA alterations were attenuated when mice were treated with

probiotic Lactobacillus strains for 3 days in advance of the 24 hours long infection [264].

Furthermore, B. breve UCC2003 has been shown previously to exert beneficial effects against C.

rodentium infection [319, 320]. A few strains of B. bifidum have also been implicated in

preventing EHEC colonization in vitro and improving IBD in an animal model [31, 212].

Nonetheless, B. bifidum MIMBb75 administration was ineffective in preventing or mitigating C.

rodentium associated pathology in the current study. The ineffectiveness of B. bifidum

MIMBb75 could be attributed to the implementation of daily intra-gastric gavage, which is a

stressful event to animals and can lead to intestinal barrier dysfunction, as evidenced by the

abnormally high intestinal permeability in health control mice [379]. Another possibility is that

105

B. bifidum MIMBb75 does not confer beneficial effects on infectious colitis. A double-blind,

placebo-controlled RCT showed that supplementation of this probiotic strain improved IBS

symptoms [23]. Unlike IBD or infectious colitis, IBS is a functional disorder with no apparent or

a low grade chronic inflammation. It is possible that B. bifidum MIMBb75 has strain-specific

benefits on functional disorders but not in prominent inflammatory pathological conditions.

Generally speaking, findings in this project reveal a potential mechanism underlying the

crosstalk between the host and the pathogen C. rodentium such that miRNA-mediated post-

transcriptional regulation may contribute to the host hyperplasic response during the infection.

The study also shows that daily gavage, a procedure commonly used to study probiotic-host

interaction may not be ideal since it alters the intestinal barrier function. B. bifidum did not

mitigate C. rodentium-induced colitis and did not interfere with the expression of intestinal

miRNA affected by C. rodentium at the time point considered. Time-dependent effects should be

evaluated. Also, it would be of interest to investigate the miRNA response in mice infected with

C. rodentium and treated with probiotics proven to be beneficial in this context, such as B. breve

UCC2003 [320], L. rhamnosus R0011 and L. acidophilus R0052 [160, 323].

6.1 Strengths, Limitations and Future Directions

This is the first study using a highly sensitive and reproducible expression profiling technique,

NanoString, to study pathogen-host interaction at the level of posttranscriptional regulation. With

this advanced technology, miRNA response to a colonic pathogen was examined for the first

time in a comprehensive manner. This is important as miRNAs work cooperatively to regulate

gene expression. Moreover, a highly adhesive probiotic strain, which preferentially colonizes the

large intestine, was utilized for studying the effect of bifidobacteria species on pathogen

infection of the colon.

Though, there are also some limitations that need to be considered. First of all, in both Study 1

and 2 whole thickness tissues were used for gene expression analysis. Therefore it is not possible

to isolate the cellular origins of the expression signals detected. Taking into account that there is

a substantial mucosal infiltration of immune cells, it is uncertain whether the differentially

expressed miRNAs or mRNAs represent the expression pattern of epithelial cells or immune

106

cells during infection. Indeed, some deregulated miRNAs found in Study 1 such as miR-146a

and miR-155, have also been shown to be differentially expressed in macrophages and other

immune cells upon various enteric pathogen infections [39, 40, 265]. Nevertheless, studies

currently available in the literature also used whole-thickness tissues, such that RNA was

extracted from the whole-thickness of ileal tissue in the studies of miRNA expression upon

Listeria infection [38, 264]. To pinpoint epithelial-specific responses, future studies could

consider employing other sampling approaches such as laser capture microdissection that allow

direct examination of a specific cell type. To isolate C. rodentium-specific responses, future

studies should also compare baseline miRNA expression before infection to that at day 10 p.i..

Another potential limitation of both studies pertains to the use of a non-purified standard chow

diet. It was shown previously that diet formulation can have significant impact on gut microbiota

composition and even diseases outcome [381]. Specifically, Ooi et al. compared the effects of

three diets on DSS and C. rodentium induced colitis and found that DSS-induced colitis was

most severe in mice fed with the high calcium containing Teklad diet (TD) designed for breeding

vitamin D receptor knockout mice, followed by the ones fed with standard chow, and was least

severe in mice fed with a synthetic purified diet (PD); notably, the TD-fed mice experienced a

delayed eradication of both primary and secondary C. rodentium infection with a persisted higher

fecal load compared to PD-fed mice. Although both PD and TD are purified diets, it was

suggested that the protective effect of PD could be due to its high percentages of simple sugars

(glucose and sucrose) which has implications in CD [382] and was shown to be favoured by the

commensal bacteria Bacteriodes thetaiotaomicron to outcompete C. rodentium [110, 381].

Standard Chow diet on the other hand is not a synthetic diet; it contains a relatively high level of

folic acid [383], and compared to sterile purified AIN-93G diet it contains about 20 times more

bacteria-derived endotoxin lipopolysaccharide which has proinflammatory effects [384].

Knowing the importance of diet composition, it is important to utilize a purified diet with well-

defined composition to ensure that the study is conducted in the most controlled setting. Thus,

future studies would be benefit from employing a purified diet to control for potential variability

in nutritional composition of a non-synthetic diet.

Furthermore, Study 1 evaluated the expression of several genes that were postulated to be

regulated by miRNAs based on bioinformatic analysis with complementary literature search as

107

well as cross-matching with published microarray data. In order to have a more comprehensive

view of miRNA-mediated gene expression regulation during C. rodentium infection, global

transcriptome expression profiling could be performed and the data could be used for cross-

matching with the bioinformatically identified putative targets. This would increase the chances

of identifying relevant genes for follow up. In addition, expression of the putative miRNA-

modulating gene targets was evaluated at the mRNA level. Discrepancy could exist between a

gene and its corresponding protein levels. This is particularly relevant in miRNA studies because

a miRNA can suppress gene expression by either translational repression or degradation [242].

Therefore, the action of a miRNA does not necessarily reduce the number of transcripts of its

target. Thus, it is important to examine target expression at the protein level using protein assays

such as Western blot analysis, in situ hybridization, and ELISA. Future studies should also

consider functionally confirming that Bim is a direct target of miR-17-92-related clusters by

performing in vitro gain-of-function transfection experiments in Caco-2 cells.

Last but not least, Study 2 examines the effect of a probiotic strain on host miRNA response

during C. rodentium infection, which includes four experimental groups: sham control, C.

rodentium-infection, infection with B. bifidum pretreatment, and infection with B. bifidum post-

treatment. However, the inclusion of a group that received probiotic intervention without being

infected might be important for the isolation of effects solely attributable to B. bifidum. For

example, a previous study showed that miR-148a was upregulated after 2 days but not 14 days of

B. bifidum treatment in healthy mice [272]. In the present study miR-148a was not differentially

expressed in B. bifidum-treated infected mice. Without the B. bifidum-only group, it is unclear

whether the inhibitory effect of the infection outcompetes the stimulatory effect of B. bifidum, or

if B. bifidum did not have an impact on miR-148a at the time point considered. In addition, the

probiotic was administered to mice daily by intra-gastric gavage. While this is a commonly used

protocol, this administration procedure likely resulted in increased intestinal permeability,

masking the effects of C. rodentium. Future studies should consider implementation of other

administration methods to reduce this type of stress, such as administering the probiotics via the

drinking water or perorally. Moreover, both study 1 and 2 would benefit from the addition of a

inflammation positive control group with chemically-induced colitis (such as DSS-treatment),

which would allow the identification of miRNA responses specific to C. rodentium that are not

caused by mucosal inflammation or damages in general. Since miRNA alterations in response to

108

other colon-specific infectious agents are unknown, the inclusion of an infectious positive

control group is currently not applicable. Furthermore, it might also be essential to monitor

intestinal permeability throughout the course of infection in the future. The in vivo FITC-dextran

assay requires performance of cardiac puncture and it is impractical to utilize for monitoring

permeability at various time points, since it will enormously increase the number of animals to

be used [385]. Other assays, such as quantification of fecal albumin, have been shown to produce

comparable results as the FITC-dextran test and could thus be preferred [386].

6.2 Implications

The first miRNA was discovered in 1993 in Caenorhabditis elegans [387] but the term miRNA

was not coined until 2001[388]. Since then, a growing body of research has been conducted to

understand the biological role of miRNA and recent research suggests that they may be

important in regulating host-bacteria interaction. Though, this field is still in its infancy.

Currently, only a limited number of studies have demonstrated the impacts of presence of

microbiota, probiotic bacteria and pathogens on host miRNA expression. Findings in the present

project provide the first comprehensive analysis of miRNA expression patterns in response to a

colonic pathogen and extend the current knowledge of miRNA regulation being a potential

mechanism underlying host-microbial crosstalk.

MiRNAs are constantly deregulated in pathological conditions, and have been recognized as

biomarkers for diagnosis and prognosis of a disease. Given the biological relevance of C.

rodentium-induced colitis to IBD and tumorigenesis, the differentially expressed miRNAs

identified in this study may serve as biomarkers for these conditions. As a matter of fact,

circulating and colonic levels of miR-19a, miR-21 and miR-146a, which were deregulated by C.

rodentium, have recently been suggested to be candidate biomarkers for differentiating subtypes

of IBD, CD and UC, based on genome-wide miRNA signature analysis [389]. A high serum

level of miR-21 was also found to be associated with poor prognosis in CRC [390], and

increased levels of miR-21 and miR-17-92a cluster in exfoliated colonocytes were suggested to

be promising fecal miRNA biomarkers for CRC [298].

109

The differentially expressed miRNAs may also serve as targets of therapeutic or nutritional

interventions. The identification of the putative miRNA regulatory network on an apoptosis

facilitator gene, Bim, may have implications in elucidating pathogenicity mechanisms of C.

rodentium-induced TMCH via miRNAs. From a therapeutic perspective, understanding the

pathogenicity of C. rodentium is important for developing potential treatments for EHEC

infection, where the use of antibiotics is ineffective and discouraged. Deciphering the regulatory

role of miRNAs in TMCH shed light for developing miRNA-based anti-tumor therapies for

CRC, aiming to reprogram the aberrant miRNA networks. From a disease prevention standpoint,

dietary interventions that aim to promote homeostasis by miRNA modulation could also benefit

from the findings in this study. For example, increased expression of miR-106, which is

overexpressed in C. rodentium infection, is associated with CRC; and an in vitro study revealed

that microbe-derived SCFA butyrate can suppress miR-106 expression in a human colon cancer

cell line, suggesting that the differentially expressed miRNAs found in the current study may be

relevant candidate targets for dietary interventions.

Moreover, the discovery of increased intestinal permeability associated with daily gavage

implicates that cautions should be taken when interpreting studies that administer probiotics

through daily gavage, as the data might be influenced by alteration of the barrier function.

Finally, findings pertaining to the efficacy of B. bifidum MIMBb75 on C. rodentium infection

may provide some insights with respect to recommendation of probiotic strains in functional and

inflammatory diseases.

110

6.3 Conclusions

(1) C. rodentium infection alters murine colonic miRNA signature and the alterations are

involved in the anti-apoptotic response of host epithelium associated with the

development of crypt hyperplasia during the infection.

(2) B. bifidum MIMBb75 administration does not alleviate intestinal damage nor normalize

selected miRNA alterations associated with C. rodentium infection.

111

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