‘A new target to reduce

Helicobacter pylori infection.’

Alexia Bosancic Bachelor of Biomedical Science

This thesis has been submitted in partial fulfilment of the requirements

for the degree of Bachelor of Health and Medical Science (Honours)

School of Medicine Deakin Univeristy

Supervisor: Dr Melanie Thomson

Co-Supervisors: Dr Tamsyn Crowley and Dr Sarah Shigdar

October 2016

II

Deakin Univeristy

Faculty of Health, Medicine, Nursing and Behavioural Science

School of Medicine

Student’s certifications

I am the author of thesis entitled:

A new target to reduce Helicobacter pylori infection

Submitted for the degree of:

Bachelor of Health and Medical Science (Honours)

And I agree to grant the Honours coordinators of the School of Medicine

permission to make this thesis available for loan or photocopying, in

whole and in part.

Signed: Alexia Bosancic Date: 10/10/2016

Name: Alexia Bosancic

Student ID Number: 213285304

Word Count: 12, 091

III

Acknowledgements

First and foremost, I would like to thank my supervisor Dr Melanie Thompson, for

giving me this opportunity. Throughout this entire year, you have incessantly

encouraged and supported me during all the highs and the lows. In this short amount

of time, you have taught me a lifetime worth of knowledge and never once doubted

my intelligence and capabilities. I’m excited to see what the future holds as this

chapter closes and the next with you has my lifelong mentor.

Additionally, I would like to thank my co-supervisors, Dr Tamsyn Crowley and Dr

Sarah Shigdar for assisting me through this project and providing knowledge within

your fields.

Thank you to Associate Professor Richard Ferrero (Hudson Institute) for proving the

Hp-SS1 WT samples, Dr Henry Windle (Trinity College, Dublin) for providing the

plasmid constructs and Teisa Holani for constructing the IROMP mutants.

Thank you to the GCEID team, Jason Hodge, Carly Botheras, Saad Maymand, Darcie

Cooper and Christine Roder for all the encouragement and help throughout the entire

year.

Thank you to my mother, Edita who was my focal motivation for applying to

undertake this project and complete honours. Since I was little I have watched you

suffer from gastric ulcer diseased caused by a refractory strain of Hp and wanted to

find a cure.

IV

Although I didn’t get too close, I hope I have contributed to maybe one day

eradicating this infection. You have been my rock and couldn’t have got this far

without you. Thank you for dealing my mood swings and stress and never giving up

on me.

Thank you to my dad, Peter and my sister, Xenia. The support and assistance you

have provided with every respect I required to complete my thesis is immeasurable

and I will forever be grateful to you both.

Thank you to my best friend Sam, for making sure I would take regularly breaks and

consoling me throughout my meltdowns. You have continuously encouraged me

throughout this year and have had a major influence on my success of this degree. I

am so grateful for you and your family.

Finally, I would like to thank my boyfriend Dylan. As I have been more than

intolerable this year with all the pressure of honours, I am grateful you were there

beside me throughout one of the most challenging years. You are one of the reasons I

stayed motivated and put everything into this, even in times of doubt. Thank-you for

everything.

V

Table of contents

Acknowledgements III

Table of contents V

List of figures VIII

List of tables X

List of abbreviations XI

Abstract 2

Chapter 1 : Introduction 51.1. Clinical Significance 51.2. Prevalence and epidemiology 71.3 Helicobacter pylori: The microbiology 91.4. Current Treatments and Failures 12

1. 4. 1 The misuse of antibiotics and the spread of resistant pathogens 131. 4. 2 Overcoming the ‘superbug’ 15

1. 5 Aptamers 171. 5. 1 The current clinical uses of aptamers 191. 5. 2 New uses – Antimicrobial aptamers? 22

1. 6 Rationale and aims 23

Chapter 2 : Materials and Methods 262.1 Computational analysis of the iron regulating proteins of Hp–SS1 and the Helicobacter species. 26

2.1.1 Phylogenetic investigation of iron regulating proteins of Hp-SS1 and the Helicobacter species 262.1.2 Sequence alignments of the IROMPs and related regulatory proteins 272.1.3 Visualisation of phylogenetic tree 282.1.4 Identification of protein-protein interactions 292.1.5 Generation of sequence logo 29

2.2 Bacterial Strains and routine cultures 302.2.1 Preparation of 10% HB + CAB + Dent 31

2.3 Hp-SS1 – Rapid diagnostic test: Stuart’s Urease Reagent 322.3.1 Preparation of Stuarts urease reagent 322.3.2. Urease test 33

VI

2.4 Purification of Hp-SS1 WT and mutant DNA 332.5 Quantification and assessment of DNA concentration 342.6 Selection of Kanamycin resistance 34

2.6.1 Concentration calculation for 25μg/mL Kan in 500mL of media 352.6.2 Preparation of 10% HB + Dent + 25μg/mL Kan 35

2.7 Confirmation of Helicobacter genus 352.7.1 Calculations for 100μM oligonucleotide stocks 352.7.2 Preparation of primer stocks (100μM) 362.7.3 Oligonucleotides used to detect the Helicobacter genus 36

2.8 Confirmation of fecA1, fecA2 and frpB1 KanR mutant knockouts 362.8.1 Oligonucleotides used to detect ΔIROMP knockouts 37

2.9 Polymerase Chain Reaction (PCR) 372.10 Separation of PCR products via gel electrophoresis 38

2.10.1 Preparation of 5M NaOH 382.10.2 Preparation of 0.5M EDTA (pH 8.0) 382.10.3 Preparation of 5X Tris Base/Boric Acid/ EDTA (TBE) 392.10.4 Preparation and dilution calculation for 5X TBE to 0.5X TBE 392.10.5 Preparation of 1% Agarose gel 402.10.6 Separation and analyses of Hp-SS1 DNA 40

2.11 Natural Transformation of Hp-SS1 422.11.1 ΔIROMP-interrupted gene plasmids of Hp-SS1 422.11.2 Dilution calculation for 7% FBS for 500mL media 422.11.3 Preparation of BB + 7% FBS 432.11.4 Preparation of 50mg/mL Kanamycin (1000X) 432.11.5 Calculations for 50mg/mL to 25μg/mL Kanamycin 432.11.6 Preparation of 10% HB + 25μg/mL Kan 442.11.7 Liquid method of natural transformation 44

2.12 Preparation for SELEX 462.12.1 Generation of the randomised library and primers 46

Chapter 3 : Results 473.1 Shared functions, co-occurrence and conserved elements amongst the Helicobacter species and of the Hp-SS1 IROMPs in question 47

3.1.1 Phylogenetic analysis of iron regulating proteins in Proteobacteria and most common to the Helicobacter species 473.1.2 Protein-protein interactions of the IROMPs of Hp-SS1 regulated by the fur gene 55

3.2 Isolation of Hp-SS1 WT and ΔIROMP clones 62

VII

3.3 PCR confirmation of Hp-SS1 ΔIROMP clones 643.3.1 Confirmation of the Helicobacter species 643.3.2 Expected and resulting products of the isolated HpSS1 WT and ΔIROMP clones 66

3.4 Natural transformation of Hp-SS1 743.5 Preparation for the SELEX technique 75

3.5.1 Generation of randomised library and primers 75

Chapter 4 : Discussion 764.1 Interpretation of the results with the association of the literature 764.2 Strengths and Limitations 894.3 Clinical Relevance 904.4 Future Direction 914.5 Conclusions 92

References 93

VIII

List of figures

Figure 1.1 The localisation of Hp. 5

Figure 1.2 Adapted from Peek (2008) demonstrating the variation of clinical outcomes dependent on the colonisation of Hp and its location within the gastric mucosa3. 6

Figure 1.3 The global prevalence of Hp infection (%), adapted from the reflux centre (2015) 7. 8

Figure 1.4 A depiction of the spiral-shaped pathogen, Helicobacter pylori. 9

Figure 1.5 The expression of IROMPs in varying conditions of iron, regulated by fur. 11

Figure 1.6 The current therapy for Hp eradication. 12

Figure 1.7 The acquirement, misuse and the spread of antibiotic resistance, adapted from centres for disease control and prevention (2015). 14

Figure 1.8 The schematic representation of aptamer drug conjugation (ApDC), adapted from Sun et al (2014). 18

Figure 2.1 Expected PCR results, demonstrating the order and content of each reaction. 41

Figure 2.2 Liquid method of natural transofmration of Hp–SS1 WT. 45

Figure 3.1 Phylum to genus analysis of common iron-regulating proteins. 48

Figure 3.2 Sequence alignment of the Ferric uptake regulator (fur) amongst the Helicobacter species. 50

Figure 3.3 The phylogenetic tree to scale (0.1), revealing the branch lengths or genetic distance of the Helicobacter species fur gene. 52

Figure 3.4 Phylogenetic analysis revealing the bootstrap levels between each node of the differing 24 Helicobacter fur genes. 54

IX

Figure 3.5 The essential interactions between the proteins expressed that function in iron homeostasis and are further regulated by fur. 56

Figure 3.6 The protein-protein interactions of the target IROMPs of Hp J99. 59

Figure 3.7 Analysis of the promoter regions of fecA1, fecA2, frpB1 and fur genes specific to Hp. 60

Figure 3.8 PCR amplification of Hp-SS1 WT and ΔIROMP KanR knockouts displayed on 1% agarose gel. 65

Figure 3.9 Insertion via homologous recombination of the KanR resistance cassette (aphA3 gene) into fecA1 of Hp-SS1. 67

Figure 3.10 Insertion via homologous recombination of the KanR resistance cassette (aphA3 gene) into fecA2 of Hp-SS1. 69

Figure 3.11 Insertion via homologous recombination of the KanR resistance cassette (aphA3 gene) into frpB1 of Hp-SS1. 71

Figure 3.12 Further attempts to attain expected fragments of all isolated Hp-SS1 WT and ΔIROMP KanR clones. 73

Figure 4.1 The consequences of single and double crossover events due to errors in homologous recombination (HR). 85

Figure 4.2 A schematic of the cell-SELEX method to target fecA1, fecA2 and frpB1 of Hp-SS1 88

X

List of tables

Table 1.1 The various uses of aptamers 21

Table 2.1 List of genes analysed in this study. 26

Table 2.2 List of Helicobacter species evaluated in this study 28

Table 2.3 List of reagents supplemented with the weights (g) to yield a

final volume of 500mLs Stuart’s urease reagent. 32

Table 2.4 Genus-specific 16S rRNA oligonucleotides used in this

experiment. 36

Table 2.5 Gene-specific oligonucleotides used in this study. 37

Table 2.6 Hp-SS1 ΔIROMP–interrupted gene plasmids used in this study.

42

Table 3.1 Complimentary data derived from the interactions of the iron

regulating proteins of Hp-J99, indicating the confidence of each

connection. 57

Table 3.2 Results from selective cultures of Hp-SS1 WT and ΔIROMP

clones attained throughout this investigation. 63

Table 3.3 Results of natural transformation of Hp-SS1 via the liquid

method. 74

Table 3.4 Forward and reverse primers randomly generated and selected

for the SELEX technique. 75

XI

List of abbreviations

°C

Δ

AGS

ApDC

BB

BLAST

Bp

C1

C2

C19H14O5S

CAB

CAS9

CH4N2O

CO2

CRISPR

DNA

DSB

dsDNA

EDTA

EMBL-EBI

ENA

FBS

Fur

g

Degrees Celsius

Mutant

Atmosphere Generation System

Aptamer-drug conjugation

Brucella Broth

Basic Local Alignment Search Tool

Base pairs

Concentration 1

Concentration 2

Phenol Red

Columbia Agar Base

CRISPR-associated protein-9 nuclease

Urea

Carbon Dioxide

Clustered Regularly Interspaced Short Palindromic Repeats

Deoxyribonucleic Acid

Double strand break

Double stranded DNA

Ethylenediaminetetraacetic Acid

European Molecular Biology Laboratory- European Bioinformatics

Institute

European nucleotide archive

Foetal Bovine Serum

Ferric uptake regulator

Gram

XII

g (RCF)

GI

H2O

HBA

HB

Hons

Hp

HPLC

Hp-SS1

HR

IDA

IROMPs

iTOL

KanR

L

mg/mL

MHB

MIC

MIT

mL

MRSA

N2

Na2HPO4

NaH2PO4

NaN3

NaOH

Relative centrifugal force

Water

Gastrointestinal

Horse blood agar

defibrinated Horse blood

Honours

Helicobacter pylori

High-performance liquid chromatography

Helicobacter pylori- Sydney Strain 1

Homologous recombination

Iron deficiency anaemia

Iron-responsive Outer Membrane Proteins

Interactive Tree of Life

Kanamycin Resistant

Litre

Milligrams per millilitre

Mueller-Hinton Broth

Minimum inhibitory concentration

Massachusetts Institute of Technology

Millilitre

Methicillin resistant Staphylococcus aureus

Nitrogen gas

Sodium phosphate dibasic

Sodium phosphate monobasic

Sodium azide

Sodium Hydroxide

1

NCBI

NGS

ng/μL

nM

O2

OD600nm

OMP

PCR

pH

PPI

Refseq

rpm

rRNA

SELEX

TBE

Tm

μg

μg/mL

μM

μL

V

V1

V2

vol/vol

wt/vol

WT

X

National Centre for Biotechnology Information

Next generation sequencing

Nanograms per microliter

Nanometer

Oxygen

Optical density measured at a wavelength of 600nm

Outer membrane protein

Polymerase Chain Reaction

Potential of Hydrogen

Proton pump inhibitor

Reference Sequence

Revolutions per minute

Ribosomal Ribonucleic Acid

Systemic evolution of ligands by exponential enrichment

Tris base/ Boric acid/ EDTA

Melting temperature

Microgram

Micrograms per microliter

Micromolar

Microlitre

Volts

Volume 1

Volume 2

Volume per volume

Weight per volume

Wild-type

Times

2

Abstract

Helicobacter pylori (Hp) infection is prevalent in 50% of the global population and is

deemed one of the leading causes of gastrointestinal disorders. Low socioeconomic

regions with poor sanitation, crowded living conditions and limited access to health

care facilities are risk factors that correlate to the incidence of Hp, infecting up to

80% of those living in developing countries.

Hp infection leads to gastritis and a fraction of those colonised will later develop

peptic ulcer disease, Mucosa Associated Lymphoid Tissue (MALT) lymphoma or

gastric cancer. Certain virulence factors, such as the Cag pathogenicity island (Cag

PAI) carried by certain strains of Hp have been correlated to the increased risk of

developing gastric cancer.

In the post-antibiotic era, common antimicrobial treatments are failing due to the

emergence of ‘Superbugs’ that carry genes that enable them to resist the action of

certain antibiotics. Hp has acquired such resistance to three of the commonly used

antibiotics, leading to refractory infections. This has minimised success rates of

eradication, causing more severe patient outcomes, urging the development of a novel

therapy.

3

One opportunity to develop a novel treatment to aid H. pylori eradication involves a

more direct alternative, targeting outer membrane virulence factors, while causing

minimal or no side effects. Aptamers are single stranded DNA or RNA molecules

that can assume a variation of structures due to their propensity to form hairpin loops

and helices, and once modified, will bind to proteins and peptides with a high affinity

and specificity. Furthermore, these molecules can be intercalated or functionalised

with a therapeutic agent, to be internalised or delivered to the diseased cell.

The Iron Responsive Outer Membrane Proteins (IROMPs) of H. pylori in particular

fecA1, fecA2 and frpB, are essential virulence factors required for iron acquisition

and persistence within the human host.

The key principle of this project was to identify a protein essential to Hp that would

be suitable for as target for aptamer based therapies. Aim 1 was to analyze the

IROMPs and other major proteins of iron-regulation within the Proteobacteria’s and

more specifically, Hp. Using a bioinformatics approach, the phylogenetic analysis of

key proteins in iron-regulation demonstrated a large portion of those common

amongst the phyla. The IROMPs of interest were confirmed to be specific to Hp and

were implicated in fur-dependent regulation, the central control of this process. In

turn, inhibiting these proteins could cause detriment to Hp.

Aim 2 involved the verification of the IROMP-interrupted kanamycin resistant

clones, required for negative selection of SELEX. Confirmation of these knockouts

could not be obtained, requiring further experimentation.

4

Aim 3 involved the SELEX technique to derive an aptamer that selects for fecA1,

fecA2 and frpB1 in Hp. As major component of this method could not be obtained

within the time frame the process could not be ensued.

Due to the major role of the IROMPs in maintaining a functional level of iron within

this cell, displayed qualities suitable for this targeted therapy. In turn, this study

demonstrated a ‘proof of principle’, an accurate process to identify outer membrane

virulence proteins in other disease-causing pathogens to derive specific aptamers that

potentially, can form the basis of novel antimicrobials.

5

Chapter 1 : Introduction

1.1. Clinical Significance

Helicobacter pylori (Hp) is a leading cause of gastrointestinal disorders due to its

localisation within the mucosal later of the gastric epithelium1 (Figure 1.1). Virulence

factors of Hp are comprised of cunning mechanisms that aid Hp to persist within the

host. For example, urease, an enzyme secreted by Hp that breaks down urea to

ammonia, forming a protective barrier from the harsh environment of the stomach1.

Figure 1.1 The localisation of Hp. As Hp colonises the mucosal layer of the gastric epithelium, causing inflammation weakening the mucosa of the stomach, predisposing into various clinical outcomes, like ulcers.

6

The association between Hp and gastrointestinal disorders is indicative of its clinical

significance, as severe conditions i.e. gastric and duodenal ulcers, gastric cancer and

mucosa-associated lymphoid tissue (MALT) lymphoma, can be secondary to the

presence of this pathogen. In addition, the pathophysiology is dependant on the

positioning within the stomach2.

Figure 1.2 Adapted from Peek (2008) demonstrating the variation of clinical outcomes dependent on the colonisation of Hp and its location within the gastric mucosa3. a) An antral predominant infection induces gastritis of lower part of stomach causing the hyper secretion of acid that result in site-specific ulceration. b) The body predominant infection produces inflammation of the mucosal lining of the entire stomach causing gastric atrophies that can lead to more severe clinical outcomes e.g. cancers. Colonisation with Hp may be asymptomatic or lead to varying degrees of dyspepsia2.

Chronic infection can progress to atrophic gastritis or inflammation of the stomach2,

in addition to, iron-deficiency and anaemia4.

7

Further complications arise from damage to the gastric epithelium that may cause

bleeding, peritonitis and leakage of the stomach contents2. The over secretion of acid

is characteristic to an antral-predominant infection of Hp (Figure 1.1a) thus,

prompting conditions of pre-pyloric and duodenal ulceration2. The colonisation of the

entire body of the stomach (Figure 1.1b) instigates gastric atrophy that can later lead

to ulceration, adenocarcinomas and (MALT) lymphoma2. The world health

organisation (WHO) has identified Hp as a ‘group 1 (definite carcinogen)’5 and

accordingly, the occurrence of severe ailments progressing to gastric cancers indicate

the need for eradication from the human host.

1.2. Prevalence and epidemiology

While Hp infects more than 50% of the population, the incidence varies on a global

scale6 (Figure 1.2).

The infection is common however, more prevalent within developing countries6.

Previous studies suggest that the infection is acquired in childhood or early in life6.

The correlation between Hp and socio-economic status is a determinant of the risk of

infection. In developing countries, carriage is associated with low income,

overcrowded & rural living conditions, poor sanitation and minimal access to health

care8. In westernised or developed countries, the improved standard of living

contributes to the decreased incidence of Hp infection however, in isolated areas of

increased prevalence, immigration is thought to be responsible8.

8

Figure 1.3 The global prevalence of Hp infection (%), adapted from the reflux centre (2015) 7. The incidence of Hp increases in developing countries, as up to 80% of those living in regions of low socio-economic status, e.g. parts of Africa and Asia are infected. In developed countries such as, Australia, approximately 50% of the population is infected with Hp. Recent epidemiologic studies have provided evidence of increased Hp prevalence

amongst Australia’s indigenous population9. The infection is as common in these

individuals as it is in developing countries as they reside in remote parts of Australia9.

The investigation found that 91% of the indigenous population were carrying Hp and

the cause of this rate is most likely as of their location, low-socio-economic status and

poor sanitary conditions9.

Interpersonal contact is presumed to be the route of spread although; the transmission

remains unknown6.

9

1.3 Helicobacter pylori: The microbiology

Helicobacter pylori (Hp) is a gram-negative bacterium that is further classified as a

curved rod10. A spiral or helical form is characteristic to the organism, including 5-8

polar-sheathed flagella found at one end11-12. H. pylori is microaerophillic,

necessitating a reduced amount of oxygen than found in the atmosphere for growth10.

Various enzymes such as, Hydrogenase, Urease, Catalase and Oxidase facilitate Hp

colonisation in the harsh environment of the gastric epithelium10. Following secretion,

these virulence factors promote an array of functions that range from, the colonisation,

damage to the gastric epithelium and the provision of crucial metabolic substrates13.

Figure 1.4 A depiction of the spiral-shaped pathogen, Helicobacter pylori. The 5-8 clusters of polar-sheathed flagella provide motility, and aids colonisation within the mucosal layer of the gastric epithelium14.

Some strains contain additional virulence factors and are more likely to cause severe

clinical outcomes15. The Vacuolating cytotoxin A (VacA) is a key protein that is

internalised into host cells, accumulating large vesicles16.

10

In addition, the Cag pathogenicity island (Cag PAI), a well-defined virulence

determinant, functions to encode the Cytotoxin-associated gene A (CagA) as well as,

the type IV secretion apparatus which when assembled, facilitates injection of CagA

toxins into host cells17. Subsequently, these molecules are causative of actin

remodelling, Interleukin-8 (IL-8) driven inflammation and host cell growth/apoptosis

inhibition, which helps Hp evade the host immune systems to aid persistence17.

Other virulence factors that have been identified in Hp are the collection of outer

membrane proteins (OMPs), adapting independently within the host in response to

changes in iron levels18. The Iron-responsive outer membrane proteins (IROMPs) are

a central focus of analysis for this project, in particular, the fecA1, fecA2 and frpB1.

As it is known, iron is crucial to all living beings, essentially operating as a co-factor

for many enzymatic activities and catalysing electron transport processes19. Iron is

complexed into various forms, to prevent it reacting within the body creating

damaging free radicals and oxidative stress, necessitating the ligand range of different

types of IROMP’s20. In Hp, the Ferric uptake regulator (Fur) protein is the central

switch in gene expression, balancing the intracellular iron concentration21. Depending

on iron availability, Fur controls the transcription of IROMPs specific to their role in

iron acquisition21. Moreover, such proteins attain iron from distinctive sources that are

demonstrated via the transport of Ferric citrate by FecA1/2 and the utilisation of

haemoglobin by FrpB121. The transfer of Ferrous iron by FeoB is an exception to this

family of proteins due to their significant mechanism. This form of iron can freely

pass into the cell, however, requires transport via FeoB through the cytoplasmic

membrane21.

11

Figure 1.5 The expression of IROMPs in varying conditions of iron, regulated by fur. a) Adapted from van Villet et al (2002). In conditions of increased iron availability, ironbound fur (holo-fur) binds to the ‘fur box’ promoter region, blocking RNA polymerase (RNAP) from initiating the transcription of fecA1, fecA2 and frpB1. In conditions of low iron availability, iron-poor fur (apo-fur) is disintegrated from its dimer configuration, allowing RNAP to bind the promoter region and begin the transcription of fecA1, fecA2 and frpB119. b) Adapted from Andrews et al (2003), demonstrating the expression of IROMPS in corresponding situations, regulated by Fur22. When iron levels are sufficient or high, minimal IROMPS are expressed on the surface of the plasma membrane. In opposing conditions, numerous IROMPS are located on the outer membrane as fecA1, fecA2 and frpB1 are transcribed as required22.

In iron-replete conditions (Figure 1.4), fewer IROMPs are expressed23. This results in

the partial repression of iron uptake, as less IROMPs are transcribed, saving energy23.

In iron-restricted states, all IROMPs are expressed including FeoB23. An intense

upsurge of transcriptional rates of these genes and associated proteins ensues, so that

iron from diverse sources is imported into the cell23. This complex process

constitutes the iron homeostasis system in Hp, to the detriment of the host, during

acquisition.

12

1.4. Current Treatments and Failures

The guidelines of eradication of Hp consist of a multi-drug therapy that follows a

strict combination regimen25. A first to third line scheme is suggested, contingent on

the number of eradication failures, allowing HP to persist as a refractory disease. The

initial or first line treatments involve binary triple therapy options (Figure 1.5.)

including, a proton pump inhibitor (PPI) that suppresses acid production and two

alternative antibiotics that depend on allergy to β-lactam medications25.

Figure 1.6 The current therapy for Hp eradication. Adapted from Pinion (2011), detailing the first, second and third line strategies including multi-drug regimens of antibiotics, a proton-pump inhibitor (PPI) and in some cases Bismuth26. If eradication is ineffective, the second-line is employed. This management option

contains a triple combination, however a quadruple therapy is generally advised in

response to the failure of the first line (Figure 1.5) 25. The quadruple therapy

comprises a proton-pump inhibitor (PPI), two alternative antimicrobials in addition to

Bismuth, a metal that acts as an antacid25.

13

A second incidence of failure minimises the possibility of eradicating Hp and may

lead to multi-drug resistant strains existing within the host. A third line alternative is

offered (Figure 1.5), involving ‘empirical rescue therapy’ that is, a specific

antimicrobial-based treatment formulated once ineffective combinations are

identified27.

Hp has developed resistance to key treatment antibiotics, such as Clarithromycin,

Metronidazole & Amoxicillin27. In 2002, the Helicobacter pylori Antimicrobial

Resistance Monitoring program (HARP) study, revealed the percentage of resistant

strains to the corresponding antibiotics mentioned as 12.9%, 25.1% and 0.9% in

which a yearly increase is still being reported27. The concern is shown in more current

figures, observing 56.6% of clarithromycin resistant-strains and 59.2% for

metronidazole28. The rising prevalence of Hp infection in addition to the varying

success of multi-drug therapies that can lead to treatment failures urges the need for

novel antimicrobials.

1. 4. 1 The misuse of antibiotics and the spread of resistant pathogens

Currently, antibiotic resistance is a major problem for eradication of bacterial disease

due to the continuous rise in treatment failure and hence, disease prevalence29.

Antibiotic resistance is regarded a ‘severe public health problem’ due to the absence

of treatment alternatives, in the drug discovery pipeline30. This impending public

health crisis may take treatments for infectious diseases back to a pre-antibiotic era,

limiting treatment options for other diseases like cancer and orthopaedic issues.

14

Resistance in bacteria can be an innately occurring and attribute to acquisition by

other strains via exchange of genetic material or via mutation (Figure 1.6a). This

provides an evolutionary advantage to the resistant pathogen over susceptible

strains during the spread of disease (Figure 1.6b) 31.

Figure 1.7 The acquirement, misuse and the spread of antibiotic resistance, adapted from centres for disease control and prevention (2015). a) Broad-spectrum therapies, i.e. antibiotics, used to eradicate the infecting pathogen can abolish the protective microbiota within the gut. In the presence of resistant strains that outcompete the good bacteria, continue to proliferate and spread resistance genes, producing new refractory strains. b) The misuse of antibiotics in agriculture and medicine facilitate the spread of resistant pathogens throughout the general public.

15

The leading cause of antibiotic resistance in bacteria is misuse, i.e. for viral infections,

in human medicine and agriculture31. The compliance and duration of the course of

antibiotic treatment is another factor that contributes to this problem.

1. 4. 2 Overcoming the ‘superbug’ In the post-antibiotic era, limited options exist to overcome the increasing prevalence

of Hp to resist treatment. In 2011, the second and third line treatments were

introduced as alternative measures however, Hp continued to become increasingly

resistant to antibiotics that were once considered definitively curative32.

Newer therapeutics have since been proposed. One suggested treatment involves

sequential and salvage therapies, using a defined regimen of various antibiotics

administered at precise intervals (hour/day/weeks) to minimise insufficient durations

of the treatment course29. Although this measure weakens the contribution to

resistance, again, some individuals fail to respond to this treatment and only effectual

in patients carrying clarithromycin-resistant strains and has limited efficacy data29.

In 2014, Markus Gerhard from the Technical University of Munich proposed the first

vaccination against Hp that theoretically had the potential to overcome antibiotic

resistance33. The rationale for the vaccine arose from the comprehension of the

relationship between Hp and the human host33. Knowledge of the defence mechanisms

of Hp indicated the application of the inactivated form of gamma-glutamyl

transpeptidase to overcome these microbial evasion strategies33. Typically, the protein

promotes the blockade of the T-cell response of the host immune defence, upon

activation33.

16

In the application of immunisation, the deactivated form prompts an immune response

that disables the evasion strategies of Hp33. Hypothetically this vaccine is capable

however, due to insufficient funding and limited evidence and investigation in support

of the introduction to this novel therapeutic, the vaccine is some way from clinical use

in humans. Several previous attempts to make a sterilising vaccine for Hp have failed22.

Bio prospecting or the application of plant derivatives as curatives has also been

suggested as treatment alternatives to Hp eradication. Previous studies proposed the

combination of Catechins, attained from a plant-source and sialic acid, a component of

milk34. The grouping is reported to reverse cell injury and improve the restoration

system through the autophagy pathway34. Insufficient information is available on these

constituents regarding side effects, efficacy and the therapeutic response hence these

sources are far from a potential therapy. Another similar study, explores natural

resources such as plants, probiotics and nutraceuticals as a new treatment option35. The

investigation reveals the inefficacy of this alternative, as it simply reduces bacterial

levels permitting colonisation and internal destruction35.

Several antibiotic based therapies and regimens are constantly introduced, maintaining

their position as the primary first line treatment in eradication therapy of Hp. These

treatments have several integral complications, including the constant evolution of

antibiotic resistance and the correlated unfavourable effects, the possibility of re-

infection and the increasing expense of antibiotic therapy35.

Our gut bacteria that lives in a commensal relationship with the host, is also

eradicated with the misuse of antibiotics, creating a route of infection for

opportunistic pathogens35.

17

As part of the GI ecosystem, bile acid modifying microbes usually hinder the

proliferation of detrimental bacteria’s (such as Clostridium difficile), are eradicated

by antibiotics and permits spore germination of these bacterial pathogens35. The

problem of antibiotics is their lack of specificity as they target a cell wall common to

various microbes35. Along with antibiotic resistance and the absence of beneficial

bacteria, more of these ‘Superbugs’ are outcompeting the normal flora further

spreading resistance into the community35.

These factors provide an emphasis on the demand for a novel eradication therapy,

exposing the gap in the literature that requires urgent action.

1. 5 Aptamers

Aptamers were invented in the 1990’s and the potential of these molecules were

recognised in 2007. Aptamers are synthetically produced, single-stranded DNA or

RNA molecules that can bind to pre-elected targets i.e. proteins and peptides, with

high affinity and specificity36. These molecules can further take the form of numerous

structures to produce helices and single-stranded loops that demonstrate the

adaptability in binding to assorted targets36. The key attribute of these molecules is

their increased target selectivity37. Furthermore, Aptamer’s interrelate and bind to

their targets via physical recognition37. The Systematic Evolution of Ligands by

Exponential Enrichment (SELEX) is the process by which aptamer’s are formulated,

as the nucleic acid strands begin as a random pool and during successive sequences

are enriched throughout the iterative selection procedure37.

18

A primary practice involving these molecules is the Aptamer-Drug conjugation

(ApDC) that enables the covalent or non-covalent conjugation of aptamer sequences

directly with their pre-selected drug treatment (Figure 1.7.) 37. This results in

enhanced efficacy of the therapeutic agent to target a precise location or protein on or

within a cell37.

Figure 1.8 The schematic representation of aptamer drug conjugation (ApDC), adapted from Sun et al (2014). Aptamers are able to be covalently or non-covalent conjugated to a drug to directly be delivered to the diseased cell, enhancing the efficacy of these treatments. The current gold standard small molecule therapy for many different diseases like

cancers and autoimmune disorders, are antibody-based therapeutics37. Although they

are highly specific and recognised as non-foreign to the host, various disadvantages

of these biologically derived molecules are displayed.

19

The prospective immunogenicity and high cost of these therapeutics limit the clinical

applicability of this therapy as it can lead to allergies or immune inactivation in the

host, favouring the use of aptamers37.

1. 5. 1 The current clinical uses of aptamers

Various aptamers have been introduced into the clinic for applications in conditions

of macular degeneration, coronary artery bypass graft surgery and for several types of

cancer38. This small molecule therapy has also sparked therapeutic interest in treating

Human Immunodeficiency Virus (HIV), immune modulation, haematology,

Alzheimer’s disease and Diabetes37.

Research into this relatively novel therapeutic concept has inspired further

investigation into their diverse capabilities as highly specific therapeutic molecules

(Table 1.1.) 38. In particular, aptamers have gained serious momentum in cancer

therapies38. Current investigation has displayed the capacity of aptamer’s to

accurately differentiate between distinctive types/sub-types of cancer cells38. This

high-recognition specificity has attributed to the early diagnosis of differential forms

of tumours that decreases the risk of metastatic disease38.

Aptamers have also demonstrated a targeted response to a wide-range of receptors

that can be extensively modified for diagnosis of diseases and delivery of curative

moieties into various types of cells38.

20

Macugen or Pegaptanib sodium is the first aptamer approved by the U.S. FDA that

targets the Vascular Endothelial Growth Factor (VEGF) in macular degeneration and

diabetic retinopathy and has remained a highly effective treatment and motivation of

other targeted therapies38. As underlined in Table 1.1, these small molecules can be

chemically synthesised to formulate an applicable application to a broad range of

disorders. The increasing versatility demonstrates the capacity to apply these

molecules to diagnostic measures, biosensors and targeted treatments, conveying a

positive direction in therapeutic research.

21

Table 1.1 The various uses of aptamers adapted from Shigdar et al (2010) 40. The ability of these aptamers to be functionalised to target a diseased cell or part of provides vast potential for novel therapies of human disease.

22

1. 5. 2 New uses – Antimicrobial aptamers?

The growing concern of antibiotic resistance in bacteria is adding momentum to

research for clinical applications of aptamers as a novel antimicrobial.

Aptamers in the microbial field remains highly new as limited research on these

molecules and their action on bacterial cells have been undertaken.

Of the few relevant studies in the literature, aptamers have displayed the ability to

recognise Methicillin-Resistant Staphylococcus aureus (MRSA) strains40. This is

achieved through identification of membrane structures by the molecule, enabling the

eradication with strain-specific antibiotics40.

In addition, investigation on Salmonella choleraesuis displayed the exciting ability of

target-specific aptamer’s controlling the formation and promoting antibiotics to direct

an inhibitory effect on biofilms41. The aptamer was selected to target the flagella of

S. choleraesuis to supress immediate biofilm development41. These flagella are

required for this formation and once inhibited antibiotic treatments became more

effective41.

Supplementary bacterial inquiries of significance regarding the application of

aptamer’s have been undertaken on Salmonella typhimurium, Staphylococcus aureus,

Group A Streptococcus and Escherichia coli42. These studies have provided strong

evidence on biosensing capabilities to detect pathogens and toxins42.

23

Since the use of aptamer’s and their respective analyses has mainly remained a focus

on mammalian cells, the practice of these molecules as a novel therapeutic target for

Hp is yet to be attempted. Most aptamers that have been studied for bacterial

eradication have been more directed to biosensing and inhibitors of virulence factors

that enhance the efficacy of antibiotics. Subsequent research has yet to test for

aptamers that directly kill bacteria with or without a conjugated payload, hence this is

a first attempt in the area of microbiology.

1. 6 Rationale and aims

The infection of Hp is estimated to effect 50% of Australia, in addition to, 91% of the

indigenous population9. This infection is significantly associated to gastrointestinal

disorders causing gastritis, gastric atrophy, peptic ulcers and rare forms of cancer.

The emergence of antibiotic resistance has contributed to the diminishing effects of

typical antibacterials. Hp has acquired resistance to all three commonly used

antibiotics with rates of up to 60% of resistant strains, reporting a yearly increase. Not

only does this urge the need for a novel therapy but one that can overcome antibiotic

resistance with a more direct effect. The use of aptamers display highly desirable

qualities, as they can be functionalised to bind to specific cells, obstructing essential

processes. Given that aptamer-based therapies for bacterial infections have yet to be

investigated, this demonstrates the significance of this project.

24

The rationale of this project is based upon the following:

• The projected rates of Hp resistant strains is set to increase yearly, causing an

upsurge in more serious clinical outcomes, an effective antimicrobial is

urgently required.

• Due to the limitations of broad-spectrum therapies causing detriment to the

microbiota of the gut, analysis of the targeted therapies to demonstrate this

therapeutic would be more efficacious.

• The process of iron homeostasis has been demonstrated to aid in Hp

colonisation and in particular, a select group of proteins that function in iron-

acquisition.

• The IROMPs fecA1, fecA2 and frpB1 that have demonstrated to be essential

to Hp, are expressed in iron depleted levels, attaining differing sources of

iron, and in the process of obstructing one or all, could prevent further

colonisation.

• Aptamers have demonstrated highly advantages qualities and therapeutic

efficacy, especially in the field of cancer. As it is yet to be tried within the

microbial field and has presented increasing potential, it could form the basis

of generating new antimicrobials.

This reasoning has lead to following research question that is reflected by the aims of

this project:

Hypothesis - Can the IROMPs (fecA1, fecA2 and frpB1) of Hp be used as a suitable

target for aptamer based therapies?

25

Aim 1 - To investigate the iron responsive outer membrane proteins (ΔIROMPs)

(ΔfecA1, ΔfecA2 and ΔfrpB1) of Hp and the Helicobacter species in silico.

Aim 2 - To validate the insertional mutagenesis of the ΔIROMP proteins (ΔfecA1,

ΔfecA2 and ΔfrpB1) of Hp-SS1.

Aim 3 - To generate an aptamer with a high binding affinity and specificity for the

ΔIROMP’s of Hp-SS1 using the SELEX method.

26

Chapter 2 : Materials and Methods

2.1 Computational analysis of the iron regulating proteins of

Hp–SS1 and the Helicobacter species.

2.1.1 Phylogenetic investigation of iron regulating proteins of Hp-

SS1 and the Helicobacter species

To identify the gene orthologs that retained the function of iron regulation in

Proteobacteria and further sub-groups, the NCBI reference sequence (refseq)

protein annotations, as mentioned in Table 2.1, were run against the protein

clusters43 database (https://www.ncbi.nlm.nih.gov/proteinclusters). The

analysis provided the homogeny and separation between the proteins in

question, by measuring the maximum alignment of comparable sequences,

calculated by the Basic Local Alignment Search Tool (BLAST). Each of the

outputs were collated manually to compare the range of iron-regulating genes

amongst the phyla, and confirm those specific to Hp.

Table 2.1 List of genes analysed in this study. The corresponding identifiers were generated by the ENA and NCBI reference sequence databases.

27

2.1.2 Sequence alignments of the IROMPs and related regulatory

proteins

The Ferric uptake regulator (fur) (HPG27_401) sequence derived from

H.pylori G27 in the FASTA format was run against the 23 Helicobacter

species as listed in Table 2.2, in the protein database of Ensembl bacteria44

(http://bacteria.ensembl.org/index.html), developed by the European Molecular

Biology Laboratory- European Bioinformatics Institute (EMBL-EBI). To

generate the multiple sequence alignments, the resulting 24 FASTA sequences

and the IROMP’s of interest (fecA1, fecA2 and frpB1) in addition to fur, were

submitted to Clustal Omega45 (http://www.ebi.ac.uk/Tools/msa/clustalo/). The

output data was visualised with Jalview 246, for interactive editing, annotation

of multiple sequence alignments and detection of the conserved 7-1-7 motif,

identified as the ‘fur box’ (TAATAATnATTATTA).

28

Table 2.2 List of Helicobacter species evaluated in this study retrieved from the ENA.

2.1.3 Visualisation of phylogenetic tree

The output data in FASTA format was uploaded to the Clustal W2-phlogeny

web tool, and the results were visualised using the interactive Tree of Life

(iTOL) v3.2.447 (http://itol.embl.de/) to observe the genetic distances of the

ortholog fur genes amongst the 24 species of Helicobacter, described in Table

2.2. The fur protein of Neisseria meningitidis (AAF40662), also a global

regulator of IROMPs in this species, was included as the output group for a

point of comparison and reference to generate accurate conclusions.

29

A subsequent tree was derived from the output data and visualised with

iTOL, to identify the bootstrap values (0-100%) or the level of confidence,

where a score of 100% is representative of well-supported data as the node

appeared in all bootstrap replicates.

2.1.4 Identification of protein-protein interactions

The functional interactions of the proteins in Table 2.1 were submitted to

String v10.048 (http://string-db.org/), a database of known and projected

protein-protein interactions produced from supplementary aggregated data.

The output image specified node connections based on gene fusions,

neighbourhoods, co-occurrence, textmining, co-expression, homology, curated

databanks and experimentally determined products. The table generated,

detailed the queried interactions, annotated with scores that indicated a

confidence rating (i.e. 0-1), where a score of 1 is of the maximum confidence.

2.1.5 Generation of sequence logo

The output data in FASTA format was uploaded to WebLogo 349

(http://weblogo.threeplusone.com/create.cgi) to generate a graphical depiction

of the specified multiple sequence alignment (fecA1, fecA2, frpB1 and fur). A

‘stack’ of symbols (A, T, G, C) in each position of the sequence with differing

heights defined the frequency of conservation and further highlighted the

consensus sequence.

30

2.2 Bacterial Strains and routine cultures

The subtype used in this study is a mouse-colonising strain that originated from a

42-year-old woman of Greek descent. The H. pylori Sydney Strain 1 (Hp-SS1)

was initially described by Lee et al. (1997), providing a standardised mouse model

for therapeutic development, complex screening and analyses of pathogenesis50.

The Hp-SS1 wild-type (WT) samples were donated by Associate Professor

Richard Ferrero in addition, the ΔIROMP mutant kanamycin resistant (KanR)

clones, were provided Dr Melanie Thomson (School of Medicine, Deakin

University) and constructed by Teisa Holani (Hons 2012). Long-term storage

conditions included stocks of Hp-SS1 in 20-30% (wt/vol) glycerol (Biochemicals,

Gymea, NSW) and Mueller-Hinton broth (MHB) (Bacto Laboratories Pty. Ltd.,

Liverpool, NSW), stored at -80°C. Frozen stocks of Hp-SS1 and mutant KanR

clones were inoculated onto horse blood agar (HBA) that contained: Columbia

agar base (CAB) (Oxoid, Ltd, Basingstoke, England) including 10% (vol/vol)

defibrinated horse blood (HB) (Serum Australis Pty. Ltd. for Oxoid Australia Pty.

Ltd., Manilla, NSW) in addition to, the selective supplement, Dent (5.0mg

Vancomycin, 2.5mg Cefsulodin, 2.5mg Trimethoprim, 2.5 Amphotericin B)

(Oxoid, Ltd) and incubated for 10-12 days at 37°C. Hp-SS1 and mutant ΔfecA1,

ΔfecA2 and ΔfrpB1 clones were routinely cultured in three separate technical

replicates.

Experimentation including the application of Hp-SS1 WT and KanR mutants was

conducted in the Class II Biosafety Cabinet (LAF Technologies, Pty, Ltd.,

Bayswater North, Victoria) to ensure maximum safety and sterility.

31

To mimic the microaerophillic condition, all solid and liquid cultures of Hp-SS1

were cultivated in the atmosphere generation system (AGS), producing a low

oxygen environment by converting oxygen to carbon dioxide simultaneously. The

AGS includes a sealed AneroJar® and a CampyGen® sachet (5% 02, 85% N2, 10%

CO2) generating a final concentration of 5% 02, (Oxoid, Ltd, Basingstoke,

England) that was replaced upon exposure to additional 02 or every 2-3 days as

required. Furthermore, all cultures were incubated in a still air, thermostatically

controlled incubator (Labec Incubator, Laboratory Equipment Pty. Ltd.,

Marrickville, NSW) at 37°C.

2.2.1 Preparation of 10% HB + CAB + Dent

The selective media, referred to as 10% HB + Dent, provided optimal growth

conditions for the cultivation of Hp-SS1 WT and KanR mutant clones. The

preparation comprised of CAB with 10% (vol/vol) HB and supplemented with

Dent. CAB in addition to, other media bases, were sterilised in the Pratika B20

(20L) basic autoclave (Siltex Australia, East Bentleigh, VIC) including 30 minutes

of optimisation and 5 minutes of drying time at 110°C. As recommended for the

primary isolation of Hp, CAB was utilised as a base for blood-containing media.

The HB provides essential nutrients and serum factors such as iron, supplying the

complex growth requirements of Hp and its fastidious classification. The Dent

supplement includes various antibiotics that Hp is resistant to, inhibiting the

growth of other microbes and antifungals, such as amphotericin, limiting potential

contamination whilst directly selecting for Hp.

32

Following the protocols by Oxoid (Catalogue number: CM0331), the media was

prepared, poured and dried in the Fumehood: Laminar flow workstation (AES

Environmental, Pty, Ltd., Minto, NSW) to maintain a controlled and sterile

environment.

2.3 Hp-SS1 – Rapid diagnostic test: Stuart’s Urease Reagent

2.3.1 Preparation of Stuarts urease reagent

Preparation of the urease solution included the addition of the specified reagents

listed in Table. 2.3, to 500mL of Milli-Q water (Merck Millipore, Bayswater,

VIC). The mixture was filter sterilised by the Nalgene filtration product (Thermo

Fisher Scientific Inc., Waltham, USA) and paired with the rocker 300DC vacuum

(Rocker Scientific Co., Ltd., New Taipei City, Taiwan) to separate solid particles

and maintain sterility. The solution was stored at 4°C and concealed in aluminium

foil as the dim conditions inhibit spontaneous reactivity to light.

Table 2.3 List of reagents supplemented with the weights (g) to yield a final volume of 500mLs Stuart’s urease reagent.

33

2.3.2. Urease test

The urease test is a rapid diagnostic method utilised to differentiate Hp, on the

basis of urease production. Prior to and following the conclusion of each

experiment, a urease test was undertaken to detect the presence of the urease

enzyme, an essential virulence factor for Hp. A positive result was used as an

indirect indicator that the cultured organism was likely to be Hp.

Urease Test: 1-2 colonies of Hp-SS1 WT or mutant clones were resuspended in

200μL of Stuart’s urease reagent51. An instant colour variation from yellow

(negative) to bright pink/purple (positive), indicative of the breakdown of urea to

ammonia and carbon dioxide, confirmed the production of urease and hence, likely

the presence of Hp-SS1.

2.4 Purification of Hp-SS1 WT and mutant DNA

To attain DNA in a purified form, the DNA from the isolated colonies were

extracted and stored for long-term use. The DNeasy blood and tissue kit (50)

(Qiagen, Hilden, Germany) was used to purify the DNA of Hp-SS1 and mutant

clones, as per the Quick start- DNA extraction protocol (Catalogue number:

69504) stated by the manufacturer. The purified stocks were stored in labelled

eppendorf tubes at -20°C.

34

2.5 Quantification and assessment of DNA concentration

Nanodrop spectroscopy was utilised prior to the commencement of an experiment,

to identify the concentration in addition to, the purity of DNA that was extracted.

The Nanodrop detects the DNA concentration and calculates the absorbance

reading by utilising the 0.2nm pathlength (260/280nm ratio).

To measure the concentration of each purified Hp-SS1 sample, the ND_1000

Nanodrop spectrophotometer (NanoDrop Technologies Inc., Wilmington, USA)

was blanked using 1.7μL of nuclease-free water (Promega, Madison, USA) to

obtain a reading of 0ng/μL. The process was repeated using 1.7μL of each Hp-SS1

sample, cleaning the pedestal in-between readings. The Nanodrop connected to a

desktop computer that included the complimentary software, ND_1000: V3.81

provided the DNA concentrations.

2.6 Selection of Kanamycin resistance

To confirm the insertion of the kanamycin resistance cassette, Kanamycin was

supplemented to the media at a final concentration of 25μg/mL. Confirmation of

KanR was confirmed if Hp-SS1 was cultivated on the media. Hp-SS1 from the

second culture was re-inoculated on the 10% HB + Dent + 25μg/mL Kan plates

and incubated at 37°C.

35

2.6.1 Concentration calculation for 25μg/mL Kan in 500mL of media

The concentration of the stock solution of Kanamycin required a dilution of

10ng/mL to 25μg/mL for 500mL of media. The calculations were as follows:

C1V1 = C2V2

25μg/mL x 500mL = 10,000μg/mL x V2

V2= 12,500/10,000 = 1.25mL

2.6.2 Preparation of 10% HB + Dent + 25μg/mL Kan

The 10% HB + Dent + 25μg/mL Kan was produced as per the instructions for

‘2.2.1 Preparation of 10% HB + CAB + Dent’, with the addition of 1.25mL

(25μg/mL) of Kanamycin.

2.7 Confirmation of Helicobacter genus

As all Helicobacter species possess a common 16S rRNA gene sequence, a set of

genus specific oligo’s, as described in Table 2.4, were utilised to detect the

conserved fragment as observed in Figure 2.1.C.

2.7.1 Calculations for 100μM oligonucleotide stocks

As the oligonucleotide stocks were received in their dehydrated form, Milli-q

water was added according to the nmoles in each tube. The calculations were as

follows:

Forward = 10 x 31.2μM = 312μL

Reverse = 10 x 36.7μM = 367μL

36

2.7.2 Preparation of primer stocks (100μM)

To the dehydrated forward and reverse oligonucleotides, 312μL and 367μL of

nuclease-free water (Promega, Madison, USA) were added, respectively. The

contents were vortexed thoroughly to ensure the oligo’s were completely

dissolved. The 100μM forward and reverse stocks were stored at -20°C.

2.7.3 Oligonucleotides used to detect the Helicobacter genus

The 16S rRNA oligonucleotides as described in Table 2.4, designed by Dr Waleed

Abu Al-Soud (Molecular Biology, University of Copenhagen, Denmark) et al,

were obtained from Sigma-Aldrich Co. (Missouri, USA) and used as the target

template for analysis.

Table 2.4 Genus-specific 16S rRNA oligonucleotides used in this experiment.

2.8 Confirmation of fecA1, fecA2 and frpB1 KanR mutant

knockouts

The original cloning oligonucleotides constructed and donated by Dr Henry

Windle (School of Medicine, Trinity College, Dublin) were used to confirm the

insertion of KanR cassettes within the putative ΔIROMP’s in question via PCR.

37

2.8.1 Oligonucleotides used to detect ΔIROMP knockouts

The oligonucleotides as described in Table 2.5, were obtained from Integrated

DNA technologies (IDT) Inc., (Iowa, USA) and used for further inquiry.

Table 2.5 Gene-specific oligonucleotides used in this study.

2.9 Polymerase Chain Reaction (PCR)

The DNA of Hp-SS1 WT and ΔIROMP knockouts were amplified using PCR in

25μL final reactions, succeeding numerous optimisations. The reactions included:

1μL of the applicable forward and reverse oligonucleotides detailed in Table 2.4

and 2.5, 8.5μL nuclease-free water (Promega, Madison, USA), 12.5μL GoTaq®

Green Master Mix (Promega, USA) and 2μL of Hp-SS1 DNA template. The

negative control was prepared with 10.5μL nuclease-free water, substituting the

addition of DNA to attain an equivalent reaction volume. The reactions were

amplified by the Applied Biosystems™ ProFlex™ PCR system (Life

Technologies Corporation, California, USA) following a set of standard

parameters for each PCR cycle.

38

The amplification conditions included: an initial denaturation cycle at 94°C for 5

minutes, following 35 cycles of additional denaturation at 94°C for 30 seconds,

annealing at 55°C for 30 seconds and extension at 72°C for 30 seconds. An

extension cycle at 72°C for 10 minutes finalised the reaction. The PCR samples

were stored at 4°C.

2.10 Separation of PCR products via gel electrophoresis

Gel electrophoresis was used to separate and analyse the relative size of Hp-SS1

WT and ΔIROMP mutant DNA.

2.10.1 Preparation of 5M NaOH

To prepare 200mL stock solution of 5M NaOH, 40g of NaOH (Sigma-Aldrich

Co., Castle Hill, NSW) was added to 200mL Milli-q water and stirred thoroughly

until the contents were dissolved. The solution was stored at room temperature.

2.10.2 Preparation of 0.5M EDTA (pH 8.0)

To prepare 1L of 0.5 EDTA (pH 8.0), 186.1g EDTA Disodium salt dihydrate

(AMRESCO Inc., Ohio, USA) was added to 800mL of Milli-q. While agitating the

contents with a magnetic stirrer and measuring the pH, NaOH was added in

minimal volumes and repeated until the solution reached a volume of

approximately 1L or a pH of 8.0. The solution was stored at room temperature.

39

2.10.3 Preparation of 5X Tris Base/Boric Acid/ EDTA (TBE)

To prepare a 1L solution of 5X TBE, 54g Tris, Ultra Pure base (Astral Scientific

Pty. Ltd., Taren Point, NSW), 27.5g Boric acid (G-Biosciences for Geno

Technology Inc., Saint Louis, USA) and 20mL of the pre-made 0.5M EDTA was

added to 800mL of Milli-q water. Once the contents were completely dissolved,

200mL of Milli-q water was added to adjust the final volume to 1L. The solution

was stored at room temperature.

2.10.4 Preparation and dilution calculation for 5X TBE to 0.5X TBE

TBE was diluted from 5X to 0.5X, for the preparation of 1% agarose in addition to

the subcell for gel electrophoresis. The calculations were as follows:

C1V1 = C2V2

0.5 x 1000mL = 5 X V2

V2 = 500/5 = 100mL

To prepare a 1L solution of 0.5X TBE, 100mL pre-made 5X TBE is added to

900mL of Milli-q water. The solution is stored at room temperature.

40

2.10.5 Preparation of 1% Agarose gel

To prepare 1% Agarose gel, 1g Agarose (Bio-Rad laboratories Inc., California,

USA) was added to 100mL of pre-made 5X TBE buffer and microwaved for 2

minutes at 80°C to dissolve the contents. 10μL SYBR® Safe DNA gel stain

(Invitrogen for Thermo Fischer Scientific Inc., Waltham, USA) was added to the

agarose solution and poured into a gel tray that contained a 20-well comb. The

agarose was left to solidify for approximately 10-15 minutes. The tray including

the gel were positioned into the Subcell GT (Bio-Rad laboratories Inc., California,

USA), and filled with 0.5X TBE to the maximum point indicator of the cell. The

comb was removed to reveal the wells.

2.10.6 Separation and analyses of Hp-SS1 DNA

The PCR reactions were separated via gel electrophoresis on a 1% Agarose gel in

0.5X TBE buffer. Each well included: 8μL PCR reaction and 2μL 6X DNA

loading dye (Thermo Fischer Scientific Inc., Waltham, USA) for both positive and

negative samples, as specified in Figure 2.1. To determine the size of the

fragments, the first and last well included: 8μL GeneRuler DNA ladder mix

(50μg) (Thermo Fischer Scientific Inc., USA) and 2μL 6X DNA loading dye

(Thermo Fischer Scientific Inc., USA). The samples were loaded and separated at

100V for 30-45 minutes. The gels were visualised using the ChemiDoc-It imaging

system (UVP, LLC., California, USA) and the VisionWorks™ LS analysis

software (UVP, LLC., USA).

41

Figure 2.1 Expected PCR results, demonstrating the order and content of each reaction. a. The visualised fecA1 and fecA2 products, b. displayed the corresponding frpB1 products and c. presented the genus confirmation of all isolated WT and mutants.

42

2.11 Natural Transformation of Hp-SS1

As H. pylori is a naturally competent bacterium, possessing the ability to absorb

extracellular DNA from its surroundings, the ΔIROMP-interrupted gene plasmids

as detailed in Table 2.6, were intended to be retained via homologous

recombination to produce a stable genomic mutation.

2.11.1 ΔIROMP-interrupted gene plasmids of Hp-SS1

The ΔIROMP-interrupted gene plasmids as described in Table 2.6, were provided

by Dr Melanie Thomson (School of Medicine, Deakin University) and constructed

by Teisa Holani (Hons 2012) 53 via the liquid method of natural transformation.

Table 2.6 Hp-SS1 ΔIROMP–interrupted gene plasmids used in this study.

2.11.2 Dilution calculation for 7% FBS for 500mL media

The preparation of FBS was diluted from 100% stock to 7% for 500mL of media.

The calculations were as follows:

C1V1 = C2V2

7% x 500mL = 100% x V2

V2 = 3500/100 = 35mL

43

2.11.3 Preparation of BB + 7% FBS

To prepare 500mL of BB + 7% FBS, 14g Brucella broth (BB) (BD: Bacto

Laboratories, Pty. Ltd., Mount Pritchard, NSW) was added to 465mL Milli-q

water and autoclaved at 110°C for 35 minutes. Once the broth cooled, 35mL (7%)

HyClone® Foetal bovine serum (FBS) (Thermo Fischer Scientific Inc., Waltham,

USA) was added and then stirred. The broth was stored at 4°C.

2.11.4 Preparation of 50mg/mL Kanamycin (1000X)

To prepare 10mL 50mg/mL Kanamycin stock, 0.5g Kanamycin sulfate (Bio Basic

Inc., Markham, Canada) was added to 10mL Milli-q water. The contents were

stirred to dissolve the Kanamycin. The stock solution was stored in 1mL aliquots

at -20°C.

2.11.5 Calculations for 50mg/mL to 25μg/mL Kanamycin

The stock concentration of Kanamycin required a dilution of 50mg/mL to

25μg/mL for 500mL of media. The calculations were as follows:

C1 V2 = C1 V2

25μg/mL x 500mL = 50,000μg/mL x V2

V2 = 12,500/50,000 =0.25mL

44

2.11.6 Preparation of 10% HB + 25μg/mL Kan

The 10% HB + 25μg/mL Kan was prepared as per the instructions for ‘2.2.1

Preparation of 10% HB + CAB + Dent’ minus the selective supplement, Dent and

the addition of 0.25mL (25/μg/mL) Kanamycin.

2.11.7 Liquid method of natural transformation

Natural transformation as depicted in Figure 2.2, commenced with 2 x Hp-SS1

WT + BB + 7% FBS cultures, including solution 1, where the tip inoculated with

Hp-SS1 was discarded and solution 2, incubated with the tip. At 0 hours, utilising

the WPA CO8000 cell density meter (Biochrom Ltd., Cambridge, UK) the OD600nm

was measured, reading 0.01 and 0.04 respectively, then incubated overnight at

37°C on 140rpm. At 16 hours the OD600nm absorbance reading was measured and

the bacteria appeared to be in the log phase of growth, 10 x 1.25mL (5 x solution 1

and 5 x solution 2) was centrifuged at 4300g for 5 minutes. The supernatant was

removed and the pellet was resuspended in 20μL from the 5 plasmid stocks as

described in Table 2.6, in both solution 1 and 2, and incubated a 37°C for 30

minutes. The suspensions were plated onto 10% HB + Dent plates to select for Hp-

SS1 and incubated for 24 hours at 37°C. The plates were scraped and re-inoculated

onto 10% HB + 25μg/mL Kan and incubated at 37°C for 5 days, to select for the

Hp-SS1 ΔIROMP mutant knockouts.

45

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ctio

n of

the

met

hod.

In

tot

al,

12-1

4 da

ys w

as r

equi

red

to e

ffec

tivel

y co

mpl

ete

this

pro

cedu

re a

nd

visu

alis

e th

e re

sulti

ng tr

ansf

orm

ed m

utan

ts.

46

2.12 Preparation for SELEX

2.12.1 Generation of the randomised library and primers

Two random sequences of 20bp were produced using the random DNA sequence

generator (http://www.faculty.ucr.edu/~mmaduro/random.htm). To evaluate the

melting temperature (Tm), GC content and length, the primers were submitted to

the ThermoFisher Scientific multiple primer analyser

(https://www.thermofisher.com/au/en/home/brands/thermo-scientific/molecular-

biology/molecular-biology-learning-center/molecular-biology-resource-

library/thermo-scientific-web-tools/multiple-primer-analyzer.html), confirming

that the two sequences possessed a Tm of approximately 52°C, allowing a

difference of 0.5-1°C. The MFEprimer-2.0: Dimerchecker

(http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/index.cgi/check_dimer) was

used to confirm there were no primer dimers between the two generated

sequences. These primers supplemented with high-performance liquid

chromatography (HPLC) purification and were sent to GeneWorks (Thebarton,

SA) to produce the randomised library, based on the two 20bp sequences.

47

Chapter 3 : Results

Aim 1: To investigate the iron responsive outer membrane proteins

(ΔIROMPs) (ΔfecA1, ΔfecA2 and ΔfrpB1) of Hp and the

Helicobacter species in silico.

3.1 Shared functions, co-occurrence and conserved elements

amongst the Helicobacter species and of the Hp-SS1 IROMPs in

question

3.1.1 Phylogenetic analysis of iron regulating proteins in

Proteobacteria and most common to the Helicobacter species

To assess and re-confirm the significance of iron-regulating proteins amongst the

Proteobacteria and further sub-groups, a general phylogenetic inquiry was

undertaken.

The process of iron-regulation and the key proteins involved, were analysed

against related taxonomic groups to distinguish the genes conserved further

down the lineage and closer to Hp. The protein clusters database provided a set

of proteins within Proteobacteria, previously allocated to a specialised group of

iron-regulating genes that were conserved throughout various taxonomic groups.

As revealed in Figure 3.1, feoB, tolB, exbD and exbB were present in most of the

genealogy, involving various transporter and translocation functions in iron-

regulation.

48

The fur protein, displayed conservation through the Helicobacteraceae family and

at the species level, IROMPs and siderophore-mediated transporters were tightly

conserved.

Figure 3.1 Phylum to genus analysis of common iron-regulating proteins. The IROMPs of interest, fecA1, fecA2 and frpB1 demonstrated conservation within the Helicobacter species and maintained the projected notion of occurrence in Hp. The elemental key regulator reported to function in the expression and repression of certain iron-acquisition proteins in varying conditions of iron availability was conserved within the Helicobacteraceae family, supporting this role in Hp.

49

The IROMPs fecA1, fecA2 and frpB1 amongst other outer membrane transporter

proteins, included a portion of those specific to the Helicobacter species, as

demonstrated in Figure 3.1. The verification of the incidence of the IROMPs of

interest in Hp ensued further analysis to utilise these proteins as a suitable target.

As the fur protein had been experimentally reported to repress siderophore

synthesis and through the utilisation of ferrous iron (Fe2+), act as a co-repressor,

the conservation of fur in the Helicobacter species was additionally explored to

assess it’s potential as therapeutic target.

The sequence alignment of each species-specific fur protein as displayed in Figure

3.2, produced increasing evidence of their ancestral derivation. The alignment,

generated by Clustal omega, demonstrated multiple analogous configurations,

contributing to an estimated frequency of more than 50% similarity, revealing

highly conserved regions. The relative sizes of the 24 individual fur proteins,

demonstrated a common origin that ranged from a small-scale of 390bp, 450bp,

453bp (Hp-fur), 456bp, 459bp, 462bp and 480bp, with a maximum variation of

90bp that were viewed external to the conserved areas. This motivated further

phylogenetic analysis, to determine the distance values between each fur gene of

the differing species of Helicobacter.

50

Figu

re 3

.2 S

eque

nce

alig

nmen

t of t

he F

erri

c up

take

reg

ulat

or (f

ur) a

mon

gst t

he H

elic

obac

ter s

peci

es.

Gen

erat

ed w

ith C

lusta

l om

ega

and

visu

alise

d w

ith Ja

lvie

w 2

, the

hig

hly

cons

erve

d pr

otei

n ex

pose

d va

rious

sequ

ence

sim

ilarit

ies w

ithin

the

disti

nctiv

e or

gani

sms,

conf

irmin

g th

e an

cestr

al li

neag

e an

d m

aint

aini

ng it

s fun

ctio

n as

a g

loba

l re

gula

tor o

f iro

n.

51

Following the analysis of the conserved regions of fur, a phylogenetic evaluation

was undertaken to determine the evolutionary relationship amongst the 24 proteins

of each Helicobacter species. In addition, the figure produced and visualised by

iTOL, aimed to verify the genetic distances between these fur genes.

The sequence alignment was uploaded to Clustal W2, providing a representative

figure of the common ancestors of the descendants in question. A node represented

each species and the genetic distances of the 24 Helicobacter fur proteins by

branches, connecting node-node or node-tip. A reference group or outgroup was

included as the comparative control, which satisfied the specific parameters.

Neisseria meningitidis (N. meningitidis) was included due to its evolutionary

relation to the ingroup, arising from the same phyla of the Proteobacteria’s

however, diverging from the Betaproteobacteria class. As fur was specifically

being investigated, an additional motive for selecting N. meningitidis as the point of

reference was due to their utilisation of fur, as the global regulator of iron.

The output data derived presented a parsimonious relationship between the

Helicobacter species as observed in Figure 3.3. The phylogenetic tree was rooted at

N. meningitidis to display the derivation of this ancestor in addition to, the branch

lengths or values that were assumed proportional to the number of nucleotide

substitutions per site. As revealed in Figure 3.3, the fur genes of the

individual Helicobacter species displayed minimal to no variation e.g. H. bilis and

H. trogontum, demonstrating rapid speciation of this species.

52

Figu

re 3

.3 T

he p

hylo

gene

tic tr

ee to

scal

e (0

.1),

reve

alin

g th

e br

anch

leng

ths o

r ge

netic

dist

ance

of t

he H

elic

obac

ter s

peci

es fu

r gen

e.

The

bran

ch v

alue

s in

dica

te th

e nu

mbe

r of

sub

stitu

tions

per

site

(i.e

. 0.1

am

ino

acid

sub

stitu

tions

per

seq

uenc

e po

sitio

n) r

epre

sent

ed b

y th

e sc

ale

bar.

The

exte

nt o

f ge

netic

va

riatio

n w

as fu

rther

obs

erve

d by

the

leng

th o

f the

bra

nche

s, as

the

leng

thie

r bra

nche

s im

plie

d m

ore

dive

rgen

ce h

as o

ccur

red,

as

disp

laye

d by

the

outg

roup

(N

. men

ingi

tidis

). A

s ex

pect

ed, t

he H

elic

obac

ter

spec

ies

wer

e fa

r m

ore

clos

ely

pres

ente

d, o

bser

ving

littl

e to

no

chan

ge in

var

ious

dau

ghte

r lin

es d

ue to

rap

id s

peci

atio

n. T

he p

arsi

mon

y, a

n op

timal

par

amet

er, w

as d

emon

stra

ted

as th

ere

wer

e fe

w e

volu

tiona

ry d

evia

tions

for a

ll se

quen

ces

to o

rigin

ate

from

a c

omm

on a

nces

tor.

Furth

erm

ore,

the

bran

ch le

ngth

s w

ere

scor

ed b

etw

een

0-1,

whe

re 0

is m

ost s

igni

fican

t and

any

val

ue g

reat

er th

an 0

.854

1 is

ass

umed

an

infin

ite d

ista

nce.

53

To further validate this information, a subsequent phylogenetic analysis was

produced to identify the bootstrap values or the level of confidence between each

node and ultimately exclude the fur gene as a potential target.

The confidence intervals were obtained by running an analysis of the output data

against a random sub-set data, via the ClustalW2 web tool. Visualised using iTOL,

the values derived were representative of the percentage of bootstrap replicates in

which that particular node had appeared. A value of 100% demonstrated a well-

supported node, as it had emerged within all replicates. Furthermore, a bootstrap

value of ≥95% suggested that the topology of such node could be considered

‘correct’.

As observed in Figure 3.4, the majority of nodes displayed an indefinite support for

the previous distance values derived by the Clustal W2 web tool. As most of the

daughter lineages displayed values of more than 95%, it could be established that

theses scores were highly supported and were in fact precisely related due to rapid

speciation. The outgroup, in addition to some species of Helicobacter e.g. H.

winghamensis & H. apodemus, demonstrated lower values, as they failed to appear

more readily within the bootstrap replicates. This could further be clarified by their

increased sequence variation of the individual fur proteins, demonstrating a broader

evolutionary time scale of derivation. Furthermore, as the two subsequent

phylogenetic analyses provided minimal variation of the fur gene amongst the

differing Helicobacter species, it could thus be excluded as a potential target to

minimise the probability of potential off-target effects, as it was not specific to only

H. pylori.

54

Figure 3.4 Phylogenetic analysis revealing the bootstrap levels between each node of the differing 24 Helicobacter fur genes. The phylogenetic tree demonstrated more than 50% of nodes that further presented almost indefinite support of the previous distance data derived. A confidence value of ≥95% was assumed to demonstrate well-supported data as they appeared with high frequency amongst the bootstrap replicates analysis derived from the Clustal W2 web tool. Scores that displayed percentages of ≤95% represented less confidence, in which these values were only observed in daughter lineages and the output group due to their increased variation genetically.

55

3.1.2 Protein-protein interactions of the IROMPs of Hp-SS1 regulated

by the fur gene

The succeeding portion of the investigation was to focus on the outer membrane

proteins of Hp, and their interactions with the global regulator of iron, fur.

A query of the major proteins that function in iron homeostasis within Hp and their

corresponding interactions regulated via fur was undertaken utilising the String 2.0

web tool. The documented proteins of the H. pylori strain J99 (NCBI I.D: 85963),

that is, the human version of the mouse-adapted strain of Hp utilised throughout this

experiment, were uploaded to the String 2.0 database. This generated an illustration

of the known connections amongst these proteins, that was ultimately based on the

fundamental evidence of such interactions available, the confirmation of BLAST

hits by means of homology, the information collated from these entries and the

networks centred on their interactions and scores.

As observed in Figure 3.5, the notion that fur is the global regulator of iron is

maintained by its position within this depiction in addition to, the ample connections

of other genes functioning in iron homeostasis. As the genes that were located on

the outer membrane of Hp were of interest, fecA1, fecA2 and frpB1 were specifically

investigated within this figure. The three IROMPs, displayed connections of gene

co-occurrence, gene neighbourhood, co-expression, textmining and had been

experimentally determined, which was additionally verified by Figure 3.5 and the

associated legend. These were essential attributes to identify a suitable target, as if

one or all of these were therapeutically hindered, it would disrupt the process of

iron-acquisition that is vital to Hp, leading to eradication of this infection.

56

Figure 3.5 The essential interactions between the proteins expressed that function in iron homeostasis and are further regulated by fur. The illustration derived by the String 2.0 web database, supported the notion of fur as the global regulator of iron in Hp. Furthermore, the outer membrane proteins that function in iron-acquisition, fecA1, fecA2 and frpB1 demonstrated highly favourable attributes of gene neighbourhood, gene co-occurrence, co-expression and textmining, revealed by the corresponding figure legend and colour-coded connections. The validity of this illustration was complimented by tabulated data of scores of

confidence between each protein-protein interaction.

57

Table 3.1 Complimentary data derived from the interactions of the iron regulating proteins of Hp-J99, indicating the confidence of each connection. The levels of confidence generated by the String 2.0 database from the evidence available regarding the corresponding protein interactions provided support of such connections. The confidence values ranged from 0-1, where a score of 1 was recognised as the highest support for these interactions. Values of ≥0.700 were considered of high confidence, which was revealed in more than 50% of queried connections.

58

These scores, as detailed in Table 3.1, were not indicative of strength or specificity

of each queried connection however, they signified a measure of confidence e.g. the

probability of an interaction to be supported, given the evidence available to the

String database. The values ranged between 0-1, where a score of 1 denoted the

highest level of confidence thus, values ≤0.500 were considered of no significance

between the associated connections.

As exposed in Table 3.1, 75% of these connections displayed values of varying

significance (≥0.600) where, 50% of these values were considered of high-

maximum confidence. Amongst these values, were the connections between the

IROMPs of interest, which are displayed in bold in Table 3.1, supporting their

interactions and motivating further inquiry.

An individual analysis utilising the String 2.0 database, provided a subsequent

illustration of only the IROMPs of interest to further assess their distinct

connections.

As presented in Figure 3.6, the IROMPs in question in addition to their associated

connections displayed textmining and gene co-occurrence. This further validated the

conception of targeting these outer membrane proteins as to obstruct iron-

acquisition thus, inhibiting further colonisation of Hp. As it had been previously

explored that these particular iron-acquisition proteins were essential to the

colonisation of Hp and demonstrated within the Mongolian gerbil model, this notion

was further authenticated bioinformatically.

59

Figure 3.6 The protein-protein interactions of the target IROMPs of Hp J99. The subsequent analysis derived from the String 2.0 web tool further validated a strong association between the IROMPs of interest, demonstrating favourable attributes e.g. gene co-occurrence and textmining, to be therapeutically targeted. Previous analyses of the specified IROMPs, fecA1, fecA2 and frpB1, stated the

presence of a ‘fur box’ promoter region, required for fur to bind and impede the

expression of these iron-acquisition proteins. Derived from the extensive analysis of

the promoters of these genes, the reported ‘fur box’ was expected to expose a core

sequence encompassing a specific 7-1-7 motif with a dyad symmetry. Pich et al,

recently demonstrated that iron-bound fur (holo-fur) binds with high affinity to a

palindromic repeat (5’-TAATAATnATTATTA-3’) located in proximity of the -35

and -10 promoter elements, which further revealed fur-dependant repression of the

IROMPs of interest.

To confirm this notion, a sequence alignment was generated via Clustal Omega, of

the three IROMPs in addition to the fur gene specific to Hp. The data visualised

with Jalview, presented the expected ‘fur box’ consensus, as detected in Figure 3.7.

60

Figure 3.7 Analysis of the promoter regions of fecA1, fecA2, frpB1 and fur genes specific to Hp. a) The sequence alignment computed by Clustal Omega, revealed the presence of the ‘fur box’ consensus sequence within the IROMPs of interest and fur, indicated by arrows, within the expected -35 and -10 promoter elements displayed within boxes. The fundamental site for fur-dependant regulation and more specifically, the repression of these iron-acquisition genes was identified, demonstrating the importance of these IROMPs as potential targets. b) The sequence logo generated by Weblogo 3 of the subsequent alignment, exposed differing stacks of bases, at varying heights depicting the frequency of each (the taller the base, the more frequently observed or conserved), displaying the predicted endogenous 7-1-7 motif required for holo-fur dependent regulation that further validated the anticipated occurrence.

61

As observed in Figure 3.7a, analysis of the promoter regions of the three IROMPs and

the fur gene specific to Hp, demonstrated the presence of the imperfect 7-1-7 inverted

repeat, highlighted by arrows. The expected consensus sequence was identified 35bp

upstream of the transcriptional promoter sites and the expected -10 elements, which

were indicated by boxes. This investigation provided confirmation of fur-dependent

regulation amongst the IROMPs in addition to, the self-regulation of fur, as it

possessed the required endogenous repeat.

The secondary analysis of the sequence logo generated via the Weblogo 3 tool,

complimented the previous inquiry, as the projected 7-1-7 motif was observed within

Figure 3.7b. The logo included stacks of varying heights that demonstrated the

frequency of these bases, revealing the 5’-TAATAATnATTATTA-3’ sequence

reported as the ‘fur box’. This further validated the presence of the required ‘fur box’

promoter region required for fur-dependant regulation. In addition, this evidence

reinforced the significance of the IROMPs, fecA1, fecA2 and frpB1 as they were

regulated by fur and if therapeutically targeted would impede iron-acquisition that

would ultimately be detrimental to Hp.

62

Aim 2: To validate the insertional mutagenesis of the ΔIROMP proteins

(ΔfecA1, ΔfecA2 and ΔfrpB1) of Hp-SS1.

3.2 Isolation of Hp-SS1 WT and ΔIROMP clones

To ensue further analysis and confirm the insertion of the KanR cassette (aphA3 gene)

within the specified IROMPs, it was necessary to isolate the Hp-SS1 WT and mutant

IROMP clones.

The samples were isolated from frozen stocks onto 10% HB + Dent and incubated

within an AGS at 37°C for 10-12 days to attain optimal growth. As observed in Table

3.2 accompanied by a descriptive legend, the Hp-SS1 WT displayed heavy

colonisation, in addition to, differing levels of growth observed from the various

clones of the individual IROMP mutants. The growth attained was sufficient to ensue

further analyses.

63

Table 3.2 Results from selective cultures of Hp-SS1 WT and ΔIROMP clones attained throughout this investigation.

Legend: - 0 colonies + 1-20 colonies

++ 21-40 colonies +++ 41-60 colonies ++++ 61-80 colonies +++++ 81-≥100 colonies

* Fungal contamination

64

The three ΔIROMPs and their respective clones that displayed growth, were re-

inoculated onto 10% HB + Dent + 25μg/mL Kan, to investigate the insertion of the

KanR cassette. The ΔIROMP clones, fecA1 clones 2, 3 and 5, fecA2 clones 3 and 4,

and frpB1 clones 1-8 all displayed comparative levels of growth on the selective

media including Kanamycin. This suggested the insertion of the KanR cassette,

ensuing definitive analyses.

Each sample that obtained colonisation of the Hp-SS1 WT and ΔIROMPs were

purified using the DNeasy blood and tissue kit, to extract their genomic DNA for the

assessment of DNA quality and for the analysis of the projected WT and mutant

clones.

3.3 PCR confirmation of Hp-SS1 ΔIROMP clones

3.3.1 Confirmation of the Helicobacter species

To ascertain a genus-level confirmation, the WT and mutant clones were verified via

PCR and visualised on a 1% agarose gel.

The genomic DNA (gDNA) of the respective WT and ΔIROMP clones were amplified

by the 16s rRNA genus specific primers, H276F and H676R to observe an expected

fragment of 376bp (Appendix 1).

The WT and mutant IROMPs that were successfully isolated and purified were

detected at the expected fragment of 376bp, displaying optimal concentrations of DNA

for further experimentation, as displayed in Figure 3.8.

65

Figure 3.8 PCR amplification of Hp-SS1 WT and ΔIROMP KanR knockouts displayed on 1% agarose gel. The samples that displayed sufficient colonisation were isolated and purified to attain pure gDNA. The purified products were amplified using 16s rRNA oligonucleotides, H276F and H676R to visualise an expected fragment at 376bp. The samples displayed the anticipated 376bp fragments with optimal DNA concentrations. The ΔfrpB1 clone 2 failed to be detected, in addition to ΔfecA2 clone 5, displaying a faint band however, within the expected 376bp region and could still be confirmed as the Helicobacter species. The ΔfrpB1 clone 2 failed to be detected and this sample could not be confirmed as

belonging to the Helicobacter species (Figure 3.8). The ΔfecA1 clone 5 displayed a

faint band however; it was still visible at the expected 376bp fragment and confirmed

to be of the Helicobacter species (Figure 3.8). As these samples were the only

Helicobacter species to be tested within this lab, it was further concluded that they

were in fact Hp-SS1.

66

3.3.2 Expected and resulting products of the isolated HpSS1 WT and

ΔIROMP clones

The cloning primers described in Table 2.5 used to amplify a short strand of the

2304bp WT fecA1 gene, expecting a fragment of 1159bp (1403bp-244bp), as observed

in Figure 3.9a. With the insertion of the 1597bp KanR cassette, the ΔfecA1 clones were

predicted to generate a band of 2756bp (1159+1597), indicative of successful

transformation, as displayed in Figure 3.9b.

The corresponding experimentation produced the expected 1159bp fragment for the

WT fecA1 sample, which was confirmatory of Hp (Figure 3.9c) The isolated ΔfecA1

KanR clones 2, 3 and 5 failed to appear at the expected 2756 bp fragment thus, the

insertion of the KanR cassette could not be detected within this gene (Figure 3.9c). In

addition the ΔfecA1 clone 2 displayed a band of 1159bp, characteristic of the WT. This

result was unexpected due the colonisation of Hp on plates including Kanamycin,

suggesting the insertion of the KanR cassette.

67

Figure 3.9 Insertion via homologous recombination of the KanR resistance cassette (aphA3 gene) into fecA1 of Hp-SS1. a) Expected fragment of WT fecA1. The cloning primers amplified a short strand of the 2304bp fecA1 gene, expecting a confirmatory fragment of 1159bp (1403bp-244bp) to appear on the gel. b) Expected fragment of the ΔfecA1 clones. The insertion of the 1597bp KanR cassette, anticipated the appearance of a 2756bp fragment (1159+1597), validating the successful process of homologous recombination required for negative selection within the SELEX technique. c) PCR amplification of the isolated Hp-SS1 WT and fecA1 KanR clones 2, 3 and 5 displayed on 1% agarose via gel electrophoresis. The WT sample visualised in well 2 of the gel, displayed the predicted 1159bp. The ΔfecA1 clone 2 unexpectedly appeared as a WT (1159bp). In addition, ΔfecA1 clones 3 and 5 failed to appear on the gel thus, all fecA1 mutants failed to detect the expected fragment of 2756bp.

68

The cloning primers previously mentioned, that were utilised to attain a restricted

fragment of the 2364bp fecA2 gene of Hp-SS1, was expected to display a 1045bp band

(1235bp-190bp) within the gel (Figure 3.10a). Following the insertion of the 1597bp

KanR cassette, the ΔIROMP indicative of the successful homologous recombination

was anticipated to display a band of 2642bp (1045bp+1597bp) on the gel (Figure

3.10b).

The resulting analysis, confirmed the presence of the fecA2 gene of Hp-SS1, as it

displayed a band of 1045bp as expected (Figure 3.10c). The isolated ΔfecA2 KanR

clones 3 and 4 could not be detected on the gel, although they successful colonised on

media containing Kanamycin. Again, the respective ΔIROMP KanR clones could not

be included within the SELEX technique as the negative selection, as the location of

the cassette remained unknown.

69

Figure 3.10 Insertion via homologous recombination of the KanR resistance cassette (aphA3 gene) into fecA2 of Hp-SS1. a) Expected fragments for WT fecA2. The cloning primers were expected to produce a 1045bp fragment indicative of the 2364bp WT fecA2 gene (1235bp-190bp). b) Expected fragment of the ΔfecA2 clones. Following the insertion of the 1597bp KanR cassette, an alternative fragment of 2642bp (1045bp+1597bp) was expected, representative of the interrupted ΔfecA2 clones. c) PCR amplification of the isolated Hp-SS1 WT and fecA2 KanR clones 3 and 4 displayed on a 1% agarose via gel electrophoresis. The WT fecA2 was observed at the appropriate size within the gel, displaying a fragment of 1045bp (1235bp+190bp). The ΔfecA2 KanR clones 3 and 4 failed to display a fragment of 2642bp (1045bp+1597bp) and the insertion of the KanR cassettes could not be verified.

70

The gene specific primers of Hp were utilised to attain a fragment of 1077bp

(1396bp-319bp) indicative of the 2376bp frpB1 gene (Figure 3.11a). Following the

insertion of the 1597bp KanR cassette, the ΔfrpB1 clones were predicated to observe a

band of 2674bp (1077bp+1597bp), representative of the successful process of

homologous recombination (Figure 3.11b).

The respective analysis provided the confirmation of the WT frpB1 gene, which

displayed the expected 1077bp fragment (3.11c). The ΔfrpB1 KanR clones 1-8 did not

appear on the gel and therefore, the insertion of the aphA3 gene could not be detected

(Figure 3.11c).

71

Figure 3.11 Insertion via homologous recombination of the KanR resistance cassette (aphA3 gene) into frpB1 of Hp-SS1. a) Expected fragments of WT frpB1. The respective cloning primers were utilised to produce a fragment of 1077bp (1396bp-319bp), indicative of the 2376bp WT frpB1 gene. b) Expected fragment of the ΔfrpB1 KanR clones. Following the insertion of the 1597bp KanR cassette, a fragment of 2674bp (1077bp+1597bp) was expected to be observed and indicative of natural transformation. c) PCR amplification of the isolated Hp-SS1 WT and frpB1 KanR clones 1-8 displayed on 1% agarose via gel electrophoresis. The WT frpB1 gene displayed the appropriate fragment of 1077bp. The ΔfrpB1 KanR clones 1-8 could not be detected as no bands appeared within the applicable lanes of the gel.

72

Subsequent analysis of varied PCR parameters and reaction combinations were

undertaken to detect the insertion of the KanR cassette within the differing ΔIROMP

clones.

As observed in Figure 3.12, the succeeding attempts to amplify and visualise the

expected fragments indicative of the insertion of the KanR cassette within these genes

had failed. The identical patterns continued to appear within each gel attempted of

differing PCR parameters and reaction combinations. The Hp-SS1 WT IROMPs

continued to display their respective fragments of the expected sizes (Figure 3.12a +

b). The ΔIROMP KanR clones failed to be detected within each attempt (Figure 3.12a

+b).

73

Figure 3.12 Further attempts to attain expected fragments of all isolated Hp-SS1 WT and ΔIROMP KanR clones. a) The amplified fragments of fecA1 and fecA2 KanR clones, repetitively demonstrated expected WT fragments or failed to appear on the gel. b) The amplified fragments of frpB1 KanR clones 1-8, displaying the WT expected fragment.

74

3.4 Natural transformation of Hp-SS1

The Hp-SS1 insertion of aphA3 within Hp-SS1 WT IROMPs was attempted to attain

negative selection for SELEX, via homologous recombination of natural

transformation.

As observed in Table 3.3 complimented by a descriptive legend, the attempt to re-

produce these clones had failed, as no growth was observed within the selective

media including Kanamycin plates. This hindered further progression of deriving the

IROMP-specific aptamers.

Table 3.3 Results of natural transformation of Hp-SS1 via the liquid method. The attempt to re-produce the IROMP KanR knockouts failed, as no growth was observed on the selective media.

Legend: - 0 colonies + 1-20 colonies

++ 21-40 colonies +++ 41-60 colonies ++++ 61-80 colonies +++++ 81-≥100 colonies

* Fungal contamination

75

Aim 3: To generate an aptamer with a high binding affinity and

specificity for the ΔIROMP’s of Hp-SS1 using the SELEX method.

3.5 Preparation for the SELEX technique

3.5.1 Generation of randomised library and primers

The primers consisting of two randomly generated 20bp sequences were attained as

observed in Table 3.4. The primers were produced with HPLC purification that

provided approximately 85% purity, that is optimal for the SELEX technique. In

addition, the melting temperatures were required to be at approximately 52°C with

0.5-1°C of variation, as attained with these primers (Table 3.4).

The randomise library was produced based on these primers (Table 3.4), generated by

GeneWorks.

Table 3.4 Forward and reverse primers randomly generated and selected for the SELEX technique.

As the insertion of aphA3 within fecA1, fecA2 and frpB1 could not be detected, the

process of SELEX was hindered due to the absence of negative selection.

76

Chapter 4 : Discussion

4.1 Interpretation of the results with the association of the literature As the primary aim of this project was to identify a suitable target for aptamer-based

therapies, the detailed analysis based on previous experimentation and the findings of

these, were of increasing significance. The phylum to genus analysis of the reported

genes that function in iron homeostasis in Hp motivated the exclusion of exbB, exbD,

feoB and tolB as potential targets of this highly selective therapy (Figure 3.1). The

novel therapeutic could cause more detriment to the human microbiome if genes that

were readily observed throughout differing species were targeted54. Yao et al (2016)

recently demonstrated that not only do broad-spectrum antibiotics eradicate the

infecting pathogen, they additionally decimate the beneficial bacteria of the gut

microbiome, developing a variety of immune and metabolic disorders54. The use of

broad-spectrum antibiotics for gastrointestinal infections early in childhood has

furthermore been associated to changes of the microbiome that have been identified

as short-term risk factors of secondary infection, in addition to, obesity and celiac

disease within the long term or later in life54. This evidence motivated the selection of

a more highly conserved virulence factor of Hp.

As fur is the global regulator of iron, an essential process of Hp, a comprehensive

investigation of this gene was ensued. Theoretically, to target a key protein of iron

regulation, a process essential for colonisation would present desired effects. The

analysis of fur, displayed conservation throughout the Helicobacteraceae family, in

addition to, all 24 differing species of Helicobacter.

77

The genus-specific investigation demonstrated more than 50% of conserved bases

within the divergent fur genes (Figure 3.2) and almost no variation between them

(Figure 3.3) suggestive of rapid speciation. This promptly disqualified fur as a

potential therapeutic target with the addition of supplementary information.

The frequency of the fur gene within all Helicobacter species was not the only motive

for exclusion. Current treatments for infectious diseases are failing due to antibiotic

resistance and additionally causing detriment to beneficial bacteria that once had a

protective function within the gut. As current literature regarding the investigation of

novel antibacterial therapies has swayed toward alternatives to antibiotics, such as,

the aforementioned-targeted therapies55, the concept of internalising therapy within

the cell of Hp became far more complex when additionally explored. There are

currently numerous complications within target-based screening processes. The

understanding of physical-chemical features of cell permeability remains inadequate.

Further research is necessitated to identify such characteristics of functional

properties within therapeutic molecules that possess the ability to penetrate and

accumulate within the cells of pathogenic bacteria56. As targeted therapies have only

been proposed as of late due to the increasing prevalence of antibiotic resistance,

minimal information and investigation has been pursued57. Two independent teams

led by Luciano Marraffini from The Rockefeller University and Timothy Lu from the

Massachusetts Institute of Technology (MIT) both employed by CRISPR (Clustered

Regularly Interspaced Short Palindromic Repeats) have developed a new method to

re-sensitise resistant strains of bacteria to typical antibiotics57.

78

As most antibacterial compounds being developed are solely concentrated on gram-

positive bacteria, the gram-negative pathogens continue to be exceedingly under-

addressed, motivating this novel therapeutic57.

The CRISPR-associated protein-9 nuclease (Cas9) functions to cleave double-

stranded DNA (dsDNA) at the specific site of acquired or mutated sequences causing

resistance thus, activating the double strand break (DSB) of the repair machinery57.

This causes chromosomal degradation, leading to bacterial cell death, restoring

susceptibility of the once efficacious antibiotic57. Further investigation of this

development displayed numerous limitations, as only a limited number of cells were

successfully destroyed in addition to, issues of cellular internalisation57. This lead to

the notion of targeting genetic material, e.g. sequence-specific regions of virulence

proteins, that are expressed on the outer membrane of bacterial cells.

Typical virulence factors that increase colonisation and persistence of the host

include, adhesion, invasion, immune response inhibitors and toxins found on the

outer membrane of the pathogen58. As previously mentioned, the process of iron

homeostasis is of major importance to all-living organisms and bacteria is no

exception. Due to Hp’s need to regulate sufficient levels of iron, this process has been

implicated in causing further detriment to the infected host via the process of iron-

acquisition facilitated by outer membrane proteins. Flores (2014) demonstrated that

infection of Hp caused the redistribution of free iron to the gastric epithelium, the site

of colonisation, producing Iron deficiency anaemia (IDA) within the host59. In

addition, the frpB1 outer membrane protein (OMP) of Hp, displayed over expression

on the cells surface when haem was the only obtainable source of iron60.

79

This identified the presence of motifs required to bind haemoglobin or haem60,

supporting Hp as a cause of IDA. This ensued further investigation of three particular

OMP’s fecA1, fecA2 and frpB1, previously implicated in iron-acquisition processes

and demonstrated to be essential for the colonisation of Hp.

As the presence of these IROMPs were exclusive to Hp, their interaction with fur was

explored to determine the effect on the pathogen, when targeted specifically. The

illustration of the combined interactions of the iron-regulating proteins, confirmed

previous analyses of fur functioning as the master regulator of gene expression

(Figure 3.5 and Table 3.1). In particular, the string analysis displayed the connection

of gene co-occurrence, co-expression and neighbourhood between the three IROMPs

of interest, in addition to, increasing scores of confidence supporting this data,

indicative of a process maintained by complex regulatory mechanisms. The specific

string investigation of fecA1, fecA2 and frpB1 further verified co-occurrence of these

genes (Figure 3.6). Pich and Merrell (2013) demonstrated the significance of iron-

related processes and the resultant ability of Hp colonisation in the presence and

absence of the three IROMPs60. Additionally, they exposed several-fur regulated

genes including fecA1, fecA2 and frpB1 that were essential for colonisation within

the Mongolian gerbil model of Hp infection60, the gold standard for human gastric

disease61. This was established via the generation of a defective or mutant fur,

demonstrating that colonisation was not affected by a single gene, but by the

deregulation of the subsequent fur-regulated loci of these IROMPs60. This concept

remained the centre of the succeeding portion of investigation, to observe these fur-

dependant regions within the three IROMPs.

80

The sequence alignment of the promoter regions of fur, fecA1, fecA2 and frpB1

confirmed the presence of this regulatory-region known as the ‘fur box’, contingent

for the repression of these particular IROMPs (Figure 3.7). Pich et al (2012)

identified the core sequence (5’-TAATAATnATTATTA-3’) of the ‘fur box’ and

demonstrated the defects in colonisation of Hp, due to the loss of fur regulation of the

aforementioned IROMPs62. An imperfect 7-1-7 motif was identified within two

highly conserved regions (Figure 3.7), the first located roughly 35bp upstream of the

transcriptional promoter site and the second, in the capacity of the expected -10

elements, previously proposed by Delany et al (2002) 63. This core sequence is of

preeminent significance, as it has been implicated as the site of gene expression in

varying levels of iron availability. In conditions of high iron, fur is arranged in a

dimer configuration and loaded with iron (holo-fur), binding to the core sequence and

obstructing transcription of the IROMPs63. Alternatively, when the levels are scarce

and iron is absent from fur, the dimer configuration is dilapidated, permitting RNA

polymerase to cohere to this region, initiating transcription of these genes63. In

addition, the presence of the ‘fur box’ within the fur gene exposes the capacity of

self-regulation in differing conditions of iron, controlling the expression of fur-

dependent genes as necessitated64. The specified IROMPs are transcribed for the

acquisition of free iron from various sources65. The fecA1 and fecA2 genes transport

iron (III)-dicitrate66 and the frpB1 gene binds haem66, demonstrating the capacity to

acquire variable sources of iron and underpinning the importance of maintaining iron

homeostasis within Hp. This process evidently validates the significance of iron-

regulation exposed by these intricate mechanisms that respond to the cellular stress of

opposing levels iron availability.

81

The specified IROMPs have an immeasurable function in the survival of Hp and

retain advantageous parameters, e.g. positioned on the outer membrane and are vital

to persist, for selective therapies. This demonstrates the potential of restoring the

microbiome, overcoming antibiotic resistance and most importantly, eradicating the

infection of Hp.

Once the target was selected, the subsequent phase was to isolate and confirm the

KanR knockouts of the pre-elected genes of Hp for negative selection of the SELEX

technique. The isolation of Hp-SS1 demonstrated substantial growth of the WT and

various ΔIROMP KanR clones however, a large portion of the ΔfecA1 and ΔfecA2

clones failed to grow (Table 3.2). As they were constructed in 2012, the leading

factor of weakened viability was most likely the cause of improper laboratory

practices such as, permitting the frozen stocks to thaw, which is detrimental to such

samples. The isolation of each sample would have been an ideal outcome but

conversely, a sufficient number of clones were attained to progress.

The isolated ΔIROMP KanR clones were re-inoculated onto selective media including

Kanamycin to evaluate the insertion of the KanR cassettes. Each sample resulted in

comparable growth to the initial isolation (Table 3.2), supporting the insertion of the

KanR cassette within the specified genes of interest.

The genus-specific confirmation of the isolated Hp-SS1 WT and mutants was used as

a diagnostic measure to ensure the experimentation of Hp, avoiding contaminants and

negative influences.

82

Once purified, the reactions including the 16s rRNA forward and reverse primers, in

addition to, the corresponding DNA templates, confirmed the presence of

Helicobacter species. The analysis aimed to detect a 376bp fragment, indicative of

Helicobacter and as the only bacteria of this genus utilised within this particular lab,

could further be verified as Hp.

As observed in Figure 3.8, most of samples displayed the expected 376bp band

within the gel, supporting the incidence of the Helicobacter species and more

specifically Hp. Moreover, the ΔfecA1 clone 5 displayed a faint appearance, though

the 376bp fragment could still be detected. In addition, ΔfrpB1 clone 2 appeared

absent within the gel, leading to the investigation of the source contributing to these

results. Analysis of PCR components causative of faint or lacking bands provided

logical explanations. Lee at al (2012) defined the optimal procedures of gel

electrophoresis underpinning errors within laboratory procedures that contribute to

this display67. In addition, the appearance of faint or absent bands was causative of

insufficient quality and concentrations of DNA67, which remained consistent with

ΔfrpB1 clone 2, as nominal growth was observed and purified from this sample. The

ΔfecA1 clone 5 displayed substantial growth and thus, inconsistent with this

conception however, coherent with DNA degradation, causative of the faint

appearance as reported by Lee68. As these variables contributed to only a small

percentage of the samples, the confirmation of the insertion of the KanR cassette was

pursued.

83

The utilisation of gene-specific cloning primers facilitated the investigation and

visualisation of particular fragments, indicative of WT or mutant Hp. The amplified

products of Hp-SS1 fecA1, fecA2 and frpB1 WT’s, displayed the expected

confirmatory fragments of 1159bp, 1045bp and 1077bp, respectively (Figures 3.9,

3.10 and 3.11). The interruption of ΔfecA1, ΔfecA2 and ΔfrpB1 clones with the

1597bp KanR or aphA3 cassette, estimated affirmative fragments of 2756bp, 2642bp

and 2674bp within the corresponding gels (Figure 3.9b, 3.10b and 3.11b).

Unfortunately, the mutants remained absent on the gel and consequently, the insertion

of the KanR cassette failed to be detected within these genes (Figure 3.9c, 3.10c and

3.11c). Surprisingly, ΔfecA1 clone 2 appeared as a WT, presenting a band of 1159bp

(Figure 3.9c). Due to this technical irregularity and resultant failures, numerous

attempts to alter PCR parameters and reactions were undertaken to obtain

confirmation of these mutants. Each attempt displayed patterns identical to the

fragments previously attained (Figure 3.12a+b). The purified DNA products had

demonstrated sufficient DNA concentrations within the majority of these samples

(Figure 3.8). In addition, the isolates displayed the capacity to grow on the selective

media that included Kanamycin and unexpectedly, ΔfecA1 clone 2 was confirmed to

be a WT (Figure 3.9c). This excluded the possibility of experimental inaccuracies and

sparked a comprehensive inquest to identify probable explanations such as, errors in

homologous recombination or naturally occurring Kanamycin resistance.

A review of the literature confirmed the absence of evidence and accounts of

naturally occurring Kanamycin resistance in Hp and consequently, errors within the

process of homologous recombination (HR) remained the most feasible

rationalisation.

84

Several studies have demonstrated this notion, ascertaining the propensity of

homologous recombination to be flawed. Guirouilh-Barbat et al (2014) detailed the

potential faults and consequences of homologous recombination, a process that was

once considered error-free69. DNA replication during homologous transformation can

promote the expression of gene conversion due to genetic mutations that cause re-

arrangements, such as, deletions and translocations69. As observed in Figure 4.1,

double crossovers of the donor fragment including homologous regions for site-

specific transformation, can result in alternative outcomes. In this scenario, the

replication of the resistance cassette within the fecA1 gene is the expected result

(Figure 4.1a), as the newly transformed gene remains functional. Mutations arising

during homologous recombination may appear functional as the required region is

replicated however, this can give rise to different sites as the cassette has been

transcribed within another portion of the gene (Figure 4.1b). This causes the loss of

primer binding sites and during confirmatory analysis can appear as though the

transformation was unsuccessful69. Single crossover events not only transform the

selected region but the entire sequence of the remaining plasmid (Figure 4.1c+d)

within the site of the chosen gene. This produces an extensive fragment and thus,

incapable of displaying a fragment on the gel due to its size69. Although the view

concerning the success rate of homologous recombination remains divided within the

literature, new evidence of errors arising within this process frequently reappears.

The most simplistic explanation for the failure to detect these knockouts may be

determined by the construction of the plasmids utilised for natural transofmration

(Table 2.6) prior to homologous recombination. During the generation of the original

plasmid construct, the primer site was most likely lost due to corruption or removal

arising from various mutations previously mentioned.

85

Preceding efforts to detect the insertion of the KanR cassettes within fecA1, fecA2

and frpB1 utilising identical plasmid constructs, displayed likewise complications and

remained unverified62. The precise location of the transformed KanR cassette needs to

be identified to attain the confirmation of this process. A secondary attempt to attain

negative selection for the SELEX technique was ensued.

Figure 4.1 The consequences of single and double crossover events due to errors in homologous recombination (HR). a) The expected double crossover event. The functional double crossover during HR produces the insertion of the resistance cassette within the site-selected region of fecA1. b) The occurrence of mutations during the double crossover event. This displays the expected sequence and acquires the associated resistance however, complicates detection of the specified insertion in fecA1 due to such factors of gene conversion and loss of primer sites. c) Single crossover event of the homologous region 1 (HR1). This results in the transcription of the resistance cassette and the remainder of the plasmid, complicating detection of this process. d) The single crossover event of the homologous region 2 (HR2). The consequences display identical complications to the single crossover of HR1 however, the sequence is transcribed from HR2 following the remainder of the plasmid.

86

The attempt to re-produce the IROMP KanR mutants for negative selection was a

failure (Table 3.3). Following the liquid method of natural transformation and

sufficient intervals of optimal incubation conditions, growth of Hp-SS1 mutant

IROMPS on selective media including Kanamycin, was deficient (Table 3.3). This

motivated the analysis of the natural ability of DNA uptake in Hp. Natural

competence of some bacteria is the capacity to actively transport foreign DNA into

the cell that is naturally transformed, altering the genotype70. The review of

corresponding literature identified Hp as one of these competent pathogens, as the

occurrence of transformation is common71. Mell and Redfield (2014) proposed

complications of DNA uptake within gram-negative bacteria due to the transportation

of rigid and highly charged dsDNA70. Currently, DNA uptake and the detailed

mechanics of this process is highly limited and poorly defined72. In addition,

numerous analyses of the action of natural selection in regards to natural competence

remain divisive. Moore et al (2014) demonstrated that the transformation efficiency

in Hp is highly variable73. Although this pathogen has been identified as naturally

competent, Hp is selective in DNA uptake and transformation. The transformation

efficiency is low in the presence of increasing lengths and decreasing similarity of

DNA sequences, in addition to, the initial growth phases of Hp and in deficient levels

of CO273. Due to the variable nature of natural transformation in Hp, the failure of re-

producing these knockouts could have been causative of the suggested loss of primer

binding sites within the construction of the plasmids. These constructions including

the KanR cassette could have caused elongation or disruption, loosing similarity of the

sequence, obstructing this process. Alternatively, the attempt of HR via natural

transformation during premature growth phases, in addition to, insufficient conditions

of CO2 caused by a defective AGS system could have caused the observed failure.

87

Although resolutions arose from this analysis, the IROMP KanR mutants required for

negative selection were unobtainable due to time constraints. Consequently, the

SELEX method to derive the IROMP-specific aptamers could not be pursued.

Preparation for the initial starting point of the SELEX method included the selection

of two 20bp randomly generated primers, as observed in Table 3.4. In addition, these

sequences formed the basis of the randomised DNA library, generated by

GeneWorks. The cell-SELEX method consists of succeeding rounds of target

binding, selection and amplification, demonstrating the significance of all portions of

this process (Figure 4.2). As aptamer-based therapies are readily being investigated

for various human diseases, the generation of the randomised library and primers

have presented major significance. Recent analyses have demonstrated the limitations

of library designs, lacking certain parameters and in turn, yielding non-specific

aptamers74. The initial library of primers usually consists of 10-15-1016 random

sequences75. Since the randomised library is so large, in many cases, it entirely

saturates the significant sequence area75. The binding positions of transcription factors

are typically <20bp long, each occupying sequence variants in which this protein is

able to bind is exemplified in a large number within the randomised library75.

Consequences arise that can generally be neglected in SELEX, for example, loss of

sequence variants due to chance fluctuations known as stochastic effects75.

Accordingly, these effects are not readily being taken into consideration during

numerical simulations, in addition to, theoretical models of the cell-SELEX method75.

Optimal parameters have been proposed to improve binding factors, as synthetic

randomly generated libraries are incompetent in reaching prerequisite qualities76.

88

Figure 4.2 A schematic of the cell-SELEX method to target fecA1, fecA2 and frpB1 of Hp-SS1 adapted from Wu et al (2015). The randomised library is incubated with selected IROMPs and the resulting bound or unbound sequences are divided. The bound aptamers are retained for negative selection. Sequences that are bound to the control cells are washed or removed, attaining aptamers specific to the targeted IROMPs that are further amplified. One round of SELEX includes target binding, selection and amplification and several rounds are repeated. The final round of SELEX and the resulting aptamers are sequenced and monitored for therapeutic validation.

The design should consist of an equal distribution of all four bases78. This is based on

the understanding of short motifs within the aptamer, normally mediating cohesion to

the target molecule78. The incidence of an equal distribution of motifs has provided

evidence that it increases the sequence area, producing a higher possibility of

deriving an aptamer with the desired binding properties78.

89

Recently, Blind and Blank (2014) have provided the advantages of using next

generation sequencing (NGS) and bioinformatics techniques to help monitor and

optimise the generation of the randomised libraries78. Furthermore, a wide range of

applications have been proposed due to the limitations of SELEX, e.g. High-fidelity

SELEX74, minimal primer and primer-free SELEX79 and the addition of primer

purifications prior to the generation of randomised libraries. These processes aim to

minimise the loss of desired aptamer properties to produce highly specific

therapeutics. The oligonucleotides derived in this study for SELEX (Table 3.4) were

supplemented with HPLC purification to eliminate truncated synthesis products and

enhance purity80. The generated library based on these primers did not undertake such

testing due to time constraints and if utilised in succeeding experimentation will need

to be further explored.

4.2 Strengths and Limitations

Most of the available mouse models of Hp infection have demonstrated variable-poor

colonisation66. Currently, the only strain that presents high-colonising ability is Hp-

SS166 and was appropriately utilised throughout this investigation. The majority of

significant research experiments have employed Hp-SS1 to mimic the colonisation

and pathogenesis of the human variation. This motivated the subsequent investigation

to eradicate Hp infection with precision, excluding possibilities of failure,

misrepresentation and inaccurate research findings.

90

The major limitation of this study was the inability to confirm the insertion of the

KanR cassette within the IROMPs of interest and the succeeding failure to re-produce

them. As a major component of the cell-SELEX method could not be obtained

within the time frame of the project, the generation of high affinity aptamers could

not be ensued. Negative selection includes a cell line minus the target markers that

contributes to the specificity arising from this process80. Aptamers that show high

affinity and bind to these negative cell lines are extracted, deriving only the ones that

demonstrate desired qualities81. If this process is completed without negative

selection, a high probability of deriving non-specific aptamers would result.

As this method is expensive and time consuming, to incorporate a negative cell line

that essentially presents a 50% chance of carrying the KanR cassette within the

IROMPS, would be impractical. This formed the rationalisation behind withdrawing

further experimentation.

4.3 Clinical Relevance

As Hp continues to evolve producing more severe clinical outcomes and refractory

strains, this potential therapy demonstrates a highly efficacious method of

eradication. This study has verified the importance of iron-regulation and the

associated outer membrane virulence factors, detailing the impairment of Hp to

colonise if the IROMPs were therapeutically targeted. In addition, it has offered an

alternative approach to generate new therapies for other infections caused by strain-

resistant bacteria, e.g. Methicillin resistant Staphylococcus aureus (MRSA). The

increasing advantages of aptamer-based therapies have motivated their production for

all sorts of human disease and particularly for cancers.

91

This ideal therapy remains inadequate within the microbial field aside from few

inquiries based on bio-sensing abilities. This project has filled the gap within the

literature of targeting essential proteins via aptamer-based therapies that increasingly

demonstrate advantages due to their highly selective qualities.

4.4 Future Direction

Short-term directions include the processes of obtaining essential components to

ultimately derive these aptamers. The re-production and validation of the IROMP-

interrupted KanR clones is required for negative selection to progress further. In

addition, an alternative direction would be to undertake whole genome sequencing for

the unconfirmed knockouts to eliminate the generation of new primers and verify the

insertion and location of the KanR cassette. Once negative selection is obtained, the

process of cell-SELEX would be ensued to derive the aptamers specific to fecA1,

fecA2 and frpB1 in Hp-SS1.

Long-term objectives are focused around the therapeutic abilities of the derived

aptamers. As Hp colonises the acidic conditions of the stomach, the stability of these

aptamers including their capacity to remain functional in comparable states will need

to be assessed using minimum inhibitory concentration (MIC) assays that include

hydrochloric acid. Depending on the functionality of these aptamers within acidic

conditions, an investigation is required to assess the therapeutic action of these

molecules. This exploration will determine if the aptamer has the ability to directly

impede the iron-acquisition process of the IROMPs or if they will need to be

conjugated to a drug, e.g. antibiotics, to aid in this process via MIC assays.

92

4.5 Conclusions

This study aimed to ensue a detailed investigation of potential therapeutic targets and

in particular the IROMPs of Hp to derive an effective therapy using aptamers. The

process of iron-acquisition mediated by fecA1, fecA2 and frpB1 confirmed to be a

major component within iron homeostasis. In addition, these IROMPs were shown to

be essential for the colonisation of Hp and if targeted would demonstrate an assured

therapeutic effect.

The current need for targeted therapies for infections like Hp that have emerged due

to the misuse of antibiotics and the spread of resistant stains was the key motivator of

this study. Not only did it provide a promising therapeutic conception for the

eradication of Hp infection, it also formed a proof of principle. This process could

potentially direct the production of targeted therapies using aptamers for other

refractory infections. The function and efficacy of aptamer-based therapies could

potentially replace antibiotics and overcome resistance as demonstrated with the

eradication of Hp infection.

93

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