Moving into the Post-Metagenomic Era of Gut

Microbiome Research

Julian R. Marchesijr@cardiff.ac.uk

School of Biosciences

Department of Surgery and Cancer

Some definitions

• Microbiota (microflora) – the qualitative and quantitative information about the different microbes present in a system – so who is there and how abundant.

• Microbiome – the functions that these microbiota have, e.g. bile metabolism – their gene catalogue.

• Metagenomics – either “gain-of-function” or DNA based approach to create gene catalogues, used to define the microbiome.

• Metaxonomics – creation of 16S rRNA gene inventories, used to define the microbiota.

• Metabolomics – a catalogue of the metabolites in a sample.

The main focus in human biology has been to explain how it functions in terms of the genome

+Disease Health

X

X

Human microbiome

• Microbiome is host specific

• Can be changed by diet, drugs and surgery

X=mutation

Ecological interactions between members of different species

+ positive (win); - negative (loss); 0 neutral Species 1/Microbiota; Species 2 = host

e.g. H. pylori or C. difficile

e.g. FMT for CDAD

Amensalism

e.g. probiotic microbes which do not reproduce in the host L. lactis.

This is the modern issue for microbiome studies

Pathologies not associated with a pathogen

The concept of amensalism involves one organism growing and in doing so inadvertently damages another, but it is not evolved to do so – collateral damage.

The study of Inflammatory Bowel Disease has been grappling with this for the last 20 years.

A disease that is multi-factorial and involves environmental factors, but for which no pathogen has been detected.

A disease which does not develop in sterile rodent models

• Colorectal cancer• Breast cancer• Metabolic syndrome• Diabetes

We can no longer use Koch’s postulate for verifying pathogenic

traits

We have no idea of the consequences of position on this continuum

The variability clouds the issue of a healthy microbiota

Bio geography complicates the story

Burkina Faso Italy

The absence of a microbiome impacts the whole host

We have a dearth of mechanisms and only

a few have been identified.

Diabetes

Inflammatory boweldisease

Obesity

Asthma/Eczema

Depression

Heart disease

Colon Cancer

Non-alcoholic fatty liver disease

All these disease have evidence for a role of the gut microbiota

Inventories of 16S rRNA genes

“Omic” approaches available to investigate the gut

Bottom up

Top down

f

Metabolic Roux-en-Y Gastric Bypass Surgery (RYGB) and gut microbiota/metabolites

Rat modelPCA of metabolite profiles PCA of bacterial profiles

RAT MODEL

Li et al., (2011) Gut

A marriage of metataxonomics and metabonomics

Time point post surgeryWeek 2 Week 8

2. Wistar Rat Study

Determination of the relative suspension growth as a measure of cytotoxicity(A) and genotoxicity (B) of fecal water extracts following treatment of L5178Y

mouse lymphoblastoid cells for 24 h.

-60

-40

-20

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-100 -50 0 50 100

PC1 (R X=24.9%)

PC

2 (R

X=

8.2%

)re

lativ

e in

tens

ity o

f obs

erve

d un

conj

ugat

ed b

ile a

cids

0

200

400

p=0.7

p=0.001 p=0.026

rela

tive

inte

nsity

of o

bser

ved

taur

ine-

conj

ugat

ed b

ile a

cids

A B

C D

Sham

RYGB

Sham

RYGBSham

RYGB

p=0.3

p=0.073 p=0.049

Week 6 Week 8 1 week pre-op Week 6 Week 81 week pre-op

1 week pre-op Week 6 Week 8

0

1

2

p=0.4

p=0.012 p=0.087

0

100

0

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300

rela

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nsity

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chol

ic a

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The Relative concentrations of observed unconjugated and taurine-conjugated bile acids

show a significant difference between sham and RYGB-operated rats at weeks 6 and 8.

Cholic acid

Unconjugated bile acids Taurine-conjugated bile acids

PCA scores plot

Altered faecal bile acid levels post RYBG

Conclusion: Our data hence suggest that BABR could be useful for the management of the impaired glucose tolerance of the metabolic syndrome, since they not only lower cholesterol levels, but also reduce obesity and improve insulin resistance.

The “normal” vaginal microbiome (non-pregnant)

‣ Represents 95% of healthy women in these ethnic groups.- 5% of women are different than

these

Ravel et al. (2011) PNAS 108 S1:4680-7.

Tuesday, April 9, 13

• 5 Community State Types (CST)

• Many lactic acid producers

• Maintain low pH

• Produce antimicrobials

Ravel et al. PNAS 2011; 108: 4680-7

• Lactobacillus spp. characteristic of “normal”, “healthy” reproductive age women.

Vaginal microbiome CSTs in our UK population

• Hierarchical clustering analysis identified 5 major CSTs

• 60% of post-partum samples are high diversity CST IV

• CST * may be a new UK specific CST

Longitudinal assessment of vaginal CSTs throughout pregnancy and post partum

Incr

ease

d d

iver

sity

So far this is one of the only scenarios where more diversity is deemed to be bad

The vaginal microbiome in Preterm Premature Rupture Of Membrane at 28 weeks gestation

16S rRNA genes from the vaginal microbiome enables stratification of PPROM patientsinto Lactobacillus- dominant (PPROM 9-15) and Lactobacillus-depleted heterogeneousgroups (PPROM 1-8).

Two separate aetiologies? Treat differently?

The dialogue between the host and microbiome in a healthy context

Disease Health

X

X

Human microbiome

X=mutation

These arrows really depict the movement of proteins and

metabolites - in other words the communication is via the

proteomes and metabonomes

Exploring the gut microbiome via activity – emergent properties

Why?

Bacterial proteases are a potential virulence factor in colorectal cancer and IBD. They have also been shown to compromise tight junction integrity.

Steck N et al., Gastroenterology. 2011 141(3):959-71.

Metaproteome HostMicrobiome

The missing link is the proteome

IBD and the degradome

The degradome and degradomics is the study of enzymes which degrade proteins i.e. proteases. So does it play a role in inflammatory bowel disease?

Casein Collagen Keratin

Use of targeted proteases inhibitors to understand the degradomein IBD and healthy samples

BacterialPlantFungal

Mammalian

IBD and the degradome cont.

The proteolytic activity varies in both cohorts

PROTEASE

INTESTINALALKALINE

PHOSPHATASE

LPS

BACTERIALALKALINE

PHOSPHATASE

INFLAMMED NASH

NOT INFLAMMED NASHX

The microbiome and the healthy person

Tight junction integrity

There are many more generic bacterial enzymes which may be involved in health

and disease.

We are now moving into a post-metagenomics era of gut microbiome research, which moves further away from metagenomics and towards multiplatform omics?

Emergent features and functions should be the focus and not necessarily gene content e.g. protease levels.

What roles does a healthy microbiome play?

SCFA production, niche exclusion and unknown roles in development.

Conclusions

Impact can be at a local level

Off tumour Dysplasia Cancer

On tumour mucosal cancer microbiota are specific to pathology and maybe prognostic

The gut microbiome also influences drug metabolism