Session 3 Lecture 1

Dynamics of the GIT Microbiome: “Microbial Darwinism”

10 14 bacteria representing ~1000 species live in a limited space: The GITThese represent mainly four phyla. When placed in perspective, all animals with backbone are only one phyla!The GIT microbiome is an ecosystem where diverse bacteria “fight for space and survival”.

Survival and proliferation within the microbiome depends on:Availability of oxygen, an optimal external pH, which differs

regionally as does the makeup of the microbiomeCompetition for space and attachment sites on enterocyctesSusceptibility/resistance to anti-microbials from other members

of the microbiome, e.g. bacitracinSusceptibility to therapeutic antibioticsAbility to metabolize complex carbohydrates to SCFAAbility to survive the host immune system

Survival also depends on the ability of bacteria to rapidly change their genome.A small genome and rapid replication allows generation of mutants 1000-fold faster than their host (see later).Genomic variants resulting from spontaneous mutation and are no longer susceptible to therapeutic antibiotics

or antibiotic produced by other members of the microbiome, or by the attacking host immune system

Bacteria can horizontally transfer genetic survival traits to other bacteria by various mechanisms [see later]

Stomach Duodenum Ileum Colon

Bacteria Avoid Death from Antibiotics and Host Lymphocytesby Rapid Genetic Change

Bacteria disguise themselves by becoming “moving genetic targets”Bacteria undergo reproduction in hours while their vertebrates hosts require yearsReproduction is the time when genetic changes from spontaneous mutation are passed to the offspring

The efficiency of this process is related to genome size

Replication is the time when new traitsare transferred

Rate of genetic change= genome size X replication time

Human 8.6 x 1012

Bacteria 1.2 x 109

Influenza 2.6 x 105

Thus, bacteria can alter their genome1000 faster than humans

Viruses can do it at least a milliontimes faster

Here comes the Smut: “Bacterial Porn”

Bacteria divide and replicate themselves and their DNA like all life forms on earth

Bacteria can also transfer genetic traits “horizontally”

Transformation

Transduction

Conjugation& Plasmid Transfer

Plasmids are replicating circular DNA that are independent of chromosomal (genomic) DNAPlasmid cannot transfer “life”, but can “hand-off” virulent/resistance traitsA pathobiont can “hand off” virulent traits to a “good” member of the microbiome that is not targeted by

the immune system or antibiotics so the trait is not eliminatedMost antibiotic resistance traits are carried by plasmids hidden in eubiotic “good” bacteria

“Enter the Jedi Lymphocyte”: How higher Vertebrates competewith the rapid genetic changes of bacteria (and viruses)

Mutualism requires “Peaceful Co-existence” : Don’t kill the microbiome, control it!

Lymphocytes somatically alter their genes to compete with bacterial genome changesEarliest versions of lymphocytes appeared in jawless fishesThe current lymphoid system appeared in jawed fishesThe lymphoid system is most sophisticated in warm-blood birds and mammals

How do lymphocytes accomplish this somatic feat?Extensive gene duplication followed by mutation of the duplicated genes,

a process called “somatic gene conversion”Somatic recombination of the many duplicated and slightly different genes (see next diagram)Subsequent somatic mutation of the somatic recombinants adds additional diversity

Jawless fish Fishes Amphibians MammalsBirdsReptiles

Lymphocyte-like

True Lymphoid Systems

Combinatorial Diversity: The First Weapon in the Arsenal of the Jedi Lymphocyte

Antibody structureComposed of two different sizedpolypeptide chains: “heavy” and “light”

The N-terminal of each contains the antibody binding site [red, yellowand green gene segments in diagram]

These regions and the genes that encode them are called “variable”VL and VH

Antibody geneticsDifferent loci encode the light andheavy chain genes of antibodies

Recombination of the duplicated VLand VH genes yields the eventualrecombinant variable region genes

40 5 91 30 6

Number of Variable Gene Segments in Humans

How does Somatic Recombination and Mutation by Jedi Lymphocytes Compare tothe Rate of Genetic Change of Bacteria and Viruses?

Recombination MathHuman heavy chain variable genes: 91 VH x 30DH x 6JH yields > 9,000 possibilities

Only 30-50% are productive so that means 3.6 x 103

Human light chain variable genes: 40 VL x 5JL yields 200 possibilitiesOnly 30-50% are productive so that mean 80

In forming the entire antibody it means 2.8 x 105

Somatic hypermutation (Second weapon of the lymphocyte)After each productive variable heavy and variable light chain rearrangement occurs, they are subjected

to a hypermutation process that generates 100-1000 more variants

How does this lymphocyte process compare to the rate of genetic changes possible by microbes?Antibody variants = 2.8 x 106

Rate of genomic change by bacteria= 1 x 103

Rate of genomic change by viruses ~1 x 106

Fortunately for higher vertebrates, the Jedi lymphocytes win and save humanity!

The Mucosal Antibody System of the GIT

Antigen is sampled by M-cells and dendritic cells(DC) and delivered to germinal centers

B cells specific for the antigen are selected froma million possibilities in the germinal center

Selected B cells leave the follicle to becomemature plasma cells (antibody factories)

Secreted IgA that is transported through the enterocyte barrier into the lumen

Antibodies bind and clump the luminalbacteria that goes to the feces

SIgA antibodies also bind non-specificiallyvia carbohydrate moeities

60% of feces is made of bacteria from the microbiome

Many are coated with SIgA antibodies (the “rotor-rooter” antibody)

Removal of especially pathobionts helps to prevent translocation and bacteremiaFecesPlasma cell

Gut lumen

SIgA transportedby enterocytes

Migratorypathways

Oral ToleranceThe GIT is exposed daily to “pounds” of foreign proteins and 1014 bacteria

However, antibodies to food proteins in healthy individuals are subclinicalin serum and stool

Mucosal antibodies are limited to SIgA in healthy individuals, and the majority are specific for pathobionts but SIgA also attaches to manybacteria through carbohydrate moeities

Intravenous injection of even a small amount of food antigen e.g. “ovalbumin”, results in a vigorous serum antibody response

However, long-term feeding of the same antigen will suppress or eliminate the serum response

This is call “oral tolerance” and is antigen-specific

Oral tolerance depends on T-cells called regulatory T-cells or Tregs

Only CD-4 T cells specific for the antigen will work

Other CD-4 cells called “helper” or TH2, are needed for antibody development

The number of CD-4 “helpers” is very low when ovalbumin is continuously ingested

The Tregs are a special CD-4 subset with certain surface markers that secrete certain immunosuppressive cytokines

Tregs Patrol the Gut mucosaThe T cell system is complex

We will consider three variantsTc are cytotoxic and kill virus-infected cellsTH1 cells are inflammatory and TH2 cells are “helper cells”Tregs especially regulate the activity of TH1 and TH2 T cells

Three different scenarios are illustrated

The Healthy GITTregs block antibody responses to food proteinsTregs block inflammation by neutrophils and Tc activity

The virus-infected MucosaBlockage of antibody production to the virus is removedTc are activated to kill virus-infected cellsInflammation is blocked

Bacterial Invasion, i.e. TranslocationAntibody responses are allowedBlockage of TH1-induced inflammation is removed to allow local

inflammation in which neutrophils remove the bacteria

Blocked pathway

Blocked pathway

Blocked pathway

Cell Cross-talk

Cell Cross-talk

Cell Cross-talk

A Faulty Treg “On-Off-Switch” can Result in an Unhealthy GIT

Cytotoxic cells do not stop with the killing of virus-infected cells and do collateral damage to adjacent enterocytes, i. e. “friendly fire”

Antibodies attack both pathobionts and “good” eubiotic bacteria

The inflammatory response is not controlled when the threat ofthe infection ends which leads to damage of the mucosal barrier

Oral tolerance to food antigens is lost

Disruption of the Microbiome can Result in an Unhealthy GIT

Pathobionts or potential pathobionts are always “lurking” among the 1014 bacteria that comprise the GIT microbiomeThese include e.g. C difficille, Staph aureus and E.coli

E.coli exist in many forms and specializes in the exchange of plasmid that include those that contain virulence genes

Most antibiotic resistant genes “hide” in plasmid carried by “good” mutualists

Salmonella and Shigellae can act as a “Trojan horses” andbreach the mucosal barrier by entering through M-cells

Disruption of the microbiome can give advantages to pathobiontsViral infection, e.g. TGE in piglets, damages the mucosal

enterocyte barrier by shearing off villi allowing pathobionts to gain access and thus potentially cause bacteremia

Antibiotic therapy can result in massive loss of “good” bacteria that normally blocks pathobionts allowing organisms like C. difficile to expand

Both viral infections and antibiotics can result in bacteremia as shown for C. difficille