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
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