IMMUNE REGULATION BY BACTEROIDES POLYSACCHARIDE CAPSULES

AbstractSymbiotic human gut bacteria perform a number of beneficial physiological processes, including fermentationof dietary fiber polysaccharides and instruction of mucosal immune development. During homeostasis, theimmune system utilizes several mechanisms to tolerate gut microbes; however, the same symbionts can causeinflammation when certain defects in the immune system exist. Gut symbionts have also evolved mechanismsto protect themselves from the host immune response, including the propensity to synthesize a variety ofdifferent surface capsular polysaccharides (CPS). Bacteroides thetaiotaomicron (B. theta) is a model gutsymbiont that degrades a wide variety of dietary, host, and microbial glycans and dedicates hundreds of genesto CPS production. The ability to express multiple different capsules, along with their complex, phase-variableregulation, is a common feature of gut Bacteroidales species, including B. fragilis and B. theta. One of themany B. fragilis CPS, termed PSA, is zwitterionic, and has been shown to modulate host cytokine levels andinduce anti-inflammatory responses that limit intestinal disease. Recently, several other diverse intestinalbacteria, besides B. fragilis, have been shown to produce zwitterionic CPS that also exhibit anti-inflammatoryproperties. Thus, it has emerged that several CPS, among just a small set studied, have evolved bioactiveproperties. In parallel, genome sequencing has revealed a bewildering diversification of genetic loci involved inCPS by human gut bacteria. The central hypothesis of this proposal is that other gut bacterial CPS (especiallynon-zwitterionic forms) have evolved and been selected under immune pressure to possess a variety ofimmunomodulatory functions. To test this premise, we have created a novel experimental system: a definedCD4+ TCRtg mouse line in which T cells are specific for a B. theta outer membrane protein, combined with aset of B. theta strains that each either express a single CPS or none at all. Our hypothesis is supported by twomajor findings that set the basis for this proposal. First, when eight individual strains that each express adifferent B. theta CPS were tested for their ability to be processed and presented by macrophages to stimulateT cells in vitro, there is a broad range of activities, from weak to strong. Second, analysis of just 14 othersequenced B. theta strains, revealed extensive CPS diversification. Strikingly, we identified a total of 49 uniquecps loci among just these 14 strains, suggesting that the ?universe? of Bacteroides CPS is large. We proposeto test our hypothesis through two aims: 1) Determine the mechanisms by which 4 individual B. theta capsulesexert their range of immunomodulatory activities, and 2) Isolate and express novel CPS genes fromBacteroides species and measure their immunomodulatory activity in vivo and in vitro. Polysaccharides are themost diverse class of biomolecules in nature and have the potential to exert potent biological effects. Thesestudies will expand the known lexicon of bacterial polysaccharide-immune system interactions and should leadto discovery of new, bioactive CPS for potential use as therapeutics.