The Role of Bacterial Amyloids In Regulating Gastrointestinal Homeostasis

Abstract

Many bacterial species exist in nature as part of highly structured multicellular communities known as biofilms. Amyloids, proteins with a conserved β-sheet quarternary structure, show high resistance to many chemical and enzymatic processes including proteinase K and SDS treatments and are produced as essential adhesins during biofilm formation. Curli fibers expressed by Enterobacteriaceae family members including E. coli and S. Typhimurium are the most studied amyloids to date. Curli-like fibers are also produced by members of the predominant phyla found in the host gastrointestinal microbiota in environmental biofilms. Curli fibers are the predominant microbial-associated molecular pattern (MAMP) on enteric bacteria recognized by the Toll-like receptor (TLR) 2/1-heterodimer complex. Interestingly, the TLR2/1 complex has been implicated as a key player in modulating gastrointestinal homeostasis. The focus of the current studies centered on the innate immune recognition of curli fibers by cells of the gastrointestinal tract and how that contributes to gastrointestinal homeostasis. In the first phase of our studies, utilizing intestinal epithelial cells polarized on semi-permeable tissue culture inserts (Transwells®), we observed that the recognition of curli fibers on Salmonella enterica serovar Typhimurium by intestinal epithelial cells led to the augmentation of the intestinal epithelial barrier in a PI3K-dependent manner. We also observed that bacterial translocation of S. Typhimurium from the apical side to the basolateral side of the Transwell system was limited when curli fibers were present. Furthermore, infection of mice with S. Typhimurium showed that translocation of bacteria from the intestinal lumen into the cecal tissue and mesenteric lymph nodes was limited in C57BL/6 mice as compared to TLR2 knockout mice. In the second phase of our studies, we sought to further investigate the effect that curli fibers exert on gastrointestinal homeostasis through the induction of immunomodulatory cytokines such as Interleukin 10 (IL10) from subepithelial lamina propria cells. IL10 has been shown to contribute to the maintenance of the intestinal epithelial barrier and IL10-deficient mice develop lethal colitis within the first 2-3 months of life. 6-8 week-old female C57BL/6 and TLR2-/- mice were given 5mg/kg of curli fibers via intraperitoneal injection. Subsequent RT-PCR analysis of the small intestine showed a significant expression of Il10 in C57BL/6 that was absent in TLR2-/- mice. Interestingly, no changes in Ifnγ or Tgfβ mRNA were observed in these mice. This response was gut-specific, as Il10 was not detected at all in the spleen. Furthermore, in a chemically-induced colitis model, we observed that the administration of curli fibers to 8-week old Balb/c mice ameliorated disease severity as compared to colitic mice that received mock treatments. Interestingly, Il10 was also induced in the colons of colitic mice that received curli and which were euthanized 6 days after colitis was induced. Our results suggest that curli fibers induce IL10 production via a TLR2-dependent manner to dampen inflammation in the gastrointestinal tract. Overall, our results partially describe a novel role for curli amyloid fibers produced by commensal bacteria in modulating gastrointestinal health and homeostasis. We propose that the induction of immunomodulatory cytokine such as IL10 by amyloid fibers is an important mechanism utilized by commensal bacterial to confer beneficial effects that benefit both the host and microbe. We also propose curli fibers as a potential alternative in the treatment of inflammatory bowel disease.