The role of a bifunctional mucinase in modulating personalized gut microbiota-Vibrio cholerae interactions during infection

PROJECT SUMMARY Vibrio cholerae (Vc), the causative agent of cholera, colonizes the mucosal surface of the small intestine. Infec- tion is mediated via virulence factors to penetrate the mucus layer, attach to epithelial cells, and proliferate, all the while modulating interactions with both host cells and the gut microbiota. The human gut microbiota is highly diverse, and interpersonal variation in the structure and function of the microbiota drives dramatic differ- ences in Vc colonization. At the center of microbe-microbe and microbe-host interactions lies the host mucus layer, comprised of secreted mucin glycoproteins including the dominant mucin MUC-2. Mucus provides at- tachment sites and carbon sources for both pathogens and commensal members of the gut microbiota at the interface of the epithelium and the gut lumen, as provides a key physical barrier to infection. However, the role of mucin metabolism in metabolic interchanges between Vc and specific configurations of the gut microbiota, and the resulting impact on personalized Vc infection outcomes, has not been well studied. Here, we show that TagA, a secreted metalloprotease upregulated by the Vc virulence master regulator ToxT, promotes Vc growth in mucin, and that TagA is a bifunctional protein, acting as both mucinase and a mucus secretagogue that in- duces host mucin production. Using a combination of ex vivo tissue culture and in vivo gnotobiotic mouse colonization models combined with TagA mutants lacking either proteolytic activity or MUC2-inducing activity, we found that TagA's two activities have different effects on Vc fitness during infection depending on the pres- ence of specific human gut microbes. We have generated model gut microbiota characteristic of human gut microbiota states: one model microbial community similar to that of healthy individuals, which promotes Vc in- fection resistance, and another model microbiota characteristic of the dysbiotic state found in cholera endemic areas associated with high susceptibility to Vc colonization. TagA mucolytic activity is important for Vc infection resistance within the colonization-resistant microbiota, while the mucin-inducing activity of TagA FN3 leads to increased Vc infection within dysbiotic communities. Therefore, we hypothesize that Tag drives Vc metabolic interactions with specific gut microbiota leading to community-specific attachment, growth, and overall infection outcomes. We will test aspects of this core hypothesis in four specific aims. Aim 1 will elucidate mechanisms driving mucin-dependent interactions of Vc with commensal gut microbes in epithelial attachment and growth. Aim 2 will examine how personalized gut microbiota structure in cholera endemic areas modulates mucin- and TagA-dependent disease phenotypes. Aim 3 will determine how TagA-microbiota interactions drives produc- tion and metabolism of host mucins. Finally, Aim 4 will elucidate the role of proximity and spatial specificity in driving microbiota-dependent Vc disease outcomes. The ultimate goal of this application is to shed light on the role of pathogen-mediated mucin metabolism in microbial interactions during enteric infection.