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

项目总结 霍乱弧菌(VC)是霍乱的病原体，定植于小肠的粘膜表面。Infec- 感染是通过毒力因子穿透粘液层、附着于上皮细胞和增殖，所有这些都是通过毒力因子介导的。 同时调节与宿主细胞和肠道微生物区系的相互作用。人类肠道微生物区系是 微生物区系的结构和功能的高度多样性和人际差异导致了巨大的差异- 风险投资殖民的征兆。在微生物-微生物和微生物-宿主相互作用的中心是宿主粘液 层，由分泌的粘蛋白糖蛋白组成，包括主要的粘蛋白MUC-2。粘液在- 肠道微生物区系中病原体和共生成员的接触位置和碳源 上皮和肠腔的界面，AS为感染提供了一个关键的物理屏障。然而，这个角色 在VC和肠道微生物区系的特定构型之间的代谢交换中的粘蛋白代谢， 而由此产生的对个性化VC感染结局的影响，还没有得到很好的研究。在这里，我们展示了 TagA是VC毒力主控调控因子ToxT上调的一种分泌型金属蛋白酶，促进VC生长 在粘蛋白中，TagA是一种双功能蛋白质，既起粘蛋白酶作用，又起粘液促分泌剂的作用，在... 诱导宿主产生粘蛋白。使用体外组织培养和体内诺生菌小鼠的组合 与缺乏蛋白分解活性或MUC2诱导活性的TagA突变体相结合的定植模型， 我们发现，Taga的两种活动在感染过程中对VC适合性的影响取决于压力- 特定的人类肠道微生物。我们已经生成了具有人类肠道特征的肠道微生物群模型 微生物区系状态：一种与健康个体相似的模式微生物群落，促进VC在- 感染抗性，以及在霍乱流行中发现的另一种具有非生物状态特征的模式微生物区系 与VC定植高度易感性相关的地区。TagA粘液溶解活性对VC感染的重要作用 耐定植微生物体内的抗性，而TagA FN3的粘蛋白诱导活性导致 在非生物群落中，VC感染增加。因此，我们假设TAG驱动VC新陈代谢 与特定肠道微生物群的相互作用导致社区特有的依附、生长和总体感染 结果。我们将在四个具体目标中测试这一核心假设的各个方面。目标1将阐明机制 在上皮附着和生长中推动VC与共生肠道微生物的粘蛋白依赖的相互作用。 目标2将研究霍乱流行地区个性化的肠道微生物区系结构如何调节粘蛋白和 TAGA依赖的疾病表型。AIM 3将确定Taga-微生物区系相互作用如何推动生产- 寄主粘蛋白的分泌和代谢。最后，目标4将阐明邻近和空间专一性在 推动依赖微生物区系的VC疾病结局。这个应用程序的最终目标是阐明 病原体介导的粘蛋白代谢在肠道感染过程中微生物相互作用中的作用。

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.