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

项目摘要 霍乱弧菌（Vc）是霍乱的病原体，定植在小肠的粘膜表面。感染- 通过毒力因子介导，穿透粘液层，附着于上皮细胞并增殖，所有 同时调节与宿主细胞和肠道微生物群的相互作用。人类肠道微生物群 高度多样性，以及微生物群结构和功能的人际变化，导致了巨大的差异- VC殖民地。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，包括占主导地位的粘蛋白MUC-2。粘液提供- 附着位点和碳源的病原体和肠道微生物群的寄生虫成员， 上皮和肠腔的界面，为感染提供了关键的物理屏障。然而，作用 在Vc和肠道微生物群的特定构型之间的代谢交换中的粘蛋白代谢， 以及由此产生的对个性化Vc感染结果的影响尚未得到很好的研究。在这里，我们表明， 由Vc毒力主调节因子ToxT上调的分泌型金属蛋白酶TagA促进Vc生长 在粘蛋白中，TagA是一种双功能蛋白质，既作为粘蛋白酶又作为粘液促分泌素， 诱导宿主产生粘蛋白。使用离体组织培养和体内致菌小鼠的组合 与缺乏蛋白水解活性或MUC 2诱导活性的TagA突变体组合的定殖模型， 我们发现，在感染过程中，TagA的两种活性对Vc适合度有不同的影响， 特定人类肠道微生物。我们已经生成了人类肠道的模型肠道微生物群特征， 微生物群状态：一个模型微生物群落类似于健康个体，促进Vc在- 感染抗性，以及霍乱流行中发现的另一种微生物群模式的微生物群特征 与Vc定植高度易感性相关的区域。TagA粘液溶解活性对Vc感染很重要 在耐定殖微生物群内的抗性，而TagA FN 3的粘蛋白诱导活性导致 在微生态社区内增加Vc感染。因此，我们假设Tag驱动Vc代谢 与特定肠道微生物群的相互作用导致社区特异性附着，生长和整体感染 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驱动Vc与肠道微生物的粘蛋白依赖性相互作用。 目标2将研究霍乱流行地区的个性化肠道微生物群结构如何调节粘蛋白- TagA依赖性疾病表型。目标3将确定TagA-微生物群相互作用如何驱动生产， 宿主粘蛋白的作用和代谢。最后，目标4将阐明邻近性和空间特异性的作用， 驱动微生物群依赖性Vc疾病的结果。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期间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.