Regulation of protein secretion in human gut commensals

项目摘要 人类肠道中的共生细菌为它们的宿主提供了许多益处， 宿主无法获得的食物，以防止病原体入侵。许多宿主-微生物组相互作用是 由分泌到肠道中的细菌蛋白质（分泌组）介导，但这种调节和协调 这个过程还没有研究过。该提案侧重于肠道优势菌类杆菌属，以测试 假设这些生物体使用中枢机制来控制分泌蛋白的部署。一条线索 来自对大肠杆菌编码毒素的研究：两种分泌毒素在序列上没有相似性， 两种靶都使用相同的表面相关半胱氨酸蛋白酶来协调它们的分泌。的同源物 这种半胱氨酸蛋白酶（但不是其靶标）在拟杆菌中是保守的，这表明 这些酶调节蛋白质分泌。该提案将使用创新的N-末端蛋白质组学 一种确定拟杆菌中蛋白酶底物库的方法， 同源物在物种内和物种之间共享目标。在gnotobiotic小鼠中进行的补充研究将确定 蛋白酶加工对体内分泌组的贡献。这项提案还将利用遗传学， 厌氧微生物学和gnotobiotics，以阐明环境线索和信号转导途径 调节蛋白酶的表达。这将决定这些蛋白酶如何协调波的释放 分泌的蛋白质来响应肠道中的环境信号。如果成功，这些研究将揭示 微生物组中分泌组调节的基本机制及其对宿主微生物组的影响 相互作用和生物工程治疗酶在肠道中的递送。 本建议书还提出了一个详细的培训计划，将允许候选人发展新的技能 职业发展所需的。这项研究将在高度合作和 多学科微生物科学研究所（MSI）在耶鲁大学。赞助商有既定的轨道 导师的记录，实验室和MSI提供最先进的设施。同行的多样化专业知识 MSI的化学家、生物化学家和遗传学家将支持候选人的研究 目标.指导委员会将为项目提供技术指导，并支持候选人的 职业发展。候选人还将在Poorvu完成指导和教学培训 教学中心和耶鲁博士后协会，他将把这种培训， 通过指导学生在当地的科学展览项目中进行实践。通过向耶鲁大学的同行展示他的研究成果 在大学和会议中，候选人将培养他的沟通技巧，并建立一个网络， 同事总之，这个培训计划将建立所需的技能和连接，以取得成功， 独立调查员

Project Summary Commensal bacteria in the human intestinal tract provide numerous benefits to their hosts, from digestion of foods inaccessible to the host to protection against pathogen invasion. Many host-microbiome interactions are mediated by bacterial proteins secreted into the gut (the secretome), but regulation and coordination of this process has not been studied. This proposal focuses on the dominant gut genus Bacteroides to test the hypothesis that these organisms use central mechanisms to control deployment of secreted proteins. One clue comes from studies of commensal-encoded toxins: two secreted toxins with no similarities in sequence or targets both use the same surface-associated cysteine protease to orchestrate their secretion. Homologs of this cysteine protease (but not its targets) are conserved across Bacteroides, suggesting a broader role for these enzymes in regulating protein secretion. This proposal will use an innovative N-terminome proteomic approach to determine the repertoire of protease substrates in Bacteroides and establish whether protease homologs share targets within and between species. Complementary studies in gnotobiotic mice will determine the contribution of protease processing to the in vivo secretome. This proposal will also use genetics, anaerobic microbiology, and gnotobiotics to elucidate environmental cues and signal transduction pathways that regulate protease expression. This will determine how these proteases might coordinate release of waves of secreted proteins in response to environmental signals in the gut. If successful, these studies will uncover fundamental mechanisms for secretome regulation in the microbiome, with implications for host-microbiome interactions and bioengineering therapeutic enzyme delivery in the gut. This proposal also presents a detailed training plan that will allow the candidate to develop new skills required for career advancement. This research will be conducted at the highly collaborative and multidisciplinary Microbial Sciences Institute (MSI) at Yale University. The sponsor has an established track record of mentorship, and the lab and the MSI provide state-of-the-art facilities. The diverse expertise of peers and faculty at MSI, ranging from chemists, biochemists, and geneticists, will support the candidate's research goals. A mentoring committee will provide technical guidance for the project and will support the candidate's career development. The candidate will also complete training in mentoring and teaching from the Poorvu Center for Teaching and Learning and the Yale Postdoctoral Association, and he will put this training into practice by mentoring students in their local science fair projects. By presenting his work to peers at Yale University and in conferences, the candidate will foster his communication skills and build a network of colleagues. Together, this training plan will build the skillset and connections required to succeed as an independent investigator.