Mechanistic investigation of bacterial type 9 secretion system machinery and its involvement in gut metabolism and immunomodulation

总结 由肠道微生物群产生的代谢物提供多种健康益处。但 肠道微生物群利用来自宿主的底物并产生有益的 产品不太了解。此外，关于分泌蛋白组的作用的信息，即，一 由肠道微生物群分泌的细胞外蛋白的收集对健康的影响是最小的。 目前，还没有用于研究肠道微生物9型蛋白分泌系统（T9 SS）的模型系统 存在.我们初步的生物信息学分析表明，在几个细胞中存在功能性T9 SS。 拟杆菌属的人类肠道分离物。我们预测肠道类杆菌分泌的T9 SS 果胶酸裂解酶和其他分解膳食纤维并随后将其发酵成短纤维的酶， 链脂肪酸（SCFAs）。SCFA的减少导致代谢紊乱。我们预测 相对未被充分研究，但重要的是，人类肠道分离物，包括拟杆菌， 诺氏拟杆菌、流动拟杆菌、普雷沃氏菌和Distasonsis副拟杆菌分泌许多 蛋白质通过T9 SS。我们正在培育一种遗传上易于驯服的物种B。作为一种模式生物， 填补了关于肠道中T9 SS分泌组研究的知识空白。我们假设T9 SS 肠道微生物群的分泌组可能会产生一个共同的低聚糖库， 生产品种。此外，我们的目标是找到一个假定的拟杆菌感觉转导的控制器 网络，感觉丰富的膳食纤维和调节生产的SCFA生产 内切酶我们的初步数据还预测了免疫抑制性半胱氨酸蛋白酶的分泌， B的T9 SS。好吧为了检验我们的预测和假设， 显著提高我们对物种间合作，资源优化， 肠道微生物群的免疫调节。 T9 SS是最近发现的革兰氏阴性菌的蛋白输出途径 纤维杆菌属-绿双-拟杆菌超门。到目前为止，研究T9 SS的模式生物 来自人类口腔微生物群、环境分离物和感染水生动物的病原体。 通过这项提案，我们正在利用我们在T9 SS方面的专业知识，推动阻碍我们 了解肠道微生物群的分泌组。T9 SS机械的螺母和螺栓由 19种不同的蛋白质，但其结构和功能特性尚不清楚。今后，这 这些信息可以帮助我们控制肠道微生物群的T9 SS。T9 SS的核心是一个旋转电机， 为微生物的滑行运动提供动力。这种能动性使货物运输和形状的空间 微生物群落的组织。我们提出实验来填补有关机制的差距 T9 SS通过其使细菌运动和蛋白质分泌。两个重要的知识差距： T9 SS在肠道微生物群中的作用，以及（B）T9 SS的结构和功能将通过多个 使用遗传学、生物化学、生物物理学和鼠模型的分叉实验方法。 总之，我们的目标是获得T9 SS机制的机械理解，其在肠道中的分泌蛋白 微生物群及其对健康的影响。

SUMMARY Metabolites generated by the gut microbiota provide multiple health benefits. However, the mechanism via which the gut microbiota utilizes substrates from the host and generates beneficial products are not well understood. Additionally, information regarding the role of secretome i.e., a collection of extracellular proteins secreted by the gut microbiota on health is minimal. Currently, no model system for the study of Type 9 protein Secretion System (T9SS) of gut microbes exists. Our preliminary bioinformatics analysis suggests the presence of functional T9SS in several human gut isolates of the genus Bacteroides. We predict that the T9SS of gut Bacteroides secretes pectate lyase and other enzymes that breakdown dietary fibers and subsequently ferment them to short- chain fatty acids (SCFAs). Reduction of SCFAs results in metabolic disorders. We predict that the relatively understudied, yet important, human gut isolates that include Bacteroides intestinalis, Bacteroides nordii, Bacteroides fluxus, Prevotella copri, and Parabacteroides distasonsis secrete many proteins via T9SS. We are developing a genetically tractable species B. intestinalis as a model organism to fill knowledge gap regarding studies of T9SS secretome in the gut. We hypothesize that the T9SS secretome of the gut microbiota might create a common pool of oligosaccharides that enrich SCFA producing species. Additionally, we aim to find controllers of a putative Bacteroides sensory transduction network that senses the abundance of dietary fibers and regulates production of SCFA producing enzymes. Our preliminary data also predicts the secretion of immune-suppressive cysteine proteases by the T9SS of B. intestinalis. The proposed experiments to test our predictions and hypothesis will significantly enhance our understanding of interspecies cooperation, resource optimization, and immunomodulation by the gut microbiota. T9SS is a recently discovered protein export pathway of bacteria of the Gram-negative Fibrobacteres-Chlorobi-Bacteroidetes superphylum. Thus far, the model organisms for the study of T9SS are from the human oral microbiota, environmental isolates, and pathogens infecting aquatic animals. With this proposal, we are using our expertise with T9SS to push the barriers that impede our understanding of secretome of the gut microbiota. The nuts and bolts of T9SS machinery are composed of nineteen different proteins but their structure and functional properties are unclear. In future, this information can help us control T9SS of the gut microbiota. At the core of T9SS is a rotary motor that powers gliding motility of microbes. This motility enables cargo transportation and shapes the spatial organization of a microbial community. We propose experiments to fill the gap regarding the mechanism via which T9SS enables bacterial motility and protein secretion. Two important knowledge gaps: (a) the role of T9SS in the gut microbiota, and (b) the structure and function of T9SS will be filled via a multi- pronged experimental approach that uses genetics, biochemistry, biophysics, and murine models. Together, we aim to gain a mechanistic understanding of T9SS machinery, its secretome in the gut microbiota, and their impact on health.