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

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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.