Selective Pressures Exerted by the Innate Immune System on Commensals in the Zebrafish Gut

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 DESCRIPTION (provided by applicant): The significance of the impacts of the intestinal microbiota on human health and disease cannot be overstated. It is undeniable that these microbes are integral to the healthy function of the hosts they colonize. They provide a plethora of beneficial functions including ones impacting nutrition, development of the immune system, and protection from pathogens. However, many diseases are associated with changes in the composition, structure, or function of these communities, including obesity, inflammatory bowel disease, diabetes, colon cancer, and Clostridium difficile-associated diarrhea. Unfortunately, there is not currently a deep enough understanding of the host-microbe and microbe-microbe interactions that govern community structure and function to fully explicate disease development. More importantly, current knowledge is not sufficient to facilitate the development of therapies aimed at restoring healthy function of dysbiotic communities in order to ameliorate disease. Therefore, the overarching goal of this proposal is to expand our knowledge of how the host impacts community assembly and dynamics, and inform us about adaptive strategies of commensal organisms. More specifically, I propose to determine the selective pressures exerted by the host innate immune response during bacterial adaptation to the vertebrate gut. Herein I will test the hypothesis that there is an optimal immunogenicity of commensal bacteria such that adaptation in the presence of a functional innate immune system results in a shift towards this optimum level. To accomplish this, I will use the gnotobiotc zebrafish model to investigate the selective pressures exerted by the host innate immune system during adaption of a natural gut isolate. This is a simple yet powerful model system because it specifically allows for observation of interactions between the innate immune response and the selected bacterium. Two different approaches will be used to generate variant strains of Aeromonas ZOR0001 (Aer01) that have increased fitness in the zebrafish gut. In AIM 1 a forward genetic screen (Tn-seq) is used to assay the fitness contributions of the entire genetic repertoire of Aer01 to specifically identify mutations that result in increased fitness in vivo. In AIM 2, an adaptive evolution approach is used to compare genetic adaptations conferring increased fitness when evolved in WT versus immunodeficient hosts. Gain-of-fitness mutations identified by each approach will be constructed in the WT ancestral genetic background and their colonization fitness and capacity to stimulate the host immune response assayed. These two approaches allow for an unbiased identification of the genetic determinants of adaptation of Aer01, maximizing the potential to study multiple avenues of bacterial adaptation and the selective pressures exerted by the host innate immune system. The impact of this work is significant because it will provide insights into how the the innate immune system shapes commensal fitness in the vertebrate gut, which is knowledge that can be exploited to precisely manipulate host-associated microbiota and moves us closer to advanced biotherapeutic treatments for dysbiotic diseases.