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

 描述(由申请人提供)：肠道微生物区系对人类健康和疾病的影响的重要性怎么强调都不为过。不可否认，这些微生物对于它们定居的宿主的健康功能是不可或缺的。它们提供了过多的有益功能，包括影响营养、免疫系统发育和保护免受病原体侵害的功能。然而，许多疾病与这些群落的组成、结构或功能的变化有关，包括肥胖、炎症性肠病、糖尿病、结肠癌和艰难梭菌相关性腹泻。不幸的是，目前对控制群落结构和功能的宿主-微生物和微生物-微生物相互作用的了解还不够深入，无法充分解释疾病的发展。更重要的是，目前的知识不足以促进旨在恢复非生物群落的健康功能以改善疾病的治疗方法的开发。因此，这项建议的首要目标是扩大我们对宿主如何影响群落聚集和动态的知识，并告知我们共生生物体的适应策略。更具体地说，我建议确定在细菌适应脊椎动物肠道期间宿主先天免疫反应施加的选择性压力。在这里，我将测试这样的假设，即共生细菌存在最佳的免疫原性，从而在功能正常的天然免疫系统存在的情况下进行适应，导致向该最佳水平的转变。为了做到这一点，我将使用Gnotobiotc斑马鱼模型来研究宿主先天免疫系统在适应自然肠道分离株期间施加的选择性压力。这是一个简单但功能强大的模型系统，因为它特别允许 用于观察先天免疫反应和选定细菌之间的相互作用。将使用两种不同的方法来产生不同的气单胞菌ZOR0001(Aer01)菌株，这些菌株增加了斑马鱼肠道的适应性。在AIM 1中，正向遗传筛查(TN-seq)被用来分析Aer01的整个遗传库的适合度贡献，以专门识别导致体内适合度提高的突变。在AIM 2中，一种适应性进化方法被用来比较在WT中进化时赋予更高适应性的遗传适应与免疫缺陷宿主的遗传适应。每种方法确定的适应度获得突变都将构建在WT祖先的遗传背景中，并对其定植、适应度和刺激宿主免疫反应的能力进行分析。这两种方法允许对Aer01适应的遗传决定因素进行公正的鉴定，最大限度地提高研究细菌适应的多种途径和宿主先天性免疫系统施加的选择压力的潜力。这项工作的影响是重大的，因为它将提供对先天免疫系统如何塑造脊椎动物肠道共生健康的洞察，这是可以利用的知识，可以精确地操纵与宿主相关的微生物区系，并使我们更接近于针对厌生菌疾病的先进生物治疗方法。

 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.