AN INTERBACTERIAL ADAPTIVE IMMUNE SYSTEM ENCODED BY BACTEROIDALES

项目概要/摘要 细菌群落的组成对其功能至关重要。激烈的竞争互动 细菌之间可以通过改变组装或稳定性来影响群落组成， 抗细菌分子。其中一个充满竞争性相互作用的生态系统是人类的胃肠道 微生物群，其中含有大量来自拟杆菌目的细菌。这些细菌编码 VI型分泌系统（T6SS）是一种接触依赖性毒素传递途径，可介导有效的细胞间相互作用。 物种竞争编码T6SS的细菌还编码免疫蛋白，其结合并 尤其是肉毒毒素我发现类杆菌基因组具有"孤儿免疫"阵列， 在不存在T6SS或相应毒素的情况下，这些多免疫阵列被发现与 与xerD样（PAX）重组酶基因，这表明一个积极的招聘机制。我假设 PAX簇内的基因代表了来自过去直接细菌间拮抗作用的选择压力。 因此，PAX簇的功能、活性和生物发生的表征为研究PAX簇的功能、活性和生物发生提供了途径。 鉴定体内直接细菌相互作用并了解其生态后果。 这个建议的目的是增加我在细胞生物学，进化生物学， 遗传学与无菌小鼠实验的新培训，以研究这种新的适应机制 拟杆菌目内T6SS介导的细菌间拮抗作用，并了解其对肠道微生物组的影响 装配和动力学。在目标1中，我建议通过以下方式来表征PAX簇内基因的功能： 在E.在体外和体内的生长竞争实验中， 促进接触依赖性细菌间拮抗作用的条件。在目标2中，我将检验以下假设： PAX重组酶介导通过酪氨酸重组酶从PAX簇获得和切除基因 以类似于整合子的方式活动。在目标3中，我将把我对PAX集群的描述扩展到它们的 作为一种新的工具，通过对暴露后插入的新基因进行测序来破译细菌间的相互作用 含PAX的菌株对体内不同细菌群落的影响。我会和林恩·哈贾尔博士一起训练 华盛顿大学的Gnotobiotic动物核心设施，以获得使用无菌小鼠进行研究的经验 PAX簇在体内肠道微生物群中的作用。耶鲁大学的安德鲁·古德曼博士 并提供建议和智力支持。这些实验将为我的研究奠定基础。 独立的研究计划，其中生物信息学，细菌遗传学和体内实验将 结合起来，深入了解肠道微生物群内的直接相互作用以及这些相互作用的影响。 人类健康和疾病的相互作用。

PROJECT SUMMARY / ABSTRACT The composition of bacterial communities is paramount to their function. Intense competitive interactions between bacteria can influence community composition by altering assembly or stability through the production of anti-bacterial molecules. One such ecosystem rife with competitive interactions is the human gastrointestinal microbiota, which harbors an abundance of bacteria from the order Bacteroidales. These bacteria encode the type VI secretion system (T6SS), a contact-dependent toxin delivery pathway shown to mediate potent inter- species competition. Bacteria that encode the T6SS also encode immunity proteins which bind to and specifically inactivate toxins. I have found that Bacteroidales genomes possess arrays of “orphan immunity” genes in the absence of the T6SS or corresponding toxin. These polyimmunity arrays are found associated with xerD-like (PAX) recombinase genes that suggest an active mechanism of recruitment. I hypothesize that genes within PAX clusters represent selective pressure from past episodes of direct interbacterial antagonism. Therefore, characterization of the function, activity, and biogenesis of PAX clusters offers an avenue for the identification of direct bacterial interactions in vivo and an understanding of their ecological consequences. This proposal aims to augment my interdisciplinary background in cell biology, evolutionary biology, and genetics with new training in germfree mouse experimentation to investigate this new mechanism of adaptation to T6SS-mediated interbacterial antagonism within Bacteroidales and understand its impact on gut microbiome assembly and dynamics. In Aim 1, I propose to characterize the function of genes within PAX clusters through in vitro expression experiments in E. coli and growth competition experiments in vitro and in vivo under conditions that promote contact-dependent interbacterial antagonism. In Aim 2, I will test the hypothesis that the PAX recombinase mediates gene acquisition and excision from PAX clusters via tyrosine recombinase activity in a manner analogous to integrons. In Aim 3, I will extend my characterization of PAX clusters to their use as a new tool for deciphering interbacterial interactions by sequencing new gene insertions after exposure of a PAX-containing strain to different bacterial communities in vivo. I will train with Dr. Lynn Hajjar at the University of Washington Gnotobiotic Animal Core facility to gain experience in using germfree mice to study the role of PAX clusters in the gut microbiota in vivo. Dr. Andrew Goodman at Yale University will collaborate and provide advice and intellectual support. The proposed experiments will lay the foundation for my independent research program, in which bioinformatics, bacterial genetics, and in vivo experimentation will be combined to yield insight into direct interactions within the gut microbiota and the implications of these interactions for human health and disease.