AN INTERBACTERIAL ADAPTIVE IMMUNE SYSTEM ENCODED BY BACTEROIDALES

项目摘要/摘要 细菌群落的组成对它们的功能至关重要。激烈的竞争互动 细菌之间可以通过改变生产过程中的组装或稳定性来影响群落组成 抗菌分子。人类胃肠道就是这样一个充满竞争互动的生态系统。 微生物区系，其中含有丰富的拟杆菌目细菌。这些细菌编码了 VI型分泌系统(T6SS)，一种接触依赖的毒素传递途径，被证明介导了有效的相互作用。 物种竞争。编码T6SS的细菌也编码与T6SS结合的免疫蛋白 特别是灭活毒素。我发现类杆菌的基因组具有“孤儿免疫力”。 基因在没有T6SS或相应毒素的情况下。这些多重免疫阵列被发现与 带有类xerD(PAX)重组酶基因，这表明了一种活跃的招募机制。我假设 PAX簇中的基因代表了过去几次细菌间直接拮抗的选择压力。 因此，对PAX簇的功能、活性和生物发生的表征为 确定体内细菌的直接相互作用并了解其生态后果。 这项建议旨在增强我在细胞生物学、进化生物学和 遗传学与无菌小鼠实验的新训练，以研究这种新的适应机制 T6SS介导的类杆菌科细菌间拮抗作用及其对肠道微生物群的影响 装配和动力学。在目标1中，我建议通过以下方式描述PAX簇中基因的功能 在大肠杆菌中的体外表达实验和体内外的生长竞争实验 促进接触依赖的细菌间拮抗的条件。在目标2中，我将测试假设 PAX重组酶通过酪氨酸重组酶介导PAX簇的基因获取和切除 以类似于整合子的方式进行活性。在目标3中，我将把我对PAX集群的描述扩展到他们的 作为一种新的工具，通过对暴露后的新基因插入进行测序来破译细菌间的相互作用 一株含有PAX的菌株对体内不同细菌群落的影响。我将和林恩·海贾尔博士一起在 华盛顿大学灵生动物核心设施将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区系中的作用。耶鲁大学的安德鲁·古德曼博士将与 并提供建议和智力支持。提议的实验将为我的 独立研究计划，其中生物信息学、细菌遗传学和活体实验将 结合在一起，深入了解肠道微生物区系内的直接相互作用以及这些 对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.