AN INTERBACTERIAL ADAPTIVE IMMUNE SYSTEM ENCODED BY BACTEROIDALES

拟杆菌编码的细菌间适应性免疫系统

• 批准号: 10045595
• 负责人: Benjamin Ross
• 金额: $ 24.81万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-10 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045595
• 关键词: Adaptive Immune System; Amino Acids; Animals; Anti-Bacterial Agents; Antibiotic Resistance; Bacteria; Bacteroides; Bacteroides fragilis; Binding; Biochemistry; Biogenesis; Bioinformatics; Biological Assay; Biology; Biosensor; Cells; Cellular biology; Communities; Core Facility; DNA cassette; Development; Disease; Ecosystem; Engineered Probiotics; Engraftment; Escherichia coli; Excision; Exhibits; Foundations; Frequencies; Future; Gene Frequency; Generations; Genes; Genetic; Genome; Gnotobiotic; Growth; Health; Human; Immunity; In Situ; In Vitro; Integrase; Integrons; Knowledge; Mediating; Methods; Molecular; Mus; Mutation; Nature; Orphan; Pathway interactions; Probiotics; Production; Proteins; Proteobacteria; Research; Role; Sampling; Specificity; Suggestion; System; Testing; Time; Toxin; Training; Tyrosine; Universities; Washington; Work; bacterial community; bacterial genetics; design; experience; experimental study; fitness; gene function; gut bacteria; gut microbiome; gut microbiota; improved; in vitro Assay; in vivo; insight; member; microbiota; pressure; prevent; probiotic therapy; programs; recombinase; recruit; residence; tool

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.

项目摘要 /摘要 细菌群落的组成对于它们的功能至关重要。激烈的竞争互动 细菌之间可以通过生产改变组装或稳定性来影响社区组成 抗细菌分子。这样的生态系统与竞争性互动是人类胃肠道 微生物群，它可以从菌群中抽象细菌的抽象。这些细菌编码 VI型分泌系统（T6SS），这是一种接触依赖性的毒素递送途径 物种竞争。编码T6SS的细菌还编码结合和结合的免疫蛋白 我发现细菌基因组具有“孤儿免疫”阵列 在没有T6SS或相应毒素的情况下基因。这些综合阵列是相关的 与XERD样（PAX）重组酶基因一起，提示有效的募集机理。我假设这一点 PAX簇中的基因代表了直接细菌间拮抗的过去发作中的选择性压力。 因此，PAX簇的功能，活性和生物发生的表征为该途径提供了途径 鉴定体内细菌相互作用及其生态后果的理解。 该建议旨在增加我在细胞生物学，进化生物学和 通过新训练的遗传学在无用的小鼠实验中进行了研究，以研究这种适应的新机制 到T6SS介导的细菌间拮抗细菌性拮抗作用，并了解其对肠道微生物组的影响 组装和动态。在AIM 1中，我建议通过通过PAX簇中的基因功能来表征 大肠杆菌和生长竞争实验的体外表达实验在体外和体内实验 促进接触依赖性的细菌间拮抗作用的条件。在AIM 2中，我将检验以下假设 PAX重组酶通过酪氨酸重组酶从PAX簇中介导了基因的获取和切除 活动的活动方式类似于整数。在AIM 3中，我将将PAX簇的特征扩展到他们 通过对新的基因插入进行测序，用作解解细胞间相互作用的新工具 在体内对不同细菌群落的含PAX菌株的菌株。我将与Lynn Hajjar博士一起训练 华盛顿大学gnotobiotic Animal核心设施，以获取使用无用小鼠学习的经验 PAX簇在体内肠道微生物群中的作用。耶鲁大学的安德鲁·古德曼博士将合作 并提供建议和智力支持。提出的实验将为我的基础奠定基础 独立研究计划，其中生物信息学，细菌遗传学和体内实验将是 合并以洞悉肠道菌群中的直接相互作用及其含义 人类健康和疾病的相互作用。