Engineering of Complex Infectious Loci in Culture

在疾病期间，大量细菌病原体在深部组织部位内生长。病原体生长 在这些网站的结果是招募免疫细胞，试图清除入侵者，但这些细胞 通常是无效的，因为有毒的生物体阻碍了清除过程。因此， 微生物建立了一个滩头阵地，在那里它可以建立一个持续的感染或冒险传播 整个主机。根据招募细胞的性质和发生的组织损伤， 感染病灶被称为脓肿、微脓肿、肉芽肿或其组合。 流程.对于许多在宿主细胞外生长的病原体，预测会形成不同的小菌落， 导致相当多的微生物间通讯和直接靶向周围的宿主细胞， 殖民地传染病领域的一个首要问题是，最终的架构只能是 在动物感染模型中建立，并且不能在培养物中维持或分析。这项工作 他试图克服这个绊脚石。 假结核耶尔森氏菌是一种肠道致病菌，可以从肠道传播 进入区域淋巴结，肝脏和脾脏，建立由多层 嗜中性粒细胞、巨噬细胞和炎性单核细胞。细菌直接在附近灭活 中性粒细胞，但有一个补偿攻击远端巨噬细胞产生一氧化氮（NO）， 其抗菌衍生物。小菌落周围的细菌会分泌NO，保护它们的 暴露于有毒代谢物的中枢神经系统。拟议的研究计划将利用一个 生物工程凝胶微滴系统来精确地重建这场战斗。目的是分析 y.假结核与免疫细胞的相互作用，通过在凝胶内的小菌落中生长细菌 微滴，在微滴周围粘附有活化的巨噬细胞，形态学上模仿真正的 感染部位使用荧光报告读出，外周和中枢的转录谱 将分析定位的细菌，并与在没有巨噬细胞的情况下生长的细菌进行比较。 压力或非结构化环境中。该系统将用于识别细菌转录回路 使周围的细菌保持活力，并保护位于中心的亲属免受攻击。它 还将鉴定细菌对组织中发现的聚集体生长的转录反应，以及 鉴定小细菌群落的先前未表征的生理和应激反应， 分泌巨噬细胞产物。成功完成目标是长期目标的一部分， 确定细菌间的相互作用如何确保建立感染性生态位，并评估 抗微生物免疫细胞如何与病原体合作促进疾病。

A wide swath of bacterial pathogens grow within deep tissue sites during disease. Pathogen growth in these sites results in the recruitment of immune cells that attempt to clear of the invader, but these cells are often ineffective because the virulent organism blocks the clearing process. As a consequence, the microorganism sets up a beachhead where it can either establish a persistent infection or venture to spread throughout the host. Depending on the nature of the recruited cells and tissue damage that occurs, these foci of infection are referred to as abscesses, microabscesses, granulomas, or some combination of processes. For many pathogens that grow outside of host cells, distinct microcolonies are formed, predicted to result in considerable intermicrobial communication and direct targeting of host cells surrounding the colony. An overriding problem in the infectious disease field is that the resulting architecture can only be established in animal infection models and cannot be maintained or analyzed in culture. This work proposes to overcome this stumbling block. Yersinia pseudotuberculosis is an enteropathogenic bacterium that can spread from the intestine into regional lymph nodes, the liver and the spleen, establishing microcolonies surrounded by layers of neutrophils, macrophages and inflammatory monocytes. The bacterium directly inactivates nearby neutrophils, but there is a compensating attack by distal macrophages that generates nitric oxide (NO) and its antimicrobial derivatives. Bacteria on the periphery of the microcolony inactivate NO, protecting their centrally localized kin from exposure to toxic metabolites. The proposed Research Plan will exploit a bioengineered gel microdroplet system to accurately reconstruct this battle. The Aims propose to analyze Y. pseudotuberculosis interaction with immune cells, by growing bacteria in microcolonies within the gel droplets, surrounding the droplets with adherent activated macrophages, morphologically mimicking a true infectious site. Using a fluorescent reporter readout, the transcriptional profiles of peripheral and centrally located bacteria will be analyzed, and compared to bacteria growing either in the absence of macrophage stress or in a nonstructured environment. The system will be used to identify bacterial transcriptional circuits that allow peripheral bacteria to maintain viability, and which protect the centrally located kin from attack. It will also identify the bacterial transcriptional response to growth in aggregates found in tissues, as well as identify previously uncharacterized physiological and stress responses of the small bacterial community to secreted macrophage products. Successful completion of the Aims is part of the long-term goal of determining how inter-bacterial interactions ensure the establishment of an infectious niche, and to evaluate how anti-microbial immune cells collaborate with pathogens to promote disease.