Engineering of Complex Infectious Loci in Culture

在疾病期间，大量的细菌病原体生长在深层组织部位。病原菌生长 在这些位置导致免疫细胞的招募，试图清除入侵者，但这些细胞 通常是无效的，因为有毒的有机体阻止了清除过程。因此， 微生物建立了一个滩头阵地，在那里它可以建立持续感染或冒险传播 在整个宿主中。根据招募细胞的性质和发生的组织损伤，这些 感染的病灶称为脓肿、微小脓肿、肉芽肿或以下几种类型的组合 流程。对于许多生长在宿主细胞外的病原体，预测会形成不同的微集落 导致大量微生物间的交流和对周围宿主细胞的直接靶向 殖民地。传染病领域的一个首要问题是，由此产生的体系结构只能是 建立在动物感染模型中，不能在培养中维护或分析。这部作品 提出要克服这一绊脚石。 假结核耶尔森氏菌是一种可从肠道传播的肠道致病细菌。 进入区域淋巴结、肝脏和脾，建立被层层包围的微集落 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，但远端巨噬细胞有代偿性攻击，产生一氧化氮(NO)和 其抗微生物衍生品。微菌落外围的细菌使NO失活，保护他们的 集中定位的亲属免受有毒代谢物的影响。拟议的研究计划将利用 生物工程凝胶微滴系统准确地重建了这场战斗。目的是提出分析 Y.假结核杆菌与免疫细胞的相互作用，通过在凝胶内的微菌落中生长细菌 液滴，液滴周围附着着激活的巨噬细胞，在形态上模仿真实的 传染部位。使用荧光报告读数，外周和中央的转录图谱 将分析定位的细菌，并将其与在没有巨噬细胞的情况下生长的细菌进行比较 压力或在非结构化的环境中。该系统将用于识别细菌转录电路 这使得外围细菌能够维持生存能力，并保护位于中心的亲属免受攻击。它 还将确定细菌对组织中发现的聚集体生长的转录反应，以及 确定小细菌群落以前未描述的生理和应激反应 分泌的巨噬细胞产物。成功完成这些目标是实现以下长期目标的一部分 确定细菌间的相互作用如何确保建立具有感染力的生态位，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.