Contribution of innate immune cells in promoting antibiotic tolerance

项目总结 尽管抗生素暴露水平很高，但一些单个细菌细胞仍能存活多个疗程 由于抗生素耐受性而进行的抗生素治疗，这是一种与减少 新陈代谢或生长减慢。目前尚不清楚是什么推动了宿主组织内耐受细胞的形成， 以及宿主免疫系统产生的抗菌素是否有助于形成 耐抗生素细菌细胞。调查宿主衍生的压力，特别是反应性压力的影响 氧物种(ROS)和活性氮物种(RNS)，关于抗生素耐药性，我们正在探索生长 人类病原体假结核耶尔森氏菌在小鼠模型中的研究。在宿主组织内，这种微生物 复制形成细胞外细菌的克隆性集群，这些克隆集群直接与一层中性粒细胞对接， 反过来又被一层巨噬细胞所包裹。细菌外边缘的细菌亚群 微集落通过上调抗吞噬细胞III型分泌物的表达来响应中性粒细胞接触 系统(T3SS)。这些宿主细胞相关细菌是一个更大的外围层的一部分，它对 并解毒源自远端巨噬细胞的扩散性一氧化氮(NO)。这个系统清楚地允许我们 区分单个细菌细胞的空间位置，并确定宿主细胞是否相互作用，或 与它们产生的抗菌剂相互作用，有助于形成耐药细菌 人口。 我们假设，耐抗生素细菌由于先天原因出现在宿主组织中。 免疫细胞来源的ROS和RNS。我们最近开发了一种新的报告系统来检测抗生素 (多西环素)暴露在宿主组织中的单个细菌细胞内，并将使用该工具：1)定义 免疫细胞来源的ROS和RNS在促进抗生素耐药中的作用2)决定途径 被寄主组织内耐多西环素的亚群利用。此外，我们还开发了一种 创新的基于琼脂糖滴的系统来模拟宿主组织内的生长，这将使动力学 分析，并提供对细菌群落如何与免疫细胞相互作用的更全面的理解 在活体内。宿主组织内细菌异质性的程度在很大程度上还没有被探索，这使这成为一种 疾病过程的新的、未被充分研究的方面，也是我的实验室研究的重点。识别和 了解促进耐受细菌亚群形成的线索很重要 对新疗法设计的影响，需要针对所有亚群有效地消除 宿主组织内的细菌。

PROJECT SUMMARY Despite high levels of antibiotic exposure, some individual bacterial cells survive multiple courses of antibiotic treatment due to antibiotic tolerance, which is a transient phenotypic change associated with reduced metabolism or slowed growth. It remains unclear what drives the formation of tolerant cells within host tissues, and whether the antimicrobials generated by the host immune system may contribute to the formation of antibiotic tolerant bacterial cells. To investigate the impact of host-derived stresses, specifically reactive oxygen species (ROS) and reactive nitrogen species (RNS), on antibiotic tolerance, we are probing the growth of the human pathogen, Yersinia pseudotuberculosis, in a mouse model. Within host tissues, this microbe replicates to form clonal clusters of extracellular bacteria that directly interface with a layer of neutrophils that are, in turn, enveloped by a layer of macrophages. The bacterial subpopulation at the outer edge of the microcolony responds to neutrophil contact by upregulating expression of the anti-phagocytic, type III secretion system (T3SS). These host cell-associated bacteria are part of a larger peripheral layer, which responds to and detoxifies diffusible nitric oxide (NO) originating from distal macrophages. This system clearly allows us to distinguish the spatial location of individual bacterial cells, and to determine if host cell interactions, or interactions with the antimicrobials they produce, are contributing to the formation of antibiotic tolerant bacterial populations. We hypothesize that antibiotic tolerant bacteria emerge within host tissues due to innate immune cell-derived ROS and RNS. We have recently developed a novel reporter system to detect antibiotic (doxycycline) exposure within individual bacterial cells in host tissues, and will use this tool to: 1) define the roles of immune cell-derived ROS and RNS in promoting of antibiotic tolerance, 2) determine the pathways utilized by doxycycline-tolerant subpopulations within host tissues. In addition, we have also developed an innovative agarose droplet-based system to model growth within host tissues, which will enable kinetic analyses, and provide a more thorough understanding of how bacterial communities interact with immune cells in vivo. The extent of bacterial heterogeneity within host tissues has been largely unexplored, making this a novel, understudied aspect of the disease process, and the focus of research in my lab. Identifying and understanding the cues that promote the formation of tolerant bacterial subpopulations has important implications for the design of novel therapeutics, which need to target all subpopulations to effectively eliminate bacteria within host tissues.