Contribution of innate immune cells in promoting antibiotic tolerance

项目摘要 尽管高水平的抗生素暴露，一些个别的细菌细胞存活多个疗程， 由于抗生素耐受性导致的抗生素治疗，这是一种与减少的 新陈代谢或生长减缓。目前还不清楚是什么驱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剂是否有助于 耐抗生素的细菌细胞。研究宿主衍生应激的影响，特别是反应性应激， 氧物种（ROS）和活性氮物种（RNS）对抗生素耐受性的影响，我们正在探索 人类病原体假结核耶尔森氏菌的小鼠模型。在宿主组织中，这种微生物 复制以形成细胞外细菌的克隆簇，其直接与 被一层巨噬细胞包裹着细菌亚群在外缘的 小菌落通过上调抗吞噬细胞III型分泌的表达来响应中性粒细胞接触 系统（T3SS）。这些与宿主细胞相关的细菌是更大的外周层的一部分， 并使源自远端巨噬细胞的可扩散的一氧化氮（NO）解毒。这个系统显然可以让我们 区分单个细菌细胞的空间位置，并确定宿主细胞相互作用，或 与它们产生的抗菌剂的相互作用，有助于形成抗生素耐受性细菌 人口。 我们假设，抗生素耐受性细菌出现在宿主组织内，由于先天性 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 本发明的目的是提供一种用于检测宿主组织中的单个细菌细胞内的多西环素（多西环素）暴露的方法，并且将使用该工具来：1）确定多西环素的浓度。 免疫细胞来源的ROS和RNS在促进抗生素耐受性中的作用，2）确定途径 由宿主组织内的多西环素耐受亚群利用。此外，我们还开发了 基于琼脂糖液滴的创新系统，用于模拟宿主组织内的生长， 分析，并提供更全面的了解细菌群落如何与免疫细胞相互作用 in vivo.宿主组织内细菌异质性的程度在很大程度上尚未被探索，这使其成为一个新的研究领域。 这是一个新的、未被充分研究的疾病过程，也是我实验室的研究重点。识别和 了解促进耐受性细菌亚群形成的线索， 新疗法的设计，需要针对所有亚群，以有效地消除 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.