Identifying the pathways associated with bacterial antibiotic persistence within host tissues

项目总结 抗生素敏感细菌的残留亚群可保留在宿主组织内 抗生素治疗。这些幸存的细菌被称为持久细胞，它们对高剂量的药物有短暂的耐受性 抗生素含量高，治疗后可引起严重的复发感染。关键的是，目前的治疗 战略不以持之以恒的人为目标。为了完全根除所有细菌细胞，治疗时间延长，增加 患者和临床成本。长期接触抗生素会增加抗生素耐药性，进一步强调 需要提高治疗效果。改进的治疗策略将同时针对所有 细菌种群的成员，包括顽固者。然而，持久化的研究主要是在 在宿主组织内的培养和相关的伴胞细胞特异性药物靶点在很大程度上是未知的。细菌 在宿主组织中的表现非常不同，在那里营养限制和抗微生物宿主防御激活了很强的 细菌病原体的应激反应途径。我们预测持久者利用独特的，潜在的新奇， 在宿主环境中的生存策略。 为了研究细菌在宿主组织中的抗生素持久性，我们建立了一种小鼠模型。 多西环素治疗假结核耶尔森菌脾深部组织感染。强力霉素是一种有效的 治疗人类耶尔森氏菌感染，但需要7天连续治疗，已纳入 进入我们的老鼠模型。在抗生素治疗之前，假结核杆菌复制形成成簇的 直接与中性粒细胞接触的胞外细菌，中性粒细胞又被一层 单核细胞。在多西环素治疗的最初4小时，我们观察到活细菌显著减少。 数字，这与中性粒细胞渗入脾的浪潮有关。然而，一种残留的细菌 在7天的治疗过程中，亚群(~10%)仍留在脾内。细菌细胞恢复生长， 当抗生素浓度下降时会导致致命性，将这些细胞定义为持久性细胞。 我们假设，与中性粒细胞和单核细胞的相互作用容易使持久者 在抗生素治疗中幸存下来，长期接触抗生素会促进额外的转录 以及宿存细胞内的基因变化。利用我们的荧光报告系统检测活的， 对于小鼠脾内暴露于多西环素的细菌，我们将：1)鉴定转录、蛋白质组和 抗生素治疗的小鼠体内存活细菌特有的基因变化，2)确定特定的 细菌靶标对抗生素在宿主中的持久性至关重要，3)决定单核细胞或中性粒细胞 相互作用促进了抗生素的持久性。我们假设激活的中性粒细胞最初会减少细菌 负担，我们将确定逃避中性粒细胞介导的杀伤是否促进宿存细胞的存活。 识别宿主组织中的宿存细胞生存策略将提供关键信息，以促进 这将有助于开发更有效的针对细菌感染的治疗策略。

PROJECT SUMMARY Residual subpopulations of antibiotic-susceptible bacteria can remain within host tissues following antibiotic treatment. These surviving bacteria are called persister cells, which are transiently tolerant to high levels of antibiotic, and can cause serious relapsing infection after treatment. Critically, current treatment strategies do not target persisters. To fully eradicate all bacterial cells, treatments are prolonged, increasing patient and clinical costs. Prolonged antibiotic exposure can promote antibiotic resistance, further emphasizing the need to improve treatment efficacy. Improved treatment strategies would simultaneously target all members of the bacterial population, including persisters. However, persisters have been primarily studied in culture, and relevant persister cell-specific drug targets within host tissues are largely undefined. Bacteria behave very differently in host tissues, where nutrient limitation and antimicrobial host defenses activate strong stress response pathways in bacterial pathogens. We predict persisters utilize distinct, potentially novel, survival strategies within the host environment. To study bacterial antibiotic persistence within host tissues, we established a mouse model of doxycycline treatment of Yersinia pseudotuberculosis splenic deep tissue infection. Doxycycline is an effective treatment for human Yersinia infection, but requires 7 days continuous treatment, which has been incorporated into our mouse model. Prior to antibiotic treatment, Y. pseudotuberculosis replicate to form clusters of extracellular bacteria that directly interface with a layer of neutrophils that are, in turn, enveloped by a layer of monocytes. In the initial 4h of doxycycline treatment, we observe a significant decrease in viable bacterial numbers, which correlates with a wave of neutrophil infiltration into the spleen. However, a residual bacterial subpopulation (~10%) remain in the spleen throughout the 7-day treatment. Bacterial cells resume growth and cause lethality when antibiotic concentrations wane, defining these cells as persisters. We hypothesize that interactions with neutrophils and monocytes predispose persisters to survive antibiotic treatment, and prolonged antibiotic exposure promotes additional transcriptional and genetic changes within persister cells. Utilizing our fluorescent reporter system to detect viable, doxycycline-exposed bacteria within the mouse spleen, we will: 1) identify the transcriptional, proteomic, and genetic changes specific to surviving bacteria within antibiotic-treated mice, 2) determine whether specific bacterial targets are critical for antibiotic persistence in the host, and 3) determine if monocyte or neutrophil interactions promote antibiotic persistence. We hypothesize activated neutrophils initially reduce the bacterial burden, and we will determine if evasion of neutrophil-mediated killing promotes persister cell survival. Identifying persister cell survival strategies within host tissues will provide critical information to advance the field and enable the development of more efficacious therapeutic strategies against bacterial infections.