Identifying the pathways associated with bacterial antibiotic persistence within host tissues

确定与宿主组织内细菌抗生素持久性相关的途径

基本信息

批准号：
10638788
负责人：
Kim Davis
金额：
$ 59.06万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-06 至 2027-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10638788
关键词：
Aftercare Antibiotic Resistance Antibiotic Therapy Antibiotic susceptibility Antibiotics Bacteria Bacterial Genes Bacterial Infections Binding Biological Assay Cell Separation Cell Survival Cells Clinical Critical Pathways Development Doxycycline Drug Metabolic Detoxication Drug Targeting Ensure Environment Gene Expression Gene Expression Profile Genes Genetic Genetic Engineering Genetic Transcription Growth Heterogeneity Host Defense Human Immune response In Vitro Infection Investigation Knowledge Mass Spectrum Analysis Mediating Modeling Mus Mutation Neutrophil Infiltration Nitric Oxide Nutrient Pasteurella pseudotuberculosis Pathway interactions Patients Phagocytes Pharmaceutical Preparations Population Positioning Attribute Predisposition Proteome Proteomics Public Health RNA Reactive Oxygen Species Recurrence Relapse Reporter Residual state Role Spleen Stress Survivors System Systemic infection Testing Therapeutic Tissues Transgenic Mice Treatment Efficacy Treatment Failure Validation Yersinia Yersinia infections antimicrobial biological adaptation to stress cell motility cost drug discovery effective therapy extracellular human disease improved in vivo interest member monocyte mouse model neutrophil novel pathogenic bacteria screening stressor tool transcriptome sequencing treatment duration treatment strategy

项目摘要

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.

项目概要抗生素敏感细菌的残留亚群可以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，它们可以暂时耐受高浓度抗生素水平，并可能在治疗后引起严重的复发感染。关键的是，目前的治疗策略不针对持久者。为了完全消灭所有细菌细胞，治疗时间会延长，增加患者和临床费用。长期接触抗生素会促进抗生素耐药性，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群体的成员，包括持续存在者。然而，持久性的研究主要集中在培养物和宿主组织内相关的持久细胞特异性药物靶点很大程度上是不确定的。细菌在宿主组织中的表现非常不同，营养限制和抗菌宿主防御激活强烈细菌病原体的应激反应途径。我们预测坚持者会利用独特的、潜在新颖的、宿主环境中的生存策略。为了研究细菌抗生素在宿主组织内的持久性，我们建立了小鼠模型强力霉素治疗假结核耶尔森菌脾脏深部组织感染。强力霉素是一种有效的用于人类耶尔森菌感染的治疗，但需要连续治疗7天，已纳入进入我们的小鼠模型。在抗生素治疗之前，假结核杆菌复制形成簇细胞外细菌直接与一层中性粒细胞接触，而中性粒细胞又被一层单核细胞。在强力霉素治疗的最初 4 小时内，我们观察到活细菌显着减少数字，这与中性粒细胞浸润到脾脏的波相关。但残留细菌在整个 7 天的治疗过程中，亚群 (~10%) 保留在脾脏中。细菌细胞恢复生长并当抗生素浓度减弱时会导致致命，将这些细胞定义为持续细胞。我们假设与中性粒细胞和单核细胞的相互作用使持久者倾向于在抗生素治疗中存活下来，并且长期接触抗生素会促进额外的转录以及持续细胞内的遗传变化。利用我们的荧光报告系统来检测活的、对于小鼠脾脏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小鼠体内存活细菌特异的遗传变化，2) 确定是否具有特异性细菌靶标对于抗生素在宿主体内的持久性至关重要，并且 3) 确定是单核细胞还是中性粒细胞相互作用促进抗生素的持久性。我们假设活化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会促进持久细胞的存活。确定宿主组织内的持久细胞生存策略将为推进领域并能够开发更有效的针对细菌感染的治疗策略。