Determining the mechanisms that cause persistent MRSA bloodstream infection by tracking in-host evolution

通过追踪宿主进化来确定导致持续性 MRSA 血流感染的机制

基本信息

批准号：
10352493
负责人：
MATTHEW J CULYBA
金额：
$ 23.79万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10352493
关键词：
Address Antibiotic Resistance Antibiotics Bacteremia Biochemical Pathway Biological Biological Assay Biology Blood Blood specimen Cells Citric Acid Cycle Clinical Complex Data Defect Drug Interactions Drug Modelings Drug Tolerance Environment Evolution Exposure to Future Genes Genetic Genetic Screening Genetic screening method Genome Genotype Growth Human Immune Immune Tolerance Infection Kinetics Link Measures Metabolic Pathway Metabolism Methods Modeling Molecular Mutate Mutation NADH Pathogenesis Pathway interactions Patient-Focused Outcomes Patients Phagocytes Phagosomes Pharmacotherapy Phenotype Play Population Sizes Proteins Reactive Oxygen Species Resistance Respiratory Burst Role Sepsis Single Nucleotide Polymorphism Staphylococcus aureus Staphylococcus aureus infection Stress Testing Time analytical tool antibiotic tolerance bacterial genome sequencing chronic infection design effective therapy experimental study fitness genomic locus immune clearance in vivo innovation insight macrophage methicillin resistant Staphylococcus aureus microbial mortality mutant novel strategies novel therapeutics pathogen pressure respiratory response screening trait

项目摘要

Project Summary/Abstract Bloodstream infection (BSI) due to methicillin-resistant Staphylococcus aureus (MRSA) carries ~20% mortality [1, 2]. MSRA displays tolerance to antibiotic killing [11], has a propensity to cause persistent BSI (pBSI) [3], and the duration of pBSI predicts mortality [2, 12-14]. MRSA rarely acquires frank antibiotic resistance during pBSI [3], highlighting tolerance as an important cause of poor patient outcomes. Antibiotic tolerance is a complex trait, which is distinct from resistance, and there are significant barriers to its study in vivo that have hampered progress on understanding the most important mechanisms in clinical settings. In this proposal, we advance an innovative genetic screening approach to overcome these barriers. Episodes of MRSA-pBSI that occur in different patients can be viewed as biological replicates of a naturally occurring experiment in microbial evolution. As bacterial population sizes collapse due to selection from antibiotic and immune pressure, tolerant mutants will become enriched. Mutations that arise independently in the same genetic loci at a rate that exceed chance alone, are biologically meaningful. In preliminary studies, using this “genotype-first” approach, we found evidence for in-host evolution of two genetic pathways strongly linked to antibiotic tolerance. Our central hypothesis is that mutants that arise during the treatment of MRSA-pBSI contain genetic adaptations for antibiotic and immune tolerance. We propose to identify and characterize these pathways through the following specific aims: Aim 1. Determine which genes evolving during MRSA-pBSI are associated with antibiotic tolerance and energy imbalance. Tolerance mechanisms often involve perturbations in metabolism, causing a ‘low energy’ state that leads to slow turnover of antibiotic targets [4, 5]. Such perturbations could arise through a variety of redundant pathways that converge on energy dysregulation. Alternatively, in vivo conditions may stress specific nodes in the cell’s metabolic networks and some pathways may dominate the antibiotic tolerance landscape. We will utilize our genetic screening approach to identify antibiotic tolerant mutants and determine which genes evolving during MRSA-pBSI are associated with antibiotic tolerance and energy imbalance. Aim 2. Determine if TCA cycle defects evolve during MRSA-pBSI due to a host-pathogen-drug interaction. Antibiotic tolerance can be induced by harsh environments and a leading model is that host immune pressure in the form of phagocyte-derived reactive oxygen species induces S. aureus into a drug-tolerant state by reducing flux through the tricarboxylic acid (TCA) cycle [6]. In our preliminary data, we identified TCA cycle mutants that evolved during MRSA-pBSI. If these mutants evolved by outcompeting wild-type MRSA in phagosomes, they will display a fitness advantage in this setting. We will utilize these mutants to test this model directly, by performing experiments where we infect phagocytes and measure survival and drug tolerance. This study is important for understanding the fundamental biology of persistent MRSA infection and the mechanisms underlying antibiotic tolerance in vivo. This information will inform the design of novel therapies.

项目概要/摘要耐甲氧西林金黄色葡萄球菌 (MRSA) 导致的血流感染 (BSI) 携带率约为 20% MSRA 显示出对抗生素杀灭的耐受性 [11]，有导致持续性 BSI (pBSI) 的倾向。 [3]，pBSI 的持续时间可预测死亡率 [2, 12-14]。 pBSI [3]，强调耐受性是患者预后不良的一个重要原因，这是一个复杂的问题。性状与抗性不同，并且其体内研究存在重大障碍，阻碍了在理解临床环境中最重要的机制方面取得的进展在这项提案中，我们提出了一个建议。创新的基因筛查方法可以克服这些障碍。不同的患者可以被视为微生物进化中自然发生的实验的生物复制品。由于抗生素和免疫压力的选择，细菌种群规模崩溃，耐受突变体同一基因位点中独立发生的突变将以超出偶然的速度变得丰富。在初步研究中，我们使用这种“基因型优先”的方法找到了证据。对于与抗生素耐受性密切相关的两种遗传途径的宿主内进化，我们的中心假设是： MRSA-pBSI 治疗过程中产生的突变体包含针对抗生素和免疫的遗传适应我们建议通过以下具体目标来识别和描述这些途径：目标 1. 确定 MRSA-pBSI 期间进化的哪些基因与抗生素耐受性和能量不平衡的机制通常涉及新陈代谢的扰动，导致“低能量”。导致抗生素靶点周转缓慢的状态 [4, 5]。或者，体内条件可能会强调特定的能量失调。细胞代谢网络中的节点和某些途径可能主导抗生素耐受性。将利用我们的基因筛选方法来识别抗生素耐受突变体并确定哪些基因 MRSA-pBSI 期间的进化与抗生素耐受性和能量失衡有关。目标 2. 确定 MRSA-pBSI 期间 TCA 循环缺陷是否因宿主-病原体-药物而发生恶劣环境可以诱导抗生素耐受，一个主要模型是宿主免疫。吞噬细胞衍生的活性氧形式的压力诱导金黄色葡萄球菌进入耐药状态通过减少三羧酸 (TCA) 循环的通量 [6] 在我们的初步数据中，我们确定了 TCA 循环。如果这些突变体是通过与野生型 MRSA 竞争而进化的。吞噬体，它们将在这种情况下表现出适应性优势，我们将利用这些突变体来测试该模型。直接，通过进行感染吞噬细胞并测量存活率和药物耐受性的实验。这项研究对于理解持续性 MRSA 感染的基本生物学和这些信息将为新疗法的设计提供信息。