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

• 批准号: 10613457
• 负责人: MATTHEW J CULYBA
• 金额: $ 19.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613457
• 关键词: 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; Hospitalization; Human; Immune; Immune Tolerance; Infection; Kinetics; Link; Macrophage; 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; analytical tool; antibiotic tolerance; bacterial genome sequencing; chronic infection; design; effective therapy; experimental study; fitness; genomic locus; immune clearance; in vivo; innovation; insight; methicillin resistant Staphylococcus aureus; microbial; mortality; mutant; novel strategies; novel therapeutics; pathogen; pressure; respiratory; response; screening; trait

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.

项目总结/摘要 由耐甲氧西林金黄色葡萄球菌（MRSA）引起的血流感染（BSI）携带约20% 死亡率[1，2]。MSRA显示出对抗生素杀灭的耐受性[11]，有引起持续性BSI（pBSI）的倾向 [3]pBSI的持续时间可预测死亡率[2，12-14]。MRSA很少获得坦率的抗生素耐药性， pBSI [3]，强调耐受性是患者预后不良的重要原因。抗生素耐受性是一个复杂的 性状，这是从电阻不同，有重大障碍，其研究在体内，阻碍了 了解临床环境中最重要的机制的进展。在这份提案中，我们提出了一个 创新的遗传筛查方法来克服这些障碍。发生在 不同的患者可以被看作是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， 将变得丰富。在相同的基因位点上以超过偶然的速度独立出现的突变 都有生物学意义。在初步研究中，使用这种“基因型优先”的方法，我们发现了证据， 两种与抗生素耐受性密切相关的遗传途径的宿主内进化。我们的核心假设是， 在治疗MRSA-pBSI过程中出现的突变体含有对抗生素和免疫的遗传适应性， 宽容我们建议通过以下具体目标来确定和描述这些途径： 目标1。确定在MRSA-pBSI过程中进化的哪些基因与抗生素耐受性相关， 能量不平衡耐受机制通常涉及代谢紊乱，导致“低能量” 导致抗生素靶标转换缓慢的状态[4，5]。这种扰动可能是通过各种 多余的途径汇聚在能量失调上。或者，体内条件可以是应激特异性的。 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。我们 将利用我们的遗传筛选方法来识别抗生素耐受突变体，并确定哪些基因 MRSA-pBSI过程中的进化与抗生素耐受性和能量不平衡有关。 目标2.确定在MRSA-pBSI期间TCA循环缺陷是否因宿主-病原体-药物而演变 互动抗生素耐受性可以通过恶劣的环境诱导产生，主要的模式是宿主免疫 以吞噬细胞衍生的活性氧形式的压力诱导S.金黄色葡萄球菌进入耐药状态 通过减少通过三羧酸（TCA）循环的通量[6]。在我们的初步数据中，我们确定了TCA循环 在MRSA-pBSI期间进化的突变体。如果这些突变体是通过与野生型MRSA竞争而进化出来的， 吞噬体，它们将在这种环境中显示出适应性优势。我们将利用这些突变体来测试这个模型 直接地，通过进行实验，我们感染吞噬细胞并测量存活率和药物耐受性。 这项研究对于了解持续性MRSA感染的基本生物学和MRSA的耐药机制具有重要意义。 体内抗生素耐受性的潜在机制。这些信息将为新疗法的设计提供信息。