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：
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
项目类别：
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

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.

项目摘要/摘要由于甲氧西林抗葡萄球菌（MRSA）而引起的血液感染（BSI）携带约20％死亡率[1，2]。 MSRA表现出对抗生素杀戮的耐受性[11]，有望引起持续的BSI（PBSI） [3]以及PBSI预测死亡率的持续时间[2，12-14]。 MRSA很少在 PBSI [3]，强调耐受性是患者结局不良的重要原因。抗生素耐受性是一种复杂的特质与阻力不同，并且在体内的研究有重大障碍。了解临床环境中最重要的机制的进展。在此提案中，我们提出了创新的基因筛查方法克服这些障碍。发生在MRSA-PBSI的情节中可以将不同的患者视为微生物进化中天然实验的生物学重复。随着细菌种群的大小由于抗生素和免疫压迫而崩溃，耐受突变体将变得丰富。以超过机会的速率独立于同一遗传基因座出现的突变一个人在生物学上有意义。在初步研究中，使用这种“基因型优先”方法，我们找到了证据对于两种遗传途径的宿主进化，与抗生素耐受性密切相关。我们的中心假设是在治疗MRSA-PBSI期间出现的突变体含有抗生素和免疫的遗传适应宽容。我们建议通过以下特定目的来识别和表征这些途径： AIM 1。确定MRSA-PBSI期间哪些基因与抗生素耐受性和能量不平衡。耐受机制通常涉及代谢中的扰动，导致“低能量” 指出导致抗生素靶标的转移缓慢的情况[4，5]。这种扰动可能会通过多种能量失调的冗余途径。或者，体内条件可能会强调特定的细胞的代谢网络中的节点和某些途径可能主导抗生素耐受性景观。我们将利用我们的遗传筛选方法识别抗生素耐受性突变体并确定哪些基因在MRSA-PBSI期间演变的与抗生素耐受性和能量失衡有关。 AIM 2。确定由于宿主 - 病原体 - 药物的MRSA-PBSI期间TCA周期缺陷是否演变相互作用。 HARMSH环境可以诱导抗生素耐受性，而领先的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态通过减少三核酸（TCA）周期的通量[6]。在我们的初步数据中，我们确定了TCA周期在MRSA-PBSI期间演变的突变体。如果这些突变体通过在吞噬体，它们将在此设置中显示出健身优势。我们将利用这些突变体测试此模型直接，通过执行我们感染吞噬细胞并测量存活和药物耐受性的实验。这项研究对于理解持续MRSA感染的基本生物学和体内抗生素耐受性的机制。此信息将为新疗法的设计提供信息。