A familiar stress in an unfamiliar place: cell wall stressors and bacterial countermeasures in the cytosol

陌生地方的熟悉压力：细胞壁压力源和细胞质中的细菌对策

基本信息

批准号：
10064166
负责人：
Jessica Kelliher
金额：
$ 6.49万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10064166
关键词：
Antibiotic Resistance Antibiotics Bacteria Bacterial Infections Binding Biochemical Biochemical Genetics Biological Assay CRISPR/Cas technology Cell Wall Cells Clustered Regularly Interspaced Short Palindromic Repeats Combating Antibiotic Resistant Bacteria Cytosol Face Francisella Genetic Genetic Screening Genetic Techniques Goals Growth Guanosine Triphosphate Phosphohydrolases Homeostasis Host Defense In Vitro Infection Integration Host Factors Interferons Intervention Invaded Knock-out Libraries Light Listeria monocytogenes Mediating Mediator of activation protein Modeling Molecular Target Monobactams Morbidity - disease rate Muramidase Mutagenesis Mycobacterium tuberculosis Organism Pathway interactions Penicillin-Binding Proteins Phagolysosome Pharmacology Phosphotransferases Positioning Attribute Proteins Proteomics Public Health Resistance Role Serine Shigella flexneri Signal Transduction Staphylococcus aureus Stress Techniques Testing Threonine Virulence Work antimicrobial base beta-Lactams biological adaptation to stress emerging antibiotic resistance expectation extracellular genetic approach genome-wide human pathogen in vivo insight macrophage mortality mutant novel novel strategies pathogen pathogenic bacteria phosphoproteomics response small molecule stressor targeted treatment tool

项目摘要

Project Summary Antibiotic resistance in bacterial pathogens is quickly rendering current antibiotics obsolete; thus, there is a need to discover additional molecular targets and develop novel strategies for treating bacterial infections. We have previously determined that the important pathogen and powerful model Listeria monocytogenes (Lmo) is rendered sensitive to cell wall stress in vitro, including that imposed by β-lactam antibiotics and host-derived defenses such as lysozyme, upon genetic disruption or pharmacological inhibition of PrkA, a penicillin-binding protein and serine/threonine-associated (PASTA) kinase. PASTA kinases are activated by disturbances in cell wall homeostasis and mediate a stress response through largely unidentified phosphotargets and downstream pathways. PrkA is also required for cytosolic survival of Lmo, suggesting Lmo faces cell wall stress in the host cell cytosol. While the host cytosol is known to be restrictive to non-adapted bacteria, the cell autonomous defenses (CADs) responsible for killing bacteria remain largely unidentified. In this proposal, we will test the hypothesis that PrkA phosphosubstrates mediate cell wall stress responses important for cytosolic survival and virulence using an orthogonal approach combining genetic and phosphoproteomic analyses. Furthermore, we will test the hypothesis that the host elaborates cell wall-targeting defenses against bacteria in the cytosol through a forward genetic screen and parallel untargeted proteomics approach. Preliminary phosphoproteomic and genetic suppressor analyses of wild-type and ΔprkA Lmo during β-lactam exposure in vitro revealed that PrkA phosphorylates ~50 proteins, including the broadly conserved protein of unknown function IreB, during cell wall stress. However, the importance of IreB and other putative PrkA substrates in the context of the cytosol remains unknown. In this proposal, I will elucidate the function of IreB, assess its role in Lmo cytosolic survival, and test for functional conservation in other related organisms. To comprehensively assess the role of PrkA in cytosolic stress responses, I will execute parallel ex vivo phosphoproteomic analysis and in vivo suppressor selection to identify PrkA targets relevant during infection. To identify CADs that kill non-adapted bacteria in the cytosol, I will use a genome-wide CRISPR/Cas9 mutagenesis screen and proteomic analysis of Lmo isolated from the cytosol of macrophages to identify host factors that kill non-cytosol-adapted bacteria. Putative CADs will be validated through standard genetic approaches, and initial functional characterization will be performed as necessary. Completion of the Aims herein will provide insights into bacterial adaptations to cytosolic cell wall stress and will identify host factors that kill maladapted bacteria in this compartment. These studies therefore have the potential to illuminate new avenues for both pathogen- and host-directed therapies to combat antibiotic-resistant bacteria.

项目概要细菌病原体的抗生素耐药性很快就会导致现有的抗生素过时；我们需要发现更多的分子靶点并开发治疗细菌感染的新策略。先前已确定重要的病原体和强大的模型单核细胞增生李斯特氏菌（Lmo）是在体外对细胞壁应激敏感，包括β-内酰胺抗生素和宿主来源的应激防御，例如溶菌酶，对 PrkA（一种青霉素结合剂）的遗传破坏或药理学抑制蛋白质和丝氨酸/苏氨酸相关 (PASTA) 激酶由细胞干扰激活。壁稳态并通过很大程度上未识别的磷酸靶标和下游介导应激反应 Lmo 的胞质存活也需要 PrkA，这表明 Lmo 在宿主中面临细胞壁应激。虽然已知宿主细胞质对未适应的细菌具有限制性，但细胞具有自主性。负责杀死细菌的防御机制（CAD）在很大程度上仍未确定。在本提案中，我们将测试这些防御机制。 PrkA 磷酸底物介导细胞壁应激反应的假设对细胞质很重要使用遗传和磷酸蛋白质组学相结合的正交方法测定存活率和毒力此外，我们将检验宿主精心设计细胞壁靶向防御的假设通过正向遗传筛选和平行非靶向蛋白质组学对抗细胞质中的细菌方法对野生型和 ΔprkA Lmo 进行初步磷酸化蛋白质组学和遗传抑制分析。体外暴露 β-内酰胺表明 PrkA 磷酸化约 50 种蛋白质，包括广泛保守的蛋白质 IreB 的功能未知，但在细胞壁应激过程中 IreB 的重要性和其他假定的。细胞质中的 PrkA 底物仍然未知，在本提案中，我将阐明的功能。 IreB，评估其在 Lmo 胞质存活中的作用，并测试其他相关生物体中的功能保守性。为了全面评估 PrkA 在胞质应激反应中的作用，我将进行平行的离体实验磷酸蛋白质组分析和体内抑制因子选择，以确定感染期间相关的 PrkA 靶标。为了识别杀死细胞质中非适应细菌的 CAD，我将使用全基因组 CRISPR/Cas9 从巨噬细胞胞浆中分离的 Lmo 进行诱变筛选和蛋白质组学分析以鉴定宿主杀死非胞质适应细菌的因素将通过标准遗传进行验证。方法，并根据需要进行初步功能表征。本文将深入了解细菌对细胞质细胞壁应激的适应，并将确定宿主因素因此，这些研究有可能揭示新的情况。对抗抗生素耐药细菌的病原体和宿主定向疗法的途径。