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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：
10459301
负责人：
Jessica Kelliher
金额：
$ 6.98万
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/10459301
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.

项目摘要细菌病原体中的抗生素耐药性正在迅速使当前的抗生素过时;因此，需要发现更多的分子靶点，并开发治疗细菌感染的新策略。我们先前已经确定，重要的病原体和强大的模型单核细胞增生李斯特菌（Lmo）是在体外对细胞壁应力敏感，包括β-内酰胺抗生素和宿主衍生的防御，如溶菌酶，在遗传破坏或药理学抑制PrkA，青霉素结合，蛋白和丝氨酸/苏氨酸相关（PASTA）激酶。PASTA激酶被细胞内干扰激活细胞壁内稳态，并通过大量未识别的磷酸靶点和下游磷酸靶点介导应激反应。路径。PrkA也是Lmo在胞质中存活所必需的，这表明Lmo在宿主中面临细胞壁应激细胞胞质液虽然已知宿主胞质溶胶对非适应性细菌具有限制性，但细胞的自主生长能力是不稳定的。负责杀死细菌的防御系统（CAD）在很大程度上仍然是未知的。在本提案中，我们将测试 PrkA磷酸底物介导细胞壁应激反应对胞质使用遗传和磷酸化蛋白质组学相结合的正交方法分析。此外，我们将测试宿主精心制作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， approach.野生型和ΔprkA Lmo的初步磷酸化蛋白质组学和遗传抑制基因分析体外β-内酰胺暴露显示PrkA磷酸化约50种蛋白质，包括广泛保守的在细胞壁应激过程中的未知功能蛋白IreB。然而，IreB和其他假定的 PrkA底物的背景下的胞质溶胶仍然未知。在本建议中，我将阐明 IreB，评估其在Lmo细胞溶质存活中的作用，并测试在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。磷酸蛋白质组学分析和体内抑制子选择以鉴定感染期间相关的PrkA靶标。为了鉴定杀死胞质溶胶中非适应细菌的CAD，我将使用全基因组CRISPR/Cas9 从巨噬细胞胞质溶胶中分离的Lmo的诱变筛选和蛋白质组学分析以鉴定宿主杀死非胞质溶胶适应细菌的因子。将通过标准遗传学方法验证推定CAD 方法，并将在必要时进行初步功能表征。完成目标本文将提供对细菌适应胞质细胞壁应激的见解，并将鉴定宿主因子杀死这个隔间里不适应环境的细菌。因此，这些研究有可能阐明新的病原体和宿主定向治疗对抗耐药细菌的途径。