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

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，存在 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 此前已确定的重要病原菌和强大的模型单核细胞增多性李斯特菌(LMO)是 对体外细胞壁应力敏感，包括β-内酰胺类抗生素和宿主来源的压力 防御，如溶菌酶，一旦基因中断或药物抑制PrkA，青霉素结合 蛋白质和丝氨酸/苏氨酸相关(面食)激酶。细胞内的干扰可激活面食激活酶 壁内稳态，并通过大部分未知的磷靶和下游介导应激反应 小路。PrkA也是LMO胞质生存所必需的，这表明LMO在宿主中面临细胞壁压力 细胞胞浆。虽然已知宿主细胞质对非适应细菌具有限制性，但细胞是自主的 负责杀灭细菌的防御系统(CAD)在很大程度上仍未确定。在本提案中，我们将测试 PrkA磷酸底物介导对胞质重要的细胞壁应激反应的假说 用遗传和磷蛋白质组学相结合的正交法研究生存和毒力 分析。此外，我们将测试宿主阐述细胞壁靶向防御的假设。 通过正向基因筛查和平行的非靶向蛋白质组学对抗细胞质中的细菌 接近。野生型和ΔPrkA LMO的初步磷蛋白组学和遗传抑制分析 体外β-内酰胺暴露显示PrkA可磷酸化~50个蛋白质，包括广泛保守的 未知功能的IreB蛋白，在细胞壁胁迫过程中。然而，IreB和其他假定的 胞浆中的PrkA底物仍不清楚。在这项建议中，我将阐明 IreB，评估其在LMO胞质存活中的作用，并测试其他相关生物的功能保守性。 为了全面评估PrkA在胞质应激反应中的作用，我将在体外进行平行实验 磷酸蛋白质组学分析和体内抑制子选择以确定感染过程中相关的PrkA靶点。 为了鉴定能杀死胞质中非适应细菌的Cad，我将使用全基因组的CRISPR/Cas9 巨噬细胞胞浆LMO的诱变筛选及蛋白质组学分析鉴定宿主 杀死非胞质适应细菌的因子。假定的CAD将通过标准基因进行验证 如有必要，将进行初步的功能鉴定。目标的完成 本文将深入了解细菌对胞质细胞壁应激的适应，并确定寄主因素。 杀死这个隔间里不适应的细菌。因此，这些研究有可能阐明新的 病原体和宿主指导的疗法对抗抗生素耐药性细菌的途径。

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.