PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes.
PASTA kinase-dependent control of peptidoglycan synthesis via ReoM is required for cell wall stress responses, cytosolic survival, and virulence in Listeria monocytogenes.
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DOI：
10.1371/journal.ppat.1009881
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发表时间：
2021-10
期刊：
影响因子：
6.7
通讯作者：
Sauer JD
中科院分区：
文献类型：
--
作者：
Kelliher JL;Grunenwald CM;Abrahams RR;Daanen ME;Lew CI;Rose WE;Sauer JD
致病细菌依靠蛋白质磷酸化来快速适应压力，包括宿主在感染期间施加的压力。青霉素结合蛋白和丝氨酸/苏氨酸相关 (PASTA) 激酶是信号转导系统，可感知细胞壁完整性并调节细菌生理学的多个方面以响应细胞包膜应激。胞质病原体单核细胞增生李斯特氏菌 (PrkA) 中的 PASTA 激酶是细胞壁应激反应、胞质存活和毒力所必需的，但其底物和下游信号传导途径仍未完全确定。我们在 β-内酰胺抗生素存在下结合正交磷酸化蛋白质组学和遗传分析来定义 PrkA 磷酸靶标和 PrkA 调节的途径。这些分析协同强调了 ReoM（最近被确定为影响肽聚糖（PG）合成的 PrkA 靶标）作为细胞壁应激期间重要的磷酸底物。我们发现，reoM 的删除可以将 ΔprkA 突变体的细胞壁应激敏感性和胞质存活缺陷恢复到接近野生型的水平。虽然 ΔprkA 突变体在细胞壁应激期间存在 PG 合成缺陷，但双 ΔreoM ΔprkA 突变体以与野生型相似的速率合成 PG。在系统性李斯特菌病小鼠模型中，删除 ΔprkA 背景中的 reoM 几乎完全将毒力恢复到野生型水平。然而，仅reoM的缺失也会导致毒力减弱，这表明ReoM在发病过程中的某些时刻至关重要。最后，我们证明 PASTA 激酶/ReoM 细胞壁应激反应途径在相关病原体（耐甲氧西林金黄色葡萄球菌）中是保守的。总而言之，我们的磷酸蛋白质组学分析提供了对重要模型病原体的 PASTA 激酶靶标的全面概述，并表明 PrkA 在体内的关键作用是通过调节 ReoM 来调节 PG 合成，以促进胞质存活和毒力。许多抗生素以细菌细胞壁生物合成为目标，这证明了继续研究这一过程以及细菌在感染过程中对细胞壁损伤的反应方式是合理的。青霉素结合蛋白和丝氨酸/苏氨酸相关 (PASTA) 激酶是细菌细胞壁应激反应的主要调节因子，在几种主要病原体中保守，包括单核细胞增生李斯特菌、金黄色葡萄球菌和结核分枝杆菌。我们之前表明，单增李斯特菌中的 PASTA 激酶 PrkA 对于细胞壁应激反应和毒力至关重要。在这项工作中，我们结合蛋白质组学和遗传学方法来鉴定单核细胞增生李斯特菌中的 PrkA 底物。我们表明，对两个筛选中的一种候选物 ReoM 的调节会增加细胞壁成分肽聚糖的合成，并且这种调节是发病机制所必需的。我们还证明 PASTA 激酶-ReoM 通路调节另一种重要病原体（耐甲氧西林金黄色葡萄球菌）的细胞壁应激反应。此外，我们发现 ReoM 在单核细胞增生李斯特菌体内的 PrkA 独立作用，表明在感染过程中需要对肽聚糖合成进行细致的调节。总的来说，这项研究为细菌病原体如何在感染过程中控制细胞壁合成提供了新的见解。
Pathogenic bacteria rely on protein phosphorylation to adapt quickly to stress, including that imposed by the host during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are signal transduction systems that sense cell wall integrity and modulate multiple facets of bacterial physiology in response to cell envelope stress. The PASTA kinase in the cytosolic pathogen Listeria monocytogenes, PrkA, is required for cell wall stress responses, cytosolic survival, and virulence, yet its substrates and downstream signaling pathways remain incompletely defined. We combined orthogonal phosphoproteomic and genetic analyses in the presence of a β-lactam antibiotic to define PrkA phosphotargets and pathways modulated by PrkA. These analyses synergistically highlighted ReoM, which was recently identified as a PrkA target that influences peptidoglycan (PG) synthesis, as an important phosphosubstrate during cell wall stress. We find that deletion of reoM restores cell wall stress sensitivities and cytosolic survival defects of a ΔprkA mutant to nearly wild-type levels. While a ΔprkA mutant is defective for PG synthesis during cell wall stress, a double ΔreoM ΔprkA mutant synthesizes PG at rates similar to wild type. In a mouse model of systemic listeriosis, deletion of reoM in a ΔprkA background almost fully restored virulence to wild-type levels. However, loss of reoM alone also resulted in attenuated virulence, suggesting ReoM is critical at some points during pathogenesis. Finally, we demonstrate that the PASTA kinase/ReoM cell wall stress response pathway is conserved in a related pathogen, methicillin-resistant Staphylococcus aureus. Taken together, our phosphoproteomic analysis provides a comprehensive overview of the PASTA kinase targets of an important model pathogen and suggests that a critical role of PrkA in vivo is modulating PG synthesis through regulation of ReoM to facilitate cytosolic survival and virulence. Many antibiotics target bacterial cell wall biosynthesis, justifying continued study of this process and the ways bacteria respond to cell wall insults during infection. Penicillin-binding protein and serine/threonine-associated (PASTA) kinases are master regulators of cell wall stress responses in bacteria and are conserved in several major pathogens, including Listeria monocytogenes, Staphylococcus aureus, and Mycobacterium tuberculosis. We previously showed that the PASTA kinase in L. monocytogenes, PrkA, is essential for the response to cell wall stress and for virulence. In this work, we combined proteomic and genetic approaches to identify PrkA substrates in L. monocytogenes. We show that regulation of one candidate from both screens, ReoM, increases synthesis of the cell wall component peptidoglycan and that this regulation is required for pathogenesis. We also demonstrate that the PASTA kinase-ReoM pathway regulates cell wall stress responses in another significant pathogen, methicillin-resistant S. aureus. Additionally, we uncover a PrkA-independent role for ReoM in vivo in L. monocytogenes, suggesting a need for nuanced modulation of peptidoglycan synthesis during infection. Cumulatively, this study provides new insight into how bacterial pathogens control cell wall synthesis during infection.
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