PDF(Pubmed)
Abstract：
Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen Listeria monocytogenes (Lm). Critical to Lm virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since Lm is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection.
OBJECTIVE: Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
OBJECTIVE: Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
摘要：
病原菌依靠分泌的毒力因子在易感宿主中引起疾病。然而,在革兰氏阳性细菌中，分泌蛋白激活和调节膜后易位的潜在机制仍然未知.使用蛋白质组学，我们鉴定了几种依赖于分泌伴侣PrsA2的蛋白质。我们追踪了表型，生物化学，以及生物物理分析和计算分析，以检查检测到的关键分泌毒力因子的调节，李斯特菌溶血素O（LLO），及其与细菌病原体单核细胞增生李斯特菌（Lm）的PrsA2的相互作用。Lm毒力的关键是宿主细胞的内化和胆固醇依赖性成孔毒素的后续作用，LLO,这使得细菌能够从宿主细胞吞噬体逃脱。由于Lm是革兰氏阳性生物，细胞膜和细胞壁之间的空间是溶剂暴露。因此,我们假设，随着病原体内化到吞噬体，从中性到酸性pH的下降对于调节PrsA2与LLO的相互作用至关重要。这里,我们证明了PrsA2以pH依赖性方式直接与LLO相互作用。我们表明，PrsA2在中性pH条件下保护和螯合LLO，可以观察到LLO聚集。此外,我们确定了相互作用以及最终LLO的折叠和活性所需的PrsA2的分子特征。此外，蛋白质复合物建模表明PrsA2通过其胆固醇结合域与LLO相互作用。这些发现强调了革兰氏阳性分泌伴侣调节分泌的机制，稳定性,和在与宿主细胞感染相关的条件下折叠成孔毒素。
目的：Lm是一种普遍存在的食源性病原体，可对脆弱人群造成严重疾病。在感染期间,Lm依赖于广泛的分泌毒力因子库,包括使细菌能够侵入宿主并在细胞之间传播的LLO。膜易位后，分泌因子必须在具有挑战性的细菌细胞膜-壁界面中变得活跃。然而,分泌蛋白折叠和功能所需的机制在很大程度上是未知的。Lm编码一个伴侣，PrsA2，这对分泌因子的活性至关重要。这里,我们表明，PrsA2直接关联并保护主要的Lm毒力因子，LLO,在与宿主胞质溶胶相对应的条件下，其中LLO经历不可逆变性。此外,我们确定了PrsA2的分子特征，使其能够与LLO相互作用。一起,我们的结果表明,Lm和其他革兰氏阳性细菌可能利用分泌的伴侣来调节感染过程中孔形成毒素的活性.
目的：Lm是一种普遍存在的食源性病原体，可对脆弱人群造成严重疾病。在感染期间,Lm依赖于广泛的分泌毒力因子库,包括使细菌能够侵入宿主并在细胞之间传播的LLO。膜易位后，分泌因子必须在具有挑战性的细菌细胞膜-壁界面中变得活跃。然而,分泌蛋白折叠和功能所需的机制在很大程度上是未知的。Lm编码一个伴侣，PrsA2，这对分泌因子的活性至关重要。这里,我们表明，PrsA2直接关联并保护主要的Lm毒力因子，LLO,在与宿主胞质溶胶相对应的条件下，其中LLO经历不可逆变性。此外,我们确定了PrsA2的分子特征，使其能够与LLO相互作用。一起,我们的结果表明,Lm和其他革兰氏阳性细菌可能利用分泌的伴侣来调节感染过程中孔形成毒素的活性.
