Agbavor Charles, Zimnicka Adriana, Kumar Allison, George Jada L, Torres Madeline, Prehna Gerd, Alonzo Francis, Durrant Jacob D, Freitag Nancy E, Cahoon Laty A
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Department of Pharmaceutical Sciences, University of Illinois Chicago, Chicago, Illinois, USA.
mBio. 2024 Jul 17;15(7):e0074324. doi: 10.1128/mbio.00743-24. Epub 2024 May 29.
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 (). Critical to 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 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.
is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, 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. encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major 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 and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
致病细菌依靠分泌的毒力因子在易感宿主中引发疾病。然而，在革兰氏阳性细菌中，膜易位后分泌蛋白激活和调控的机制仍 largely 未知。我们利用蛋白质组学鉴定了几种依赖于分泌伴侣蛋白 PrsA2 的蛋白质。随后，我们进行了表型、生化和生物物理分析以及计算分析，以研究检测到的关键分泌毒力因子李斯特菌溶血素 O（LLO）的调控及其与细菌病原体（）中 PrsA2 的相互作用。对毒力至关重要的是被宿主细胞内化以及随后胆固醇依赖性成孔毒素 LLO 的作用，LLO 使细菌能够从宿主细胞吞噬体中逃脱。由于（）是革兰氏阳性菌，细胞膜和细胞壁之间的空间是溶剂可及的。因此，我们假设病原体被内化到吞噬体中时从中性到酸性 pH 的下降对于调节 PrsA2 与 LLO 的相互作用至关重要。在此，我们证明 PrsA2 以 pH 依赖性方式直接与 LLO 相互作用。我们表明，在中性 pH 条件下 PrsA2 保护并隔离 LLO，此时可观察到 LLO 聚集。此外，我们确定了 PrsA2 与 LLO 相互作用以及最终 LLO 的折叠和活性所需的分子特征。而且，蛋白质复合物建模表明 PrsA2 通过其胆固醇结合结构域与 LLO 相互作用。这些发现突出了一种机制，革兰氏阳性分泌伴侣蛋白通过该机制在与宿主细胞感染相关的条件下调节成孔毒素的分泌、稳定性和折叠。
（）是一种普遍存在的食源性病原体，可对易感人群造成严重疾病。在感染期间，（）依赖多种分泌的毒力因子，包括使细菌能够侵入宿主并在细胞间传播的 LLO。膜易位后，分泌因子必须在具有挑战性的细菌细胞膜 - 细胞壁界面处变得活跃。然而，分泌蛋白折叠和功能所需的机制仍 largely 未知。（）编码一种伴侣蛋白 PrsA2，它对分泌因子的活性至关重要。在此，我们表明 PrsA2 在对应于宿主细胞质的条件下直接结合并保护主要的（）毒力因子 LLO，此时 LLO 会发生不可逆的变性。此外，我们确定了 PrsA使它与 LLO 相互作用的分子特征。总之，我们的结果表明（）以及可能其他革兰氏阳性细菌在感染期间利用分泌伴侣蛋白来调节成孔毒素的活性。
