Cytolysin-mediated translocation in S.pyogenes virulence

描述(申请人提供)：最近的研究发现了溶细胞素介导的易位(CMT)，这是革兰氏阳性病原菌化脓性链球菌中的一种新的易位途径，它利用致孔细胞溶血素-链球菌溶血素O(SLO)将化脓性链球菌的NAD-糖水解酶(SPN)转位到宿主细胞胞浆中。然而，SLO如何作用于SPN的转位以及SPN如何参与发病机制尚不清楚。 一个中心问题是CMT途径如何产生效应分子的极化分布，其中通过非专门化总分泌(SEC)途径从细菌输出的大部分SPN被指定为宿主细胞胞浆，而不是释放到细胞外环境中。我们最近的研究定义了SLO中的一个结构域，这个结构域对于效应器转位是必不可少的，但对于跨膜孔的形成是必不可少的。我们还证明了SPN和可溶性SLO单体之间的相互作用不容易被检测到，CMT具有区分SEC底物的能力，将SPN引导到宿主细胞胞浆中，同时将其他SPN引导到细胞外空间，并且SPN的产生需要一种假定的伴侣蛋白，我们称之为IPS(免疫因子for SPN)。 这些研究确立了几个重要的观点：1)对于CMT来说，仅有孔的形成是不够的；CMT是由涉及SLO的特定蛋白质-蛋白质相互作用驱动的；这些相互作用可能涉及多聚体而不是单体SLO；这些交互作用要么是与SPN本身，要么是出口渠道的组件。本项目将探索这些相互作用的性质，并将研究SPN的酶活性在发病机制中的作用。了解这些问题对于鉴定其他效应蛋白和评估CMT是否有助于其他革兰氏阳性病原体的毒力将是重要的。

DESCRIPTION (provided by applicant): Recent studies have identified Cytolysin-Mediated Translocation (CMT), a novel translocation pathway in the Gram-positive pathogen Streptococcus pyogenes that utilizes the pore-forming cytolysin Streptolysin O (SLO) to translocate the S. pyogenes NAD-glycohydrolase (SPN) into the host cell cytosol. However, how SLO functions to translocate SPN and how SPN contributes to pathogenesis are unknown. A central question is how the CMT pathway generates a polarized distribution of the effector molecule where the majority of the SPN exported from the bacterium by the non-specialized general secretory (Sec) pathway is destined for the host cell cytosol, and is not released into the extracellular milieu. Valuable insight has come from our recent studies that have defined a domain in SLO that is essential for effector translocation, but is dispensable for formation of transmembrane pores. We have also shown that interactions between SPN and the soluble SLO monomer are not readily detectable, that CMT has the ability to discriminate between Sec substrates, routing SPN into the host cell cytosol, while directing others into the extracellular spaces, and that production of SPN requires a putative chaperone protein we have named IPS (Immunity Factor for SPN). These studies establish several important points: 1.) that pore formation by itself is not sufficient for CMT; 2.) that CMT is driven by specific protein-protein interactions involving SLO; 3.) that these interactions likely involve multimeric and not monomeric SLO; and 4.) that these interactions are either with SPN itself and/or the components of an export channel. This Project will explore the nature of these interactions and will examine the role of SPN's enzymatic activities in pathogenesis. Understanding these issues will be important for the identification of additional effector proteins and for evaluating whether CMT contributes to the virulence of other Gram-positive pathogens.