Cytolysin-mediated translocation in S.pyogenes virulence

描述（由申请人提供）：最近的研究已经确定了溶细胞素介导的易位（CMT），这是革兰氏阳性病原体化脓性链球菌中的一种新型易位途径，其利用成孔溶细胞素链球菌溶血素O（SLO）易位链球菌。化脓性链球菌NAD-糖水解酶（SPN）进入宿主细胞胞质溶胶。然而，SLO如何移位SPN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，其中通过非特化的一般分泌（Sec）途径从细菌输出的大部分SPN被指定用于宿主细胞胞质溶胶，并且不被释放到细胞外环境中。有价值的见解来自于我们最近的研究，这些研究已经定义了SLO中的一个结构域，该结构域对于效应子易位是必需的，但对于跨膜孔的形成是不必要的。我们还表明，SPN和可溶性SLO单体之间的相互作用是不容易检测到的，CMT有能力区分SEC底物，路由SPN进入宿主细胞胞质溶胶，而指导其他进入细胞外空间，和SPN的生产需要一个假定的伴侣蛋白，我们命名为IPS（免疫因子SPN）。 这些研究建立了几个重要的观点：1）。孔形成本身对于CMT是不够的; 2.）CMT由涉及SLO的特定蛋白质-蛋白质相互作用驱动; 3.）这些相互作用可能涉及多聚体而不是单体SLO;和4.）这些交互是与SPN本身和/或导出通道的组件的交互。本项目将探讨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。了解这些问题将是重要的识别额外的效应蛋白，并评估CMT是否有助于其他革兰氏阳性病原体的毒力。

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.