CYTOLYSIN-MEDIATED TRANSLOCATION IN S. PYOGENES VIRULENC

描述（由申请人提供）：几乎所有属的致病性革兰氏阳性细菌都能产生成孔溶细胞素。分布最广泛的是CDC家族，其中Streptolysin O （SLO）是一个突出的成员。SLO是由化脓性链球菌产生的，化脓性链球菌是咽炎和其他严重疾病（包括猩红热、风湿热和坏死性面部炎）的重要病因。然而，SLO如何促进这些疾病的发病机制尚不清楚。在这个项目中，我们发现SLO在胞溶素介导的易位（CMT）途径中起着核心作用，CMT是一种新的途径，可将化脓链球菌nad1 -糖水解酶（SPN）通过宿主细胞膜转运到其细胞质中。CMT可能在发病机制中起重要作用，因为胞质SPN对培养细胞具有细胞毒性。然而，SLO如何转运SPN，以及SPN如何参与细胞毒性尚不清楚。CMT是一个极化过程，在这个过程中，大部分输出的SPN被送往宿主细胞的细胞质，而不是释放到细胞外环境中。此外，CMT需要SLO和SPN中的特定结构域，这些结构域是转运所必需的，而不是孔形成或糖水解酶活性所必需的。我们最近也发现，对于CMT来说，SLO成孔本身是完全没有必要的。因此，SLO和SPN如何与膜相互作用以及相互作用如何导致极化易位尚不清楚。此外，我们最近的数据表明，糖水解酶活性可能不是SPN细胞毒性作用的唯一或最重要的基础，因为缺乏糖水解酶活性的SPN变体仍然具有细胞毒性，而细胞毒性需要伴随形成SLO孔。本研究将探讨其易位和细胞毒性的机制，并将通过我们最近对SPN晶体结构的测定和对SPN酶学的详细动力学分析来进一步研究。需要解决的具体问题包括分析SPN中预测的聚糖结合域，SLO-膜相互作用在SPN摄取中的作用，不依赖糖水解酶的细胞毒性的基础，以及SLO和SPN的各种活性如何相互作用以促进毒性和调节宿主细胞信号传导。对CMT的进一步分析将揭示效应易位新途径的细节，以及不同毒素如何协同调节宿主细胞行为以产生特定的致病结果。

DESCRIPTION (provided by applicant): Pore-forming cytolysins are produced by virtually all genera of pathogenic Gram-positive bacteria. The most widely distributed group is the CDC family of which Streptolysin O (SLO) is a prominent member. SLO is produced by Streptococcus pyogenes, an important cause of pharyngitis and other serious diseases including scarlet fever, rheumatic fever and necrotizing faciitis. However, how SLO acts to promote the pathogenesis of any of these diseases is not well-understood. In this project, we have found that SLO plays a central role in the Cytolysin-Mediated Translocation (CMT) pathway, a novel pathway that acts to translocate the S. pyogenes NAD-glycohydrolase (SPN) across the host cell membrane and into its cytosol. CMT likely makes an important contribution to pathogenesis as cytosolic SPN is cytotoxic for cultured cells. However, how SLO functions to translocate SPN, and how SPN contributes to cytotoxicity is not clearly understood. CMT is a polarized process in which the majority of the exported SPN is destined for the host cell cytosol, and is not released into the extracellular milieu. Also, CMT requires specific domains in SLO and SPN that are absolutely required for translocation, but not for pore-formation or glycohydrolase activity. We have also recently found that SLO pore-formation itself is completely dispensable for CMT. Thus, how SLO and SPN interact with the membrane and each other and how this results in polarized translocation is not clear. In addition, our recent data suggest that glycohydrolase activity may not serve as the only or most important basis of SPN's cytotoxic effect as variants of SPN that lack glycohydrolase activity are still cytotoxic and that cytotoxicity requires concomitant formation of the SLO pore. The present study will investigate the mechanism of translocation and cytotoxicity and will be furthered by our recent determination of the SPN crystal structure and our detailed kinetic analysis of SPN enzymology. Specific questions to be addressed include an analysis of a predicted glycan-binding domain in SPN, the role of SLO-membrane interactions in SPN uptake, the basis for the glycohydrolase-independent cytotoxicity and how the various activities of both SLO and SPN interact to promote toxicity and to modulate host cell signaling. Further analysis of CMT will reveal details of a novel pathway for effector translocation and how different toxins synergize to modulate host cell behavior to produce specific pathogenic outcomes.