CYTOLYSIN-MEDIATED TRANSLOCATION IN S. PYOGENES VIRULENC

化脓性链球菌中溶细胞素介导的转位

• 批准号: 8018188
• 负责人: Michael G. Caparon
• 金额: $ 33.86万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8018188
• 关键词: Accounting; Address; Alleles; Binding; Binding Proteins; Carbohydrates; Cell Death; Cell membrane; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Cholesterol; Complex; Cultured Cells; Cytochalasin D; Cytokine Signaling; Cytolysins; Cytosol; Data; Disease; Endocytosis; Enzymatic Biochemistry; Epithelial Cells; Family; Genome; Glycoside Hydrolases; Gram-Positive Bacteria; Infection; Kinetics; Mediating; Membrane; Modeling; Mus; Mutation; N-terminal; NAD+ Nucleosidase; Outcome; Pathogenesis; Pathway Analysis; Pathway interactions; Pharyngitis; Play; Polysaccharides; Process; Property; Prostaglandins; Resistance; Rheumatic Fever; Role; Scarlet Fever; Signal Transduction; Soft Tissue Infections; Streptococcus pyogenes; Structure; Technology; Testing; Toxic effect; Toxin; Variant; Virulence; Wound Healing; base; cell behavior; cytokine; cytotoxic; cytotoxicity; extracellular; inhibitor/antagonist; killings; macrophage; member; mutant; novel; pathogen; perforin; porin; public health relevance; receptor; response; retrograde transport; streptolysin O; uptake

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. PUBLIC HEALTH RELEVANCE: Cytolysin-mediated translocation (CMT) is a novel pathway for toxin delivery in the Gram-positive pathogen Streptococcus pyogenes that uses the pore-forming cytolysin Streptolysin O (SLO) to translocate the streptococcal NAD+ glycohydrolase (SPN) across the host cell membrane and into its cytosol. Both SLO and cytosolic SPN then act to cause host cell death. In this project we will exploit our recent observations that pore-formation and glycohydrolase activity are dispensable for CMT and toxicity, respectively, along with our determination of the crystal structure of SPN to produce mutants of SPN and SLO that will be used to elucidate how SLO acts to translocate SPN. We will also use these mutants to investigate how SLO and SPN interact to both kill and manipulate the cytokine signaling responses of cells. This information will be important for understanding how the multiple toxins of S. pyogenes interact with each other and with host cells to unravel how this pathogen can cause such a diverse range of different diseases.

性状（由申请方提供）：几乎所有致病性革兰氏阳性菌属均产生成孔溶细胞素。分布最广泛的组是CDC家族，其中链球菌溶血素O（SLO）是主要成员。SLO由化脓性链球菌产生，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原因。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在细胞溶解素介导的易位（CMT）途径中起着核心作用，CMT途径是一种新的途径，可以使S.化脓性链球菌NAD-糖水解酶（SPN）穿过宿主细胞膜并进入其胞质溶胶。CMT可能对发病机制做出重要贡献，因为胞质SPN对培养细胞具有细胞毒性。然而，SLO如何作用于SPN易位，以及SPN如何促进细胞毒性尚不清楚。CMT是一个极化过程，其中大部分输出的SPN被指定用于宿主细胞胞质溶胶，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，这些结构域是易位所绝对需要的，但不是孔形成或糖水解酶活性所绝对需要的。我们最近还发现，SLO孔隙形成本身完全不适合CMT。因此，SLO和SPN如何与膜相互作用以及如何相互作用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，并且细胞毒性需要同时形成SLO孔。本研究将探讨易位和细胞毒性的机制，并将进一步通过我们最近确定的SPN晶体结构和我们详细的动力学分析的SPN酶学。要解决的具体问题包括一个预测的聚糖结合结构域在SPN的分析，在SPN吸收的SLO膜相互作用的作用，糖水解酶独立的细胞毒性的基础上，以及如何SLO和SPN的各种活动相互作用，以促进毒性和调节宿主细胞信号。对CMT的进一步分析将揭示效应子易位的新途径的细节，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。 公共卫生关系：溶细胞素介导的转运（CMT）是革兰氏阳性病原体化脓性链球菌中的一种新型毒素转运途径，其利用成孔溶细胞素链球菌溶素O（SLO）将链球菌NAD+糖水解酶（SPN）转运穿过宿主细胞膜并进入其胞质溶胶。然后SLO和胞质SPN两者都起作用以引起宿主细胞死亡。在这个项目中，我们将利用我们最近的观察，即孔形成和糖水解酶活性分别与CMT和毒性有关，沿着我们对SPN晶体结构的测定，以产生SPN和SLO的突变体，这些突变体将用于阐明SLO如何作用于SPN的易位。我们还将使用这些突变体来研究SLO和SPN如何相互作用以杀死和操纵细胞的细胞因子信号传导反应。这一信息对于理解沙门氏菌的多种毒素是如何产生的具有重要意义。化脓性细菌彼此之间以及与宿主细胞之间的相互作用揭示了这种病原体是如何引起如此多样的不同疾病的。