Virulence mechanisms of group A streptococcal toxins

描述(由申请人提供)：A组链球菌(化脓性链球菌或GAS)每年在美国导致数百万例咽炎和皮肤感染，并伴随着发病率和缺课。在美国，这种细菌每年还导致大约10,000例侵袭性感染，包括菌血症、坏死性筋膜炎和链球菌中毒性休克。在全球范围内，气体感染和感染后综合征急性风湿热的并发症估计每年造成50万人死亡。目前还没有疫苗可用，治疗失败、复发和感染复发仍然很常见。除极少数例外，临床分离的GAS能产生致孔溶血素O(SLO)。SLO是胆固醇依赖细胞溶蛋白的一员，这是一个由许多革兰氏阳性细菌产生的分泌蛋白家族，它们共同具有与含胆固醇的膜结合的特性，在那里它们齐聚并插入形成大孔。实验感染研究表明，SLO对GAS毒力的贡献不大；然而，动物模型不一定对影响人类宿主定植的因素敏感。人类口咽部的定植是GAS感染的中心，我们最近的研究结果表明，SLO通过其增强口咽角质形成细胞内GAS存活的能力参与了这一过程。SLO介导的孔道形成和SLO将共毒素NAD-糖水解酶运送到感染细胞的胞浆中的能力导致气体存活时间延长。如果这些毒素是人体内气体适应生存的关键贡献者，它们就是药物干预和疫苗开发的重要潜在靶点。因此，该项目的目标1是研究SLO和NADase如何促进口咽上皮细胞对杀戮的抵抗力，作为咽部气体持续存在的潜在机制。我们将确定NADase如何在转录水平和通过蛋白质-蛋白质相互作用增加SLO的表达或稳定性。我们将使用炭疽毒素为基础的NADase递送作为一种新的方法来分离NADase的细胞效应和SLO的细胞效应，SLO通常是NADase转位到宿主细胞所必需的。这个模型系统将允许直接研究NADase如何颠覆内体和自噬成熟，并促进感染角质形成细胞的气体存活。目标2将继续研究NADase的产生与过去30年侵袭性气体疾病的出现之间的关系。如果产生NADase的气体能够在巨噬细胞中存活，这些细胞可能会成为发起或扩大侵入性感染的特洛伊木马。为了研究这一假说，我们将确定SLO和NADase是否介导了人巨噬细胞对气体的杀伤抗性，NADase是如何传递给这些细胞的，以及SLO和NADase如何改变巨噬细胞内气体的运输。了解SLO和NADase如何增强人类宿主的GAS定植和促进侵袭性感染，将为治疗和预防GAS感染提供新的策略。

DESCRIPTION (provided by applicant): Group A Streptococcus (S. pyogenes or GAS) is responsible for millions of cases of pharyngitis and skin infections each year in the United States with attendant morbidity and absenteeism from school and work. The organism also causes approximately 10,000 cases annually of invasive infections in the U.S., including bacteremia, necrotizing fasciitis, and streptococcal toxic shock. Globally, GAS infections and complications of the postinfectious syndrome of acute rheumatic fever account for an estimated 500,000 deaths per year. No vaccine is yet available, and treatment failures, relapses, and recurrences of infection remain common. With rare exceptions, clinical isolates of GAS produce the pore-forming hemolytic toxin streptolysin O (SLO). SLO is a member of the cholesterol-dependent cytolysins, a family of secreted proteins produced by many Gram- positive bacteria that share the property of binding to cholesterol-containing membranes where they oligomerize and insert to form large pores. Experimental infection studies have demonstrated a modest contribution of SLO to GAS virulence; however, animal models are not necessarily sensitive to factors that influence colonization in the human host. Colonization of the human oropharynx is central to GAS infection, and our recent results implicate SLO in this process through its ability to enhance GAS intracellular survival in oropharyngeal keratinocytes. SLO-mediated pore-formation and the capacity of SLO to deliver the co-toxin NAD-glycohydrolase to the cytosol of infected cells result in prolonged GAS survival. If these toxins are critical contributors to GAS adaptation for survival in the human host, they are important potential targets for pharmacologic intervention and vaccine development. Accordingly, Aim 1 of the project is to investigate how SLO and NADase promote resistance to killing by oropharyngeal epithelial cells as a potential mechanism for GAS persistence in the pharynx. We will determine how NADase increases expression or stability of SLO at both the transcriptional level and through protein-protein interactions. We will use anthrax toxin-based delivery of NADase as a novel means to dissociate the cellular effects of NADase from those of SLO, which is normally required for NADase translocation into host cells. This model system will permit direct study of how NADase subverts endosomal and autophagic maturation and promotes GAS survival in infected keratinocytes. Aim 2 will pursue the association of NADase production with the emergence of invasive GAS disease over the past 3 decades. If NADase-producing GAS can survive in macrophages, these cells may serve as a Trojan horse to initiate or extend invasive infection. To investigate this hypothesis, we will determine whether SLO and NADase mediate GAS resistance to killing by human macrophages, how NADase is delivered to these cells, and how SLO and NADase modify intracellular trafficking of GAS in macrophages. Understanding how SLO and NADase enhance GAS colonization of the human host and promote invasive infection will inform novel strategies to treat and prevent GAS infection.