Staphylococcal Superantigen Interactions With Vaginal Epithelium

描述（由申请人提供）：本研究的长期目标是了解金黄色葡萄球菌及其超级抗原（SAg）、细胞毒素和其他外蛋白如何与粘膜表面相互作用，促进SAg渗透，导致严重的人类疾病。我们建议使用阴道上皮作为多层粘膜表面的模型，并假设在计划研究中获得的数据也适用于其他粘膜表面。先前使用离体猪阴道通透性模型的结果表明，SAg, TSST-1，少量穿过粘膜表面，但在内上皮内定位较多，可能作为毒素储存库。在1型葡萄球菌毒素存在的情况下，TSST-1穿透阴道粘膜全层的量显著增加，同时也有相当数量的TSST-1残留在上皮内。我们假设葡萄球菌细胞毒素和其他外蛋白的细胞毒性和炎症特性增强了TSST-1对上皮细胞内的渗透，其中TSST-1与未描述的上皮细胞受体结合，引发促炎细胞因子的产生。基于这些假设，我们提出了两个特定的目标：1)确定金黄色葡萄球菌细胞毒素（外蛋白）通过引起上皮细胞的细胞溶解和/或炎症作用，增强TSST-1穿过上皮表面的作用；2)确定TSST-1受体结合和TSST-1刺激上皮细胞的机制，导致细胞内信号通路的激活和随后的细胞因子的产生。我们的初步数据表明，促进TSST-1穿过粘膜屏障的最有可能的候选外蛋白是细胞毒素，包括3-毒素，我们建议通过纯化的3-毒素成分和等位基因替代敲除菌株对其进行深入研究。在这些研究过程中，我们还将研究粘膜穿透性和缺失[缺失氨基酸1至72]突变型TSST-1蛋白（13000 MW与22000 MW野生型TSST-1）的活性，该蛋白保持超抗原性，并与人类快速进展，100%致命的极端发热综合征相关。我们相信计划中的研究将阐明TSST-1穿透粘膜屏障导致大量人类疾病的机制。这些研究可能为管理严重金黄色葡萄球菌疾病提供新的治疗策略。公共卫生相关性：金黄色葡萄球菌经常引发严重的人类疾病，因为该细菌在粘膜上定植，随后分泌强效的外毒素，称为超级抗原。疾病包括中毒性休克综合征，可能有经期和非经期形式，每年在美国可能影响数千人。该应用研究了超级抗原穿透粘膜表面的机制，使金黄色葡萄球菌引起危及生命的中毒性休克综合征，并可能提出预防和管理此类疾病的新策略。

DESCRIPTION (provided by applicant): The long-term goals of this research are to understand how Staphylococcus aureus and its superantigens (SAgs), cytotoxins, and other exoproteins interact with mucosal surfaces to facilitate SAg penetration to cause serious human illnesses. We propose to use the vaginal epithelium as the model multi-layered mucosal surface, and hypothesize the data obtained in planned studies will also be applicable to other mucosal surfaces. Prior results using an ex vivo porcine vaginal permeability model indicate that the SAg, TSST-1, traverses mucosal surfaces in small amounts, but localizes in higher amounts within the inner epithelium, possibly serving as a toxin reservoir. In the presence of staphylococcal 1-toxin, TSST-1 penetrated the full thickness of the vaginal mucosa in significantly higher amounts, with considerable TSST- 1 also remaining within the epithelium. We hypothesize that cytotoxic and inflammatory properties of staphylococcal cytotoxins and other exoproteins enhance penetration of TSST-1 to inner epithelial cells where TSST-1 binds to an undescribed epithelial cell receptor, triggering pro-inflammatory cytokine production. Base on these hypotheses we propose two specific aims: 1) To determine the contribution of S. aureus cytotoxins (exoproteins) in enhancing the penetration of TSST-1 across epithelial surfaces by causing cytolytic and/or inflammatory effects on epithelial cells, and 2) To determine TSST-1 receptor binding and the mechanism of TSST-1 stimulation of epithelial cells, leading to intracellular activation of signaling pathways with subsequent cytokine production. Our preliminary data suggest that the most likely candidate exoproteins to facilitate penetration of TSST-1 across mucosal barriers are cytotoxins, including 3-toxin, which we propose to study in depth through use of purified 3-toxin components and allelic replacement knockout strains. In the course of these studies, we will also study mucosal penetration and activities of a deletion [missing amino acids 1 to 72] mutant TSST-1 protein (13,000 MW vs. 22,000 MW wild-type TSST-1) that maintains superantigenicity and is associated with a rapidly progressive, 100% fatal, extreme pyrexia syndrome in humans. We believe the planned studies will clarify the mechanisms by which TSST-1 penetrates mucosal barriers to cause large numbers of human diseases. These studies may suggest novel therapeutic strategies to manage serious S. aureus illnesses. PUBLIC HEALTH RELEVANCE: The bacterium Staphylococcus aureus often initiates serious human diseases from the organism's colonization of mucous membranes, followed by secretion of potent exotoxins referred to as superantigens. Illnesses include toxic shock syndrome that may have both menstrual and non- menstrual forms, and that may affect thousands of humans each year in the United States. This application studies the mechanism by which superantigens penetrate mucosal surfaces, allowing S. aureus to cause life threatening toxic shock syndrome, and possibly suggesting novel strategies to prevent and manage such illnesses.