Regulation of Yersinia Type III Secretion by YopK

描述(申请人提供)：许多革兰氏阴性病原体，包括耶尔森氏菌(小肠结肠炎耶尔森氏菌、假结核耶尔森氏菌和鼠疫耶尔森氏菌)使用III型分泌系统(TTSS)致病。TTSS本质上是一个将细胞毒性蛋白注入宿主细胞的分子注射器。在耶尔森氏菌中，TTSS至少将8种被称为YOPs的蛋白质输送到靶细胞中，缺乏功能TTSS的突变体是无毒的。YopK是正确调控TTSS所必需的，但这一调控的分子细节仍然是一个谜。YopK的生物信息学分析没有发现任何酶结构域或与已知蛋白质的同源性。YopK通过TTSS被注入宿主细胞，我们的数据表明YopK从宿主细胞内部调节TTSS。由于人们对YopK知之甚少，这里提出的工作代表了对该蛋白质的全面表征。使用缺失分析，我们将确定YopK中对其注射、调节TTSS及其与其他蛋白质相互作用重要的结构域。我们还将结合生物化学、流式细胞术和显微镜来研究它在宿主细胞中的作用，以确定YopK的定位和鉴定相互作用的蛋白。从这项工作中获得的见解将为未来的研究提供基础，这些研究将阐明控制YopK调控的分子细节。总之，这项研究将使用新的方法来研究一种对调节TTSS、耶尔森氏菌和许多其他革兰氏阴性病原体的中心毒力策略至关重要的蛋白质。了解这些病原体调节TTS的分子机制将为我们提供替代广泛治疗策略的靶点。公共卫生相关性：革兰氏阴性病原体导致多种疾病，并对美国卫生保健系统构成重大压力。许多革兰氏阴性菌依靠分子注射器将有毒蛋白质注入宿主细胞；因此，堵塞注射器可能是一种有效的抗菌策略。我们的工作旨在了解注射器的调节，这里获得的见解可能有助于设计基础广泛的疗法来治疗或预防多种疾病。

DESCRIPTION (provided by applicant): Many Gram-negative pathogens including the Yersinia species (Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis) use a Type III Secretion System (TTSS) for pathogenesis. The TTSS is essentially a molecular syringe that injects cytotoxic proteins into host cells. In Yersinia, the TTSS delivers at least 8 proteins known as Yops into target cells, and mutants lacking a functional TTSS are avirulent. YopK is required for proper regulation of the TTSS, but the molecular details of this regulation remain a mystery. Bioinformatic analysis of YopK does not reveal any enzymatic domains or homology to known proteins. YopK is injected into host cells by the TTSS, and our data suggest that YopK acts from within host cells to regulate the TTSS. Since little is known about YopK, the work proposed here represents a comprehensive characterization of the protein. Using a deletion analysis, we will identify domains within YopK that are important for its injection, regulation of the TTSS, and its interaction with other proteins. We will also investigate its role within host cells using a combination of biochemistry, flow cytometry, and microscopy to determine YopK localization and identify interacting proteins. Insights gained from this work will provide a foundation for future studies that will elucidate the molecular details governing YopK regulation. In summary, this study will use novel approaches to investigate a protein that is important for regulation of the TTSS, the central virulence strategy of Yersinia and many other Gram-negative pathogens. Understanding the molecular mechanisms by which these pathogens regulate the TTSS will provide us with targets for alternative broad-based therapeutic strategies. PUBLIC HEALTH RELEVANCE: Gram-negative pathogens cause a variety of diseases and present a significant strain to the US health care system. Many Gram-negatives rely on a molecular syringe to inject toxic proteins into host cells; therefore blocking the syringe could be an effective antimicrobial strategy. Our work seeks to understand regulation of the syringe, and insights gained here may be helpful in designing broad-based therapeutics for treating or preventing numerous diseases.