Inhibitors of Yersinia Yop Type III Secretion(RMI)

描述（由申请人提供）：鼠疫是由鼠疫耶尔森氏菌引起的，具有高度毒性，即使在正确诊断和给予抗生素的情况下，也经常导致致命感染。Y.鼠疫杆菌是III型分泌系统和称为Yops的毒素，其通过III型分泌系统易位到哺乳动物细胞质中。在感染的小鼠模型系统中，Yops的易位对于肺中耶尔森氏菌的生产性感染是必需的。我们已经产生了一个报告系统，其中Yop易位信号YopE融合到一个基因，β-内酰胺酶，它编码TEM。 当TEM被递送到哺乳动物细胞中时，它切割荧光底物CCF 2，使得CCF 2以不同的波长发光。我们用CCF_2-AM负载HEp-2细胞，通过荧光显微镜检测细胞的450和520发射光谱，可以很容易地检测到YopE-TEM融合蛋白的易位。我们建议将该技术应用于高通量筛选，以鉴定Yops易位的小分子抑制剂。 最终，在小分子存在下，用表达YopE-TEM融合蛋白的耶尔森氏菌菌株感染CCR-2负载的HEp-2细胞。将在荧光ELISA酶标仪中读取460与520发射的比值。暴露于抑制易位的化合物的细胞应在520发荧光。这些化合物将在几个二级筛选中进行测试，以确认化合物抑制III型分泌和易位过程。 此类抑制剂应该提供一类新型疗法来对抗肺鼠疫以及其他使用III型分泌系统来克服宿主防御的病原体，包括优先病原体名单上的其他病原体，例如沙门氏菌和Y.小肠结肠炎 这些分子可能在肺鼠疫爆发期间或在感染的最初迹象明显之后被用于预防。此外，这些抑制剂将是探测耶尔森氏菌属和其他革兰氏阴性细菌病原体编码的III型分泌系统的结构和功能的有价值的工具。

DESCRIPTION (provided by applicant): Pneumonic plague, caused by Yersinia pestis, is highly virulent and frequently causes lethal infection even when diagnosed correctly and antibiotics are given. One (1) of the key virulence features of Y. pestis is the type secretion system and the toxins, called Yops, that are translocated into mammalian cell cytoplasm by the type III secretion system. In mouse model systems of infection, the translocation of Yops is essential for productive infection of Yersinia in lungs. We have generated a reporter system in which Yop translocation signals of YopE are fused to a gene, b-lactamase, which encodes TEM. When TEM is delivered into mammalian cells, it cleaves a fluorescent substrate, CCF2, such that CCF2 emits light at a different wavelength. We can easily detect translocation of YopE-TEM fusion proteins by loading HEp-2 cells withCCF2-AM and detecting the 450 versus 520 emission spectrum of cells with by fluorescence microscopy. We propose to adapt this technology to use in a high through-put screen to identifying small molecular inhibitors of translocation of Yops. Ultimately, CCR-2 loaded HEp-2 cells will be infected with Yersinia strains expressing YopE-TEM fusion proteins in the presence of small molecules. The ratio of 460 versus 520 emissions will be read in a Fluorescence ELISA plate reader. Cells exposed to compounds which inhibit translocation should fluoresce at 520. These compounds will'be tested in several secondary screens to confirm that the compounds inhibit type III secretion and translocation processes. Such inhibitors should provide a novel class of therapeutics against pneumonic plague as well as other pathogens that use type III secretion systems to overcome host defenses, including other pathogens on the Priority pathogens list such as Salmonella, and Y. enterocolitica. Potentially these molecules could be used either prophylatically during an outbreak of pneumonic plague or after initial signs of infection are apparent. In addition, these inhibitors will be valuable tools to probe the structure and function of type III secretion systems encoded by Yersinia species and other Gram-negative bacterial pathogens.