Inhibitors of Yersinia Yop Type III Secretion(RMI)

描述（由申请人提供）：由鼠疫耶尔森氏菌引起的肺鼠疫毒性很强，即使诊断正确并给予抗生素，也经常导致致命感染。鼠疫杆菌的主要毒力特征之一是其型分泌系统和毒素Yops，这些毒素通过III型分泌系统转运到哺乳动物的细胞质中。在小鼠感染模型系统中，Yops的易位对肺中耶尔森菌的生产性感染至关重要。我们已经建立了一个报告系统，在该系统中，Yop易位信号与编码TEM的基因b-内酰胺酶融合。当透射电镜进入哺乳动物细胞时，它会切割一个荧光底物CCF2，这样CCF2就会发出不同波长的光。我们可以很容易地检测YopE-TEM融合蛋白的易位，通过将ccf2 - am加载HEp-2细胞，并通过荧光显微镜检测细胞的450和520发射光谱。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，负载CCR-2的HEp-2细胞将在小分子存在下被表达YopE-TEM融合蛋白的耶尔森菌感染。460对520的发射比将在荧光ELISA板读取器中读取。细胞暴露于抑制易位的化合物中，在520时发出荧光。这些化合物将在几个二次筛选中进行测试，以确认这些化合物抑制III型分泌和易位过程。这种抑制剂应该提供一种新的治疗方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，包括优先病原体清单上的其他病原体，如沙门氏菌和小肠结肠炎耶氏菌。这些分子可能在肺鼠疫爆发期间或在感染的初步迹象明显之后用于预防。此外，这些抑制剂将成为探测由耶尔森菌和其他革兰氏阴性细菌病原体编码的III型分泌系统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.