ROLE OF YAPS IN Y. PESTIS PATHOGENESIS

鼠疫耶尔森氏菌是一种革兰氏阴性细菌人类病原体，是鼠疫的病原体。从历史上看，鼠疫耶尔森氏菌一直是造成重大人类发病和死亡的原因。然而，鼠疫耶尔森氏菌通常是啮齿动物的病原体，并且在肠杆菌科细菌中是独一无二的，它利用吸血昆虫（跳蚤）进行传播。人类鼠疫耶尔森氏菌感染是一种急性发热性疾病，根据接种途径可能有多种不同的表现。尽管发达国家鼠疫的环境暴发已基本得到控制，但这种病原体可能被用作生物恐怖主义的媒介，以及鼠疫在一些国家重新出现，因此需要更详细地了解鼠疫耶尔森氏菌的发病机制，以便于确定疫苗开发和治疗的目标。目前尚无疫苗可用。一组很可能导致鼠疫耶尔森氏菌的蛋白质 发病机制是自转运蛋白（AT）。 AT 蛋白由三个基本结构域组成：N 端信号序列、“过客结构域”，最后是 C 端的 β 结构域，这有助于过客结构域跨外膜易位。过客结构域提供了功能活性，随着更多的蛋白质被详细研究，新的活性被归因于这些蛋白质。虽然 AT 之间的 β 结构域相对保守，但乘客结构域可能存在显着差异。计算机分析已鉴定出鼠疫耶尔森氏菌（指定为 yaps/Yaps）的 10 种 AT 蛋白。出于多种原因，雅普人对研究感兴趣。首先，虽然许多假定的 AT 蛋白已通过计算机分析被鉴定出来， 进行实验研究的相对较少，而进行详细研究的则更少。对于鼠疫耶尔森氏菌，我们的优势在于能够将复杂的分子遗传操作与实际上是该病原体天然宿主的动物模型相结合。其次，在构建 AT 的进化树时，通过序列分析得出的许多 Yaps 相对分散，因此通过研究这组 AT 来识别新功能的可能性很高。第三，对 Yaps 的分析可能有助于确定有用的治疗靶标或候选药物，以纳入即将推出的鼠疫耶尔森氏菌疫苗混合物。我们的初步数据表明，所有 10 种雅普病不仅在实验室条件下表达，而且在感染期间也表达。我们还在全毒力鼠疫耶尔森菌株中的七个雅普中构建了缺失突变体，并已开始测试其效果 这些突变对毒力的影响。四种 yap 突变体在黑死病感染模型中明显具有表型；这些结果与我们的假设一致，即 yaps 在发病机制中发挥作用。我们的长期目标是在分子水平上了解雅普个体在疾病和鼠疫耶尔森生命周期中的作用。因此，我们提出以下具体目标：（1）分析Yaps在鼠疫耶尔森氏菌中的定位和表达； (2) Yaps毒力作用分析； (3)雅普潜在功能分析。

Yersinia pestis is a Gram-negative bacterial human pathogen that is the causative agent of plague. Historically Y. pestis has been responsible for significant human morbidity and mortality. However, Y. pestis is typically a pathogen of rodents, and is unique among the Enterobacteriaceae in using a blood-sucking insect (flea) for transmission. Y. pestis infection in humans is an acute febrile disease that can have a number of different presentations depending upon the route of inoculation. Although environmental outbreaks of plague in the developed world largely have been controlled, the possible use of this pathogen as an agent of bioterrorism, as well as the re-emergence of plague in several countries, necessitates a more detailed understanding of the pathogenesis of Y. pestis to facilitate the identification of targets for vaccine development and treatment. No vaccine is currently available. A group of proteins that have a high likelihood of contributing to Y. pestis pathogenesis are the autotransporter proteins (ATs). AT proteins consist of three basic domains: a N-terminal signal sequence, a ¿passenger domain¿ and finally a β-domain at the C-terminus which facilitates translocation of the passenger domain across the outer membrane. The passenger domain provides the functional activity and new activities are being attributed to these proteins as more of them are studied in detail. While the β-domains are relatively conserved between ATs the passenger domains can vary significantly. In silico analyses have identified ten AT proteins of Y. pestis (designated yaps/Yaps). The Yaps are of interest to study for a number of reasons. First, while many putative AT proteins have been identified by in silico analyses, relatively few have been studied experimentally and even fewer have been studied in detail. With Y. pestis we have the advantage of being able to pair sophisticated molecular genetic manipulations with an animal model that is actually a natural host for this pathogen. Second, many of the Yaps by sequence analysis are relatively divergent when evolutionary trees of ATs are constructed and thus, the likelihood of identifying new functions by studying this group of ATs is high. Third, analysis of the Yaps may help identify a useful therapeutic target or candidate for inclusion in upcoming Y. pestis vaccine cocktails. We have preliminary data indicating that all ten of the yaps are expressed not only during laboratory conditions but also during infection. We also have constructed deletion mutants in seven yaps in a fully virulent Y. pestis strain and have begun testing the effect of these mutations on virulence. Four yap mutants clearly have phenotypes in a bubonic plague model of infection; these results are consistent with our hypothesis that the yaps play a role in pathogenesis. Our long term goal is to understand at a molecular level the role(s) of the individual Yaps in disease and the Y. pestis life cycle. Thus, we propose the following specific aims: (1) Analysis of the localization and expression of Yaps in Y. pestis; (2) Analysis of the role of Yaps in virulence; (3) Analysis of potential functions of the Yaps.