Identity, function and control of Francisella effectors encoded outside its pathogenicity island

摘要 方济各氏菌是革兰氏阴性细菌的一个属，它们占据着广泛的 后生动物宿主的范围。该群包括图拉氏弗朗西斯菌亚种。图拉尔人，一种高度毒力的人类 由于其低感染剂量而被归类为一级选择因素的病原体，气雾剂路线 接种和侵袭性感染过程。多项研究表明，该病毒的关键毒力因素 这些细菌是弗朗西塞氏菌致病岛(FPI)，它编码一种VI型细菌的变体 分泌系统(T6SS)。然而，FPI在寄主范围不同的Francisella spp中是保守的，并且 多变的致病能力。在这项提案所附的初步数据中，我们证明了 编码FPI T6SS底物效应蛋白的基因位于基因组的远端。我们 进一步研究发现，虽然这些效应器在弗朗西斯氏菌中的存在方式各不相同，但它们发挥着重要的、直接的作用。 在促进携带它们的细菌的毒力策略方面发挥作用。这些发现引导我们 假设以FPI编码的T6SS作为传递不同效应器的通用平台 在基因组的其他地方编码的分子。在我们的第一个目标中，我们将通过以下方式定义分子机制 图拉氏丝虫的哪一种T6SS效应物Opia促进细胞内复制。重要的是，这种模式 到目前为止，还没有确定FPI效应蛋白的作用。我们在这里表明，欧皮亚是创始的 细菌磷脂酰肌醇激酶家族的成员，在不同的病原体中具有代表性 包括弧菌、军团菌和立克次体。这些数据和其他初步发现表明， Opia通过操纵宿主细胞膜的运输来发挥作用。在我们的第二个目标中，我们寻求识别蛋白质 在Francisella中将效应器靶向T6SS所需的，以及定义 分泌器及其底物协同调节。最后，在第三个目标中，我们建议 鉴定属于多个图拉氏丝孢子菌亚种的FPI编码的T6SS的其他底物 鉴定它们在图拉氏镰刀菌亚种致病机制中的作用。图拉尔人。总的来说，建议的 研究将为人类和动物的一个重要群体的毒力机制提供洞察 病原体。

Summary The Francisella constitute a genus of Gram-negative bacteria that occupy intracellular niches within a wide range of metazoan hosts. This group includes Francisella tularensis subsp. tularensis, a highly virulent human pathogen that has been classified as a Tier 1 select agent due to its low infectious dose, aerosol route of inoculation, and aggressive course of infection. Several studies have suggested that the key virulence factor of these bacteria is the Francisella pathogenicity island (FPI), which encodes a variant of the bacterial type VI secretion system (T6SS). However, the FPI is conserved among Francisella spp with diverse host ranges and variable capacity to cause disease. In preliminary data accompanying this proposal, we demonstrate that genes encoding substrate effector proteins of the FPI T6SS can be found at distal locations in the genome. We further find that although these effectors are variably present in Francisella spp, they play important, direct roles in promoting the virulence strategies of the bacteria that harbor them. These findings lead us to hypothesize that the FPI-encoded T6SS serves as a versatile platform for the delivery of diverse effector molecules encoded elsewhere within the genome. In our first aim we will define the molecular mechanism by which one T6SS effector of F. tularensis, OpiA, facilitates intracellular replication. Importantly, the mode of action of an FPI effector protein has not been identified to date. We show herein that OpiA is the founding member of a family of bacterial phosphatidylinositol kinases with representatives in diverse pathogens including Vibrio, Legionella, and Rickettsia spp. These data and additional preliminary findings suggest that OpiA acts by manipulating host cell membrane trafficking. In our second aim, we seek to identify the proteins required for targeting effectors to the T6SS in Francisella, as well as to define the mechanism by which the secretory apparatus and its substrates are coordinately regulated. Finally, in the third aim we propose to identify additional substrates of FPI-encoded T6SSs belonging to multiple F. tularensis subspecies and to characterize their contribution to the pathogenesis of F. tularensis subsp. tularensis. Collectively, the proposed research will provide insight into the virulence mechanisms of an important group of human and animal pathogens.