Identification and Analysis of Host Factors that Support Brucella Infection

性状（由申请方提供）：羊种布鲁氏菌是一种具有全球意义的人类和动物细菌病原体。虽然介导宿主-病原体相互作用的细菌因子已经被揭示，但是对于哺乳动物细胞的细菌感染重要的宿主因子仍然不清楚。我们最近开发了一种新的果蝇S2细胞模型布鲁氏菌感染进行试点筛选宿主因子，支持这种细胞内病原体的复制。我们发现了已经被确定为对哺乳动物细胞的布鲁氏菌感染重要的基因。此外，我们发现了几个在哺乳动物细胞模型中验证的新命中。在这里，我们建议利用这些发现来进行大规模的RNAi筛选，以寻找其他宿主因子。具体而言，我们的目标是：（1）进行大规模筛选破坏或增强B的RNAi。Melitensis感染的果蝇S2细胞;（2）将命中分类为表型和功能类别，并将获得的结果与最近完成的偶发分枝杆菌和单核细胞增生李斯特菌筛选的结果进行比较。最后，我们将采用siRNA技术来检查在我们的果蝇S2细胞筛选中获得的命中的哺乳动物直系同源物是否介导哺乳动物细胞的布鲁氏菌感染。总之，这些实验将确定支持B摄取和复制的其他宿主因子。melitensis进入动物细胞，从而提供新的见解介导这一重要的宿主-病原体相互作用的分子机制。 公共卫生相关性：本提案应识别和表征介导动物细胞布鲁氏菌感染的新型宿主因子。我们希望这项工作将有利于公共卫生，有助于我们了解这种具有全球意义的病原体，并发现潜在的蛋白质靶点进行治疗干预。

DESCRIPTION (provided by applicant): Brucella melitensis is a human and animal bacterial pathogen of global significance. Although bacterial factors mediating the host-pathogen interaction have been revealed, host factors that are important for bacterial infection of mammalian cells remain obscure. We have recently exploited a novel Drosophila melanogaster S2 cell model of Brucella infection to perform a pilot screen for host factors that support the replication of this intracellular pathogen. We uncovered genes that have already been established as being important for Brucella infection of mammalian cells. In addition, we uncovered several novel hits that were validated in mammalian cell models. Here, we propose to exploit these findings to perform a large-scale RNAi screen for additional host factors. Specifically, we aim: (1) To perform a large-scale screen for RNAis that disrupt or enhance B. melitensis infection of Drosophila S2 cells; (2) To classify hits into phenotypic and functional categories, and to compare the results obtained to those from the recently completed Mycobacterium fortuitum and Listeria monocytogenes screens. Finally, we shall employ siRNA technology to examine whether the mammalian orthologs of the hits obtained in our Drosophila S2 cell screen mediate Brucella infection of mammalian cells. Taken together, these experiments will define additional host factors that support the uptake and replication of B. melitensis into animal cells, and thereby provide new insights into the molecular mechanisms mediating this important host-pathogen interaction. PUBLIC HEALTH RELEVANCE: This proposal shall identify and characterize novel host factors mediating Brucella infection of animal cells. We expect that this effort shall benefit public health by contributing significantly to our understanding of this pathogen of global significance, and by discovering potential protein targets for therapeutic intervention.