Identification and Analysis of Host Factors that Support Brucella Infection

描述（由申请人提供）：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，但对哺乳动物细胞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型的黑腹果蝇S2细胞布鲁氏菌感染模型，对支持这种细胞内病原体复制的宿主因子进行了初步筛选。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很重要的基因。此外，我们还发现了一些在哺乳动物细胞模型中得到验证的新发现。在这里，我们建议利用这些发现对其他宿主因子进行大规模的RNAi筛选。具体来说，我们的目标是：(1)对破坏或增强果蝇S2细胞的B. melitensis感染的rnai进行大规模筛选；(2)将hit分为表型和功能两类，并与近期完成的偶发分枝杆菌和单核增生李斯特菌筛查结果进行比较。最后，我们将采用siRNA技术来检验在我们的果蝇S2细胞筛选中获得的hit的哺乳动物同源物是否介导了哺乳动物细胞的布鲁氏菌感染。综上所述，这些实验将确定支持B. melitensis在动物细胞中的摄取和复制的其他宿主因子，从而为介导这一重要宿主-病原体相互作用的分子机制提供新的见解。

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.