Identification and Analysis of Host Factors that Support Brucella Infection

描述(申请人提供)：羊布鲁氏菌是一种具有全球意义的人和动物细菌性病原体。尽管宿主-病原体相互作用的细菌因素已被揭示，但对哺乳动物细胞的细菌感染起重要作用的宿主因素仍不清楚。我们最近利用了一种新的布鲁氏菌感染的果蝇黑腹果蝇S2细胞模型来进行支持这种细胞内病原体复制的宿主因素的初步筛选。我们发现了已经被证实对布鲁氏菌感染哺乳动物细胞很重要的基因。此外，我们发现了几种在哺乳动物细胞模型中得到验证的新的命中结果。在这里，我们建议利用这些发现来进行大规模的RNAi筛查，以寻找额外的宿主因素。具体地说，我们的目标是：(1)对干扰或增强蜜蜂对果蝇S2细胞感染的RNA进行大规模筛选；(2)将HIT分为表型和功能类别，并将所获得的结果与最近完成的偶发分枝杆菌和单核细胞增多性李斯特菌筛查的结果进行比较。最后，我们将使用siRNA技术来检查在我们的果蝇S2细胞筛选中获得的HITS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，这些实验将定义更多的宿主因素，支持白念珠菌在动物细胞中的吸收和复制，从而为调节这一重要的宿主-病原体相互作用的分子机制提供新的见解。 与公共卫生相关：该提案应确定并表征介导布鲁氏菌感染动物细胞的新宿主因素。我们预计，这一努力将通过极大地促进我们对这种具有全球意义的病原体的了解，并通过发现潜在的治疗干预蛋白质靶标，从而使公众健康受益。

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.