Role of IFN-gamma Induced Macrophage GTPases in Brucella Killing

性状（由申请方提供）：布鲁氏菌属是一种高度感染性细菌，通过在宿主单核细胞内建立细胞内生态位而引起人类和动物慢性疾病。人类布鲁氏菌病很少致命，但非常虚弱，需要长期使用抗生素治疗;目前没有疫苗可用于人类。根据这一信息，可以想象，这种病原体可能被用作生物战剂，因此，它已被列为B类生物恐怖主义剂。在我们采用显微镜方法的初步研究中，我们已经确定了调理剂的B强毒株。流产和B. melitensis存活和复制在一个修改后的内体样吞噬体的初步研究，采用显微镜的方法，我们已经确定，调理毒性菌株的B。流产和B. Melitensis在人巨噬细胞系THP-1和小鼠RAW 264.7巨噬细胞中的修饰的晚期内体样吞噬体中存活和复制，并且IFN-γ处理导致这些吞噬体与溶酶体的融合增加，从而防止细菌复制。显微镜和亚细胞分级分离方法显示，IFN-γ处理的巨噬细胞中的吞噬体含有增加水平的属于不同家族的GTP酶，包括LRG-47和GBP-5。LRG-47的过表达减弱了B。巨噬细胞中的流产复制，并且这种GT3与布鲁氏菌吞噬体相关。我们假设布鲁氏菌的强毒株改变巨噬细胞基因的表达，以允许产生有利于细菌存活和复制的修饰的吞噬体，并且IFN-γ处理通过诱导与吞噬体相关的GTP酶的合成来调节吞噬体-溶酶体融合来克服这一点。提出了两个目标来检验这一假设。在第一个目标中，将使用RNAi和过表达方法与显微镜方法相结合来评估IFN-γ诱导的和吞噬体相关的GTT LRG-47在降低RAW 264.7巨噬细胞和LRG-47无效巨噬细胞中布鲁氏菌存活率中的作用。在第二个目标中，将使用可比较的方法来检查GTP酶GBP-5、RabSa和Rab 7在介导IFN-γ的抗布鲁氏菌作用中的作用。了解布鲁氏杆菌如何在转录和蛋白质运输水平上改变宿主细胞的反应，允许这种病原体在细胞内存活，以及IFN-γ如何克服这一点，应该引起更集中的研究，以帮助开发治疗布鲁氏菌病的新方法。

DESCRIPTION (provided by applicant): Brucella spp, are highly infectious bacteria that cause chronic disease in humans and animals by establishing an intracellular niche within host monocytes. Human brucellosis is rarely fatal but is very debilitating and requires long-term treatment with antibiotics; no vaccine is currently available for use in humans. Based on this information it is conceivable this pathogen could be used as a biowarfare agent, and accordingly it has been classified as a Category B Bioterrorism agent. In our preliminary studies employing microscopic approaches, we have determined that opsonized virulent strains of B. abortus and B. melitensis survived and replicated in a modified late endosome-like phagosome preliminary studies employing microscopic approaches, we have determined that opsonized virulent strains of B. abortus and B. melitensis survived and replicated in a modified late endosome-like phagosome in the human macrophage cell-line THP-1 and mouse RAW 264.7 macrophages, and that IFN-gamma treatment resulted in increased fusion of these phagosomes with lysosomes, thus preventing bacterial replication. Microscopic and subcellular fractionation approaches revealed that phagosomes in IFN-gamma treated macrophages contained increased levels of GTPases belonging to different families, including LRG-47 and GBP-5. Overexpression of LRG-47 attenuated B. abortus replication in macrophages, and this GTPase associated with Brucella phagosomes. We hypothesize that virulent strains of Brucella alter the expression of macrophage genes to allow for the generation of a modified phagosome conducive for survival and replication of the bacteria, and that IFN-gamma treatment overcomes this by inducing the synthesis of GTPases that associate with phagosomes to regulate phagosome-lysosome fusion. Two aims are proposed to test this hypothesis. In the first aim, RNAi and overexpression approaches combined with microscopic approaches will be used to assess the role of the IFN-gamma-induced and phagosome-associated GTPase LRG-47 in reducing Brucella survival in RAW 264.7 macrophages and LRG-47 null macrophages. In the second aim, comparable approaches will be used to examine the role of the GTPases GBP-5, RabSa and Rab7 in mediating the anti-Brucella affects of IFN-gamma. Understanding how Brucella spp. alter the response of the host cell at the transcriptional and protein trafficking level, allowing for intracellular survival of this pathogen, and how IFN-gamma overcomes this should lead to more focused studies to aid in the development of novel approaches in the treatment of brucellosis.