IFN-gamma Induced Macrophage GTPases in Brucella Killing

IFN-γ 诱导巨噬细胞 GTP 酶杀死布鲁氏菌

• 批准号: 7078828
• 负责人: JAMES CARDELLI
• 金额: $ 29万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7078828
• 关键词: Brucella; bacteria infection mechanism; bactericidal immunity; guanosinetriphosphatases; interferon gamma; laboratory mouse; macrophage; vesicle /vacuole

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.

描述（由申请人提供）：布鲁氏菌属，是一种传染性很强的细菌，通过在宿主单核细胞内建立细胞内生态位导致人类和动物慢性疾病。人类布鲁氏菌病很少致命，但使人非常虚弱，需要长期使用抗生素治疗；目前还没有可用于人类的疫苗。根据这一信息，可以想象这种病原体可以用作生物战剂，因此它已被列为B类生物恐怖主义剂。在我们使用显微镜的初步研究中，我们已经确定了调理毒力菌株B. abortus和B. melitensis在修饰的晚期内体样吞噬体中存活和复制。初步研究使用显微镜的方法，我们已经确定了调理毒力菌株B. abortus和B. melitensis在人巨噬细胞系THP-1和小鼠RAW 264.7巨噬细胞中存活和复制在修饰的晚期内体样吞噬体中。ifn - γ处理导致这些吞噬体与溶酶体融合增加，从而阻止细菌复制。显微镜和亚细胞分离方法显示，ifn - γ处理的巨噬细胞吞噬体中含有不同家族的gtpase水平升高，包括LRG-47和GBP-5。过表达LRG-47可降低巨噬细胞中流产杆菌的复制，且该GTPase与布鲁氏菌吞噬体相关。我们假设布鲁氏菌的毒力菌株改变巨噬细胞基因的表达，允许产生有利于细菌生存和复制的修饰吞噬体，ifn - γ治疗通过诱导与吞噬体相关的gtp酶的合成来调节吞噬体与溶酶体的融合来克服这一点。为了验证这一假设，提出了两个目标。在第一个目标中，将使用RNAi和过表达方法结合显微镜方法来评估ifn - γ诱导的和吞噬体相关的GTPase LRG-47在降低RAW 264.7巨噬细胞和LRG-47缺失巨噬细胞中的布鲁氏菌存活中的作用。在第二个目标中，将使用类似的方法来检查gtp酶GBP-5， RabSa和Rab7在介导ifn - γ抗布鲁氏菌作用中的作用。了解布鲁氏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，应该会导致更有针对性的研究，以帮助开发治疗布鲁氏菌病的新方法。