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. ? ? ? ?
| Rupper, Adam C; Cardelli, James A (2008) Induction of guanylate binding protein 5 by gamma interferon increases susceptibility to Salmonella enterica serovar Typhimurium-induced pyroptosis in RAW 264.7 cells. Infect Immun 76:2304-15 |
