In vitro and in vivo analyses of Brucella abortus genes identified in RAW 264.7 macrophage infection using a GFP reporter system

Brucella abortus is a gram-negative, facultative intracellular bacterium that infects humans and domestic animals. The mechanisms by which Brucella cells evade the antibacterial defenses, resist intracellular killing and reach their replicating niche in the host cell remain mostly unknown. Therefore, understanding the interaction between the host cell and Brucella may contribute to a greater knowledge of the infection mechanisms and pathogenesis of the disease.; We investigated the B. abortus genes potentially critical for establishing infection and evaluate their significance in bacterial survival in vitro and in vivo. Using a GFP reporter system, several B. abortus genes of potential importance were identified on agar or 4 or 24 h following RAW 264.7 macrophage infection. Extracellularly producing GFP clones exhibited sequence homology with genes associated with protein synthesis and metabolism, and detoxification. B. abortus genes potentially activated in macrophages 4 h postinfection appear to be involved in the adaptation to intracellular environmental conditions, including genes for detoxification, repair, and osmotic protection. Additionally, genes for site-specific recombination, and peptidoglycan synthesis were also identified. Genes activated 24 h following infection were those involved in biosynthesis and metabolism.; Among the set of genes identified using the GFP expression system, an integrase/recombinase (xerD) and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) genes respectively involved in the process of cell division and membrane structure of other bacteria, were further studied in Brucella pathogenicity. The survival of B. abortus xerD and mtgA insertional mutant was evaluated in vitro and in vivo. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice revealed that mice infected with B. abortus xerD::kan and B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in BALB/c mice, B. abortus xerD::kan presented a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.