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ABSTRACTPrevious studies suggest that smooth Brucellaorganisms inhibit macrophage apoptosis. In contrast, necrotic cell death of macrophages infected with rough Brucellaorganisms in vitro has been reported, which may in part explain the failure of some rough organisms to thrive. To characterize these potential macrophage killing mechanisms, J774.A1 murine macrophages were infected with Brucella abortusS2308-derived rough mutant CA180. Electron microscopic analysis and polyethylene glycol protection assays revealed that the cells were killed as a result of necrosis and oncosis. This killing was shown to be unaffected by treatment with carbenicillin, an inhibitor of bacterial cell wall biosynthesis and, indirectly, replication. In contrast, chloramphenicol treatment of macrophages infected at multiplicities of infection exceeding 10,000 prevented cell death, despite internalization of large numbers of bacteria. Similarly, heat-killed and gentamicin-killed CA180 did not induce cytopathic effects in the macrophage. These results suggested that killing of infected host cells requires active bacterial protein synthesis. Cytochalasin D treatment revealed that internalization of the bacteria was necessary to initiate killing. Transwell experiments demonstrated that cell death is not mediated by a diffusible product, including tumor necrosis factor alpha and nitric oxide, but does require direct contact between host and pathogen. Furthermore, macrophages preinfected with B. abortusS2308 or pretreated with B. abortusO polysaccharide did not prevent rough CA180-induced cell death. In conclusion, Brucellarough mutant infection induces necrotic and oncotic macrophage cell death that requires bacterial protein synthesis and direct interaction of bacteria with the target cells.

Anaplasma marginaleis a prototypical highly antigenically variant bacterial pathogen dependent on the sequential generation of major surface protein 2 (Msp2) outer membrane variants to establish persistent infection. Msp2 is encoded by a single expression site, and diversity is achieved by gene conversion of chromosomally encoded msp2pseudogenes.