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Infection and Immunity, May 2006, p. 2667-2675, Vol. 74, No. 5
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.5.2667-2675.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Brucella abortus Rough Mutants Induce Macrophage Oncosis That Requires Bacterial Protein Synthesis and Direct Interaction with the Macrophage

Jianwu Pei, Joshua E. Turse, Qingmin Wu, and Thomas A. Ficht^*

Department of Veterinary Pathobiology, Texas A&M University and Texas Agricultural Experiment Station, College Station, Texas 77843-4467

Received 21 November 2005/ Returned for modification 2 January 2006/ Accepted 1 February 2006

Previous studies suggest that smooth Brucella organisms inhibit macrophage apoptosis. In contrast, necrotic cell death of macrophages infected with rough Brucella organisms 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 abortus S2308-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. abortus S2308 or pretreated with B. abortus O polysaccharide did not prevent rough CA180-induced cell death. In conclusion, Brucella rough mutant infection induces necrotic and oncotic macrophage cell death that requires bacterial protein synthesis and direct interaction of bacteria with the target cells.

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* Corresponding author. Mailing address: Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843-4467. Phone: (979) 845-4118. Fax: (979) 862-1088. E-mail: tficht{at}cvm.tamu.edu.

Editor: D. L. Burns

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Infection and Immunity, May 2006, p. 2667-2675, Vol. 74, No. 5
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.5.2667-2675.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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