Cytotoxicity of Brucella smooth strains for macrophages is mediated by increased secretion of the type IV secretion system

1 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, PR China 2 Institute of Disease Control and Prevention, Academy of Military Medical Science, Beijing 100071, PR China Some Brucella rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a virB inactivation mutant (BM virB) and an overexpression strain (BM-VIR) of a smooth Brucella melitensis strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BM virB. The quorum sensing signal molecule N -dodecanoyl- DL -homoserine lactone (C 12 -HSL), a molecule that can inhibit expression of virB , inhibited the cytotoxicity of BM and BM-VIR, but not of BM virB. These results indicated that overexpression of virB is responsible for Brucella cytotoxicity in macrophages. Transcription analysis showed that virB is regulated in a cell-density-dependent manner both in in vitro culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to in vitro stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of Brucella for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of virB or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of virB are detrimental for Brucella intracellular survival also indicated that the expression of virB is tightly regulated in a cell-density-dependent manner. Correspondence Zeliang Chen zeliangchen{at}yahoo.com Abbreviations: C 12 -HSL, N -dodecanoyl- DL -homoserine lactone; LDH, lactate dehydrogenase; T4SS, type