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Cytotoxicity in Macrophages Infected with Rough Brucella Mutants Is Type IV Secretion System Dependent

Department of Veterinary Pathobiology, Texas A&M University and Texas Agricultural Experiment Station, College Station, Texas 77843-4467,¹ Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing 100094, China²

Received 13 March 2007/ Returned for modification 19 April 2007/ Accepted 3 October 2007

Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16MmanBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (manBA) and the T4SS (virB). As predicted, deletion of virB from 16MmanBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.

Published ahead of print on 15 October 2007.

Editor: V. J. DiRita