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Infect. Immun. doi:10.1128/IAI.00379-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cytotoxicity in macrophage infected with rough Brucella is type IV secretion system-dependent

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

^* To whom correspondence should be addressed. Email: tficht{at}cvm.tamu.edu.

	Abstract

Smooth Brucella inhibit macrophage apoptosis, whereas rough Brucella induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current report, a random mutagenesis approach was used to create a mutant bank consisting of 11354 mutants by mariner transposon mutagenesis using B. melitensis rough mutant 16MmanBA as parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (10% or less of LDH release). 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 from 51 mutants were identified by DNA sequence analysis and represented interruptions in virB, encoding the type four secretion system (T4SS) (n=36); in vjbR, encoding a LuxR-like regulatory element previously shown to be required for virB expression (n=3); and some additional mutants (n=12), among which several also have predicted roles in virB expression. These results suggest that T4SS is associated with Brucella cytotoxicity in macrophage. To verify this observation, 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 the 16MmanBA and 16M resulted in a complete loss of cytotoxicity from rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme MOI>1000. Taken together, these results demonstrate that Brucella cytotoxicity in macrophage is T4SS-dependent.