Identification of genetic loci and transcriptional networks that confer virulence and survival of Brucella melitensis



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Brucella melitensis is the etiological agent of brucellosis, a zoonotic disease characterized by abortions in ruminant animals and a chronic debilitating disease in humans. Despite genome sequencing, little is known about the genetic elements behind Brucella?s ability to survive and cause disease. Regulatory networks provide the ability to adapt to changing environments by initiating expression from specific regulons to provide adjustments to metabolism and mechanisms that enhance survival. Little detail is known about transcriptional networks that exist in Brucella, but are of great interest because they could provide information about genetic loci that contribute to virulence and intracellular survival. Transposon mutagenesis identified gene loci that are indispensable for the intracellular replication of B. melitensis, including virulence genes, metabolic defects, and transcriptional regulators. Two transcriptional regulators of interest were identified, MucR and VjbR. VjbR is a LuxR homologue and is associated with the regulation of virulence genes in a density dependent manner in a number of bacterial pathogens, and is consistent with VjbR regulation of virulence genes in B. melitensis. Microarray analysis of ?vjbR and a potential activating signal C12-HSL revealed that both regulate numerous putative virulence genes, including adhesins, proteases, protein secretion/translocation components, potential effector proteins, lipoproteins, a hemolysin and stress survival aids. This analysis also revealed that C12-HSL is not an activating signal of VjbR, but instead acts to suppress VjbR activity. MucR is a transcriptional regulator shown to regulate exopolysaccharide synthesis in the closely related Rhizobiales. Microarray analysis of a ?mucR mutant in B. melitensis suggested that MucR contributes to the regulation of nitrogen metabolism and iron sequestering/storage. MucR was also found to regulate genes involved in stress response, regulating several proteases that may contribute to enhanced survival and virulence of the organism. This work identified approximately 1,000 genetic loci that may be important to the survival of B. melitensis, revealing potential virulence genes and metabolic defects. Interruption of the VjbR regulon could be a potential chemotherapeutic target for the treatment of brucellosis. Furthermore, this work describes the functions of two gene deletions that are being evaluated as novel attenuated vaccines.