Identification Of Virulence Related Targets Of The MucR Protein In Brucella Melitensis

Brucellosis is a serious zoonotic disease caused by Brucella spp,which threats human health and animal husbandry. Transcriptional regulator MucR is a virulence factor of Brucella spp., while the related mechanism of Brucella virulence of MucR and its target genes is still unknown. In this study, we used RNA-seq and chromatin Immunoprecipitation to solve these questions.To determine the optimum condition of RNA-seq and chromatin Immunoprecipitation, lacZ report gene assay and Western blot were used to detect the expression level of mucR gene at different growth stages and stress conditions. It showed that the transcriptional level and translational level of the mucR gene were increased at lag phase and early logarithmic phase, and achieved the highest level during the late logarithmic phase. This feature made us consider that the expression of MucR might be affected by quorum sensing system. Our date showed that the expression of MucR was almost the same in△vibR and16M, and it suggested that the expression of MucR was not affected by quorum sensing related protein VjbR. When Brucella melitensis16M was cultured at different stress mediums, the expression levels of MucR were also detected, and this protein was rapidly decreased at acidic medium (pH4.5). To detect if the decrease of MucR was caused by proteolysis, bacterial protein synthesis was inhibited by chloromycetin, and it demonstrated that the decrease of MucR was mainly caused by the enhancement of proteolysis. According to above results, the optimum condition of RNA-seq and chromatin Immunoprecipitation is culturing bacteria to late logarithmic phase in TSB medium.To study the MucR related mechanism of bacterial virulence, we performed RNA-seq analysis using Brucella melitensis RNA obtained from B. melitensis16M and the mucR mutant. In total, the expression levels of442genes were significantly changed in the mucR mutant, including310up-regulated genes and132down-regulated genes. According to the result of COG analysis, these differently expressed genes were involved in metabolism, cell wall/envelope biogenesis, and transcriptional regulation. Results of stress assays demonstrated that MucR was involved in tolerance to acid, iron-limitation, and cationic polypeptides. These results mentioned above should be the possible reasons of the attenuation of the mucR mutant.The target genes regulated by MucR were also detected in our study.77differently expressed genes were verified by chromatin immunoprecipitation, and14target genes were found. These target genes contained7genes involved in transcriptional regulation,2genes encoding outer membrane proteins,2metal ion transport genes,1gene involved in resistance of acidic stress,1gene related to denitrification, and1alkaline phosphatase. These results suggested that the sensitive to acidic and iron-limiting environment, changes of bacterial outer membrane properties, and different expression of genes involved in denitrification pathway were caused by different expression of MucR target genes in mucR mutant.Our study demonstrated MucR related mechanism of bacterial virulence and the target genes regulated by MucR, which provided insights for further studies of Brucella regulation network and intracellular survival mechanisms.