A novel small RNA Bmsr1 enhances virulence in Brucella melitensis M28

Abstract

Brucellosis, caused by Brucella spp., is one of the most serious zoonotic bacterial diseases. Small RNAs (sRNAs) are recognized as a key player in bacterial post-transcription regulation, since they participate in many biological processes with high efficiency and may govern the intracellular biochemistry and virulence of some pathogenic bacteria. Here, a novel small regulatory RNA, Bmsr1 (Brucella melitensis M28 small RNA 1), was identified in a virulent Brucella melitensis M28 strain based on bioinformatic analysis, reverse transcription PCR (RT-PCR), and Northern blot. The Bmsr1 expression level was highly induced after infection of macrophage cells RAW264.7 at 48 h, suggesting a role for Bmsr1 during in vitro infection. Indeed, bmsr1 deletion mutant of M28 attenuated its intracellular survival in RAW264.7 at 24 h and 48 h post-infection. In a mouse model of chronic infection, bmsr1 deletion strain displayed decreased colonization in the spleen while Bmsr1-overexpressed strain showed higher colonization levels than wild type pathogen. Isobaric tags for relative and absolute quantification (iTRAQ) revealed that 314 proteins were differentially expressed in M28Δbmsr1 compared with wild type. Functional annotation analysis demonstrated that most of those proteins are involved in biological processes and those proteins in the ribosome and nitrogen metabolism pathways were enriched. iTRAQ results combined with target prediction identified several potential target genes related to virulence, including virB2, virB9, virB10, virB11, and vjbR and many metabolism genes. Taken together, this study revealed the contribution of a novel sRNA Bmsr1 to virulence of B. melitensis M28, probably by influencing genes involved in T4SS, virulence regulator VjbR and other metabolism genes.