Comparison of transcriptional change of B. melitensis M5-90 after macrophage infection highlights the role of ribosome gene L31 in virulence.

Brucella, a facultative intracellular bacterium, can survive and replicate in various cell types such as epithelial cell, fibroblasts and macrophage. Macrophage is the most important sites for the survival of Brucella in vivo. The mechanisms of pathogenesis are difficult to address, since the unknown virulence genes are still exist. RNA-seq is available to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Here we described and analyzed the transcriptional change of avirulent strain Brucella melitensis M5-90 (B. melitensis M5-90) during macrophage infection using RNA-seq technology. We detected 601 significant changed genes of which 428 were upregulated after infection. The upregulated gene L31 which involved in ribosome KEGG pathway was selected to illustrate its effect on virulence in a vaccine strain B. melitensis M5-90 and a virulent strain B. melitensis M28. Deletion of L31 significant attenuates the spleen colonization in model of M5-90 or M28 infection mouse at 7, 21 and 35 days post-infection (P < 0.05). We further examine the role of L31 in a macrophage cell infection model, and the result showed a significant reduction of intracellular M28ΔL31 cells at 48 h post-infection (P < 0.001). In total, our study provided a view of transcriptional landscape of B. melitensis M5-90 intracellular, and found L31 gene is required for the full virulence of B. melitensis.

A novel small RNA Bmsr1 enhances virulence in Brucella melitensis M28.

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.