Key Laboratory of Veterinary Public Health of Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
Key Laboratory of Veterinary Public Health of Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou, 225009, China.
Vet Microbiol. 2018 Sep;223:1-8. doi: 10.1016/j.vetmic.2018.07.007. Epub 2018 Jul 20.
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.
布鲁氏菌病是由布鲁氏菌属引起的一种最严重的人畜共患细菌性疾病。小 RNA（sRNA）被认为是细菌转录后调控的关键因素，因为它们高效参与许多生物学过程，并可能控制一些致病菌的细胞内生物化学和毒力。在这里，根据生物信息学分析、逆转录 PCR（RT-PCR）和 Northern blot，在一种毒力强的布鲁氏菌 melitensis M28 菌株中鉴定出一种新型小调控 RNA，Bmsr1（布鲁氏菌 melitensis M28 小 RNA 1）。Bmsr1 的表达水平在 RAW264.7 巨噬细胞感染后 48 h 时高度诱导，表明 Bmsr1 在体外感染过程中发挥作用。事实上，M28 的 bmsr1 缺失突变体在感染后 24 和 48 h 在 RAW264.7 中的存活能力减弱。在慢性感染的小鼠模型中，bmsr1 缺失株在脾脏中的定植减少，而 Bmsr1 过表达株的定植水平高于野生型病原体。相对和绝对定量的同位素标记（iTRAQ）显示，与野生型相比，M28Δbmsr1 中有 314 种蛋白质表达差异。功能注释分析表明，这些蛋白质大多数参与生物过程，核糖体和氮代谢途径中的蛋白质丰富。iTRAQ 结果与靶标预测相结合，鉴定出与毒力相关的几个潜在靶基因，包括 virB2、virB9、virB10、virB11 和 vjbR 以及许多代谢基因。总之，这项研究揭示了一种新型 sRNA Bmsr1 对 B. melitensis M28 毒力的贡献，可能是通过影响 T4SS 相关基因、毒力调节因子 VjbR 和其他代谢基因来实现的。
