羊种布鲁氏菌链霉素耐受基因筛选及鉴定

Screening and identification of genes associated with streptomycin resistance in Brucella melitensis

摘要:

【背景】布鲁氏菌(Brucella spp.)是一种兼性胞内寄生菌，能够引起世界范围内的人兽共患流行病——布鲁氏菌病(以下简称布病)。链霉素是治疗布病的推荐药物，但我国已有链霉素耐受分离株(根据CLSI推荐的耐药折点)。【目的】筛选并鉴定羊种布鲁氏菌(Brucella melitensis)链霉素耐受的新基因。【方法】利用转录组筛选羊种布鲁氏菌链霉素耐受新基因，并利用同源重组、分子对接等技术预测并鉴定相关基因的功能。【结果】羊种布鲁氏菌(M5疫苗株)在含2×MIC浓度链霉素培养基上，12 h后通过耐受恢复增殖能力。转录组分析表明，细胞膜组成成分在低浓度链霉素耐受中发挥重要作用，核糖体通路相关基因表达量显著性增加[|log₂FC|≥2.0, P<0.05]，群体感应通路、Ⅳ型分泌系统相关基因表达量显著性降低(|log₂FC|≥2.0, P<0.05)。利用同源重组技术缺失组成Ⅳ型分泌系统的元件，发现缺失virB3和virB5基因后，M5疫苗株链霉素MIC值增高，回补后与亲本株差异不显著。分子对接预测发现，VirB3和VirB5能够通过氢键与链霉素结合。【结论】链霉素主要影响羊种布鲁氏菌细胞膜组分涉及的相关通路。羊种布鲁氏菌通过降低Ⅳ型分泌系统组成元件virB3和virB5基因的表达耐受链霉素。本研究为布鲁氏菌耐药株研究提供了新思路，为布鲁氏菌新药研发提供了候选靶点。

关键词:羊种布鲁氏菌;链霉素;Ⅳ型分泌系统;VirB操纵子

Abstract:

[Background] Brucella spp. are facultative intracellular pathogens causing a worldwide zoonosis, brucellosis. Streptomycin is the recommended antibiotic for treating brucellosis, while streptomycin-resistant isolates have been identified in China (according to the breakpoint recommended by CLSI). [Objective] To screen and identify new genes associated with streptomycin resistance in Brucella melitensis. [Methods] We employed RNA sequencing (RNA-seq) to mine new genes associated with streptomycin resistance in B. melitensis. Homologous recombination and molecular docking were employed to predict and identify the functions of the relevant genes. [Results] B. melitensis M5 restored its proliferation after 12 h of culture in the medium supplemented with 2×MIC of streptomycin. The results of RNA-seq revealed that cell membrane components played a role in the resistance to low-concentration streptomycin. Under streptomycin stress, the expression of genes involved in the ribosome pathway was up-regulated [|log₂FC|≥2.0, P<0.05], while that of genes involved in quorum sensing and type IV secretion system was down-regulated (|log₂FC|≥2.0, P<0.05). The MICs of streptomycin for virB3- and virB5-deleted strains were higher than that for M5, while the MICs of streptomycin for complemented strains were not significantly different from that for the wild-type strain. Molecular docking results demonstrated that VirB3 and VirB5 could bind to streptomycin through hydrogen bond. [Conclusion] Streptomycin mainly affected pathways related to cell membrane components of B. melitensis. B. melitensis develops resistance to streptomycin by down-regulating the expression of virB3 and virB5 of the type IV secretion system. This study provides new insights into the research on the antibiotic-resistant strains of Brucella spp. and offers candidate targets for the development of new agents for treating brucellosis.

Key words:Brucella melitensis;streptomycin;type IV secretion system;VirB operon