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

作　　者：周师众 袁雅琴 宁文晴 薛天骐 杨晓雯 丁家波[1] ZHOU Shizhong;YUAN Yaqin;NING Wenqing;XUE Tianqi;YANG Xiaowen;DING Jiabo(Key Laboratory of Animal Biosafe Risk Prevention and Control(North),Ministry of Agriculture and Rural Affairs,Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Key Laboratory for Avian Preventive Medicine,Ministry of Education,Key Laboratory of Jiangsu Preventive Veterinary Medicine,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,Jiangsu,China;College of Veterinary Medicine,Shandong Agricultural University,Tai’an 271018,Shandong,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,农业农村部动物生物安全风险预警及防控重点实验室(北方),北京100193 [2]扬州大学兽医学院,禽类预防医学教育部重点实验室,江苏省动物预防医学重点实验室,江苏扬州225009 [3]山东农业大学动物医学院,山东泰安271018

出　　处：《微生物学通报》2025年第2期690-702,共13页Microbiology China

基　　金：中央级公益性科研院所基本科研业务费专项(2023-YWF-ZYSQ-09);中国农业科学院科技创新工程(CAAS-CSLPDCP-202403)。

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。[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.

关 键 词：羊种布鲁氏菌 链霉素 Ⅳ型分泌系统 VirB操纵子 

分 类 号：S852.61[农业科学—基础兽医学]