流产布鲁菌LysR家族转录调控子LTTR8缺失株的构建及其生物学特性分析

Construction and Biological Characteristics Analysis of a LysR Family Transcriptional Regulator LTTR8 Mutant of Brucella abortus

布鲁菌是一种重要的人畜共患病病原,兼性胞内寄生,无典型毒力因子,毒力相关基因的研究对阐明布鲁菌持续感染的机制具有重要意义。本研究以流产布鲁菌S2308为亲本株,通过同源重组的方法,构建流产布鲁菌LysR家族转录调控子基因bab_RS24670缺失株ΔlttR8,基于广宿主质粒pBBR1MCS构建成互补株ΔlttR8-Com。免疫印迹试验表明:ΔlttR8为光滑型菌株;体外培养分析发现lttR8缺失不影响布鲁菌的生长;细胞感染试验发现lttR8缺失不影响布鲁菌黏附、入侵细胞和胞内存活的能力。耐受性试验分析发现:lttR8缺失不影响布鲁菌对过氧化氢(H2O2)和多粘菌素B(polymyxin B)的敏感性。小鼠感染试验分析发现lttR8缺失显著减弱流产布鲁菌的毒力。综上所述,本研究发现一个新的与流产布鲁菌致病性相关的转录调控子,为布鲁菌致病机制研究提供参考。

Brucellais an important zoonotic intracellular pathogen without typical virulence factors.Identification and functional analysis of virulence-related genes are of great significance to clarify the mechanism of Brucellachronic infection.In this study,B.abortusstrain S2308 was used as the parental strain to construct a LysR-family transcription regulator bab_RS24670gene mutantΔlttR8by homologous recombination.At the same time,a complemented strainΔlttR8-Com was also constructed based on the broad host plasmid pBBR1MCS.Western blotting showed that theΔlttR8 was a smooth phenotypic strain.Growth curve analysis found that the lttR8 deletion did not affectBrucellagrowth.Cell infection experiments showed that thelttR8gene was not associated withBrucellaadhesion to,invasion into cells and intracellular survival.Tolerance test showed that theΔlttR8 mutant was sensitive to hydrogen peroxide(H2O2)and polymyxin B as same as the parental S2308 strain.Virulence test revealed that theΔlttR8 significantly reduced its virulence in mouse infection in comparison with the parent S2308.In conclusion,this study found a novel transcriptional regulator associated with B.abortus,which provided a reference for the study ofBrucellapathogenicity.