创伤弧菌转录调控因子OmpR的致病相关功能鉴定

Identification of virulence-related functions of transcription factor OmpR in Vibrio vulnificus

目的研究转录调控因子OmpR在创伤弧菌中致病相关的生物学功能及分子机制。方法通过基因无痕敲除技术构建创伤弧菌ompR基因敲除株(ΔompR)。通过生长曲线测定法检测不同渗透压下菌株的耐受力,细菌泳动试验检测运动性,结晶紫染色试验检测细菌生物膜形成能力,菌落计数法测定创伤弧菌ΔompR株黏附细胞的变化,乳酸脱氢酶释放法测定创伤弧菌ΔompR株对细胞毒性的变化,以明确ΔompR株的致病性相关生物学表型;实时荧光定量RT-PCR(qRT-PCR)法检测创伤弧菌ΔompR株和野生株中毒力相关靶基因ompN、ompU、ompA、hcp2的mRNA水平变化。结果成功构建创伤弧菌ΔompR株。与野生株相比,ΔompR株在高渗透压培养基中生长缓慢,生物膜形成能力减弱,细菌运动性未有明显差异,ΔompR株对细胞的黏附率和毒性显著低于野生株。ΔompR株中渗透压调节及生物膜形成相关ompN基因mRNA水平显著下调(P<0.05),其他靶基因mRNA水平无明显变化。结论OmpR参与调控创伤弧菌高渗透压环境下生长、生物膜形成、对细胞的黏附及毒性。

Objective To study the virulence-related functions and the pathogenic molecular mechanism of the transcription factor OmpR in Vibrio vulnificus.Methods The Vibrio vulnificus ompR mutant(ΔompR)strain was constructed using a markerless gene deletion system.Changes in the tolerance to osmotic pressure,swarming mobility and biofilm synthesis were detected by growth curve,swimming assay and crystal violet assay,respectively.Colony counting methods were used to analyze the cytoadherence of theΔompR strain.The cytotoxicity of theΔompR strain was measured by lactate dehydrogenase(LDH)releasing test.Real-time fluorescent quantitative RT-PCR(qRT-PCR)was used to detect the changes in the expression of target genes including ompN,ompU,ompA and hcp2 at mRNA level in theΔompR.Results TheΔompR strain was constructed successfully.Compared with the wild-type strain,the mutant strain showed decreased tolerance to high osmotic pressure,suppressed capability of biofilm formation and reduced cytoadherence and cytotoxicity,whereas no significant difference in motility was detected.The expression of ompN gene at mRNA level in theΔompR strain was down-regulated(P<0.05),while the expression of other target genes showed no significant changes.Conclusions The transcription factor OmpR regulated the tolerance to high osmotic pressure,biofilm formation,cytoadherence and cytotoxicity in Vibrio vulnificus.