sRNA STnc440分子特征及对鼠伤寒沙门菌毒力的影响

Molecular characteristics and effects of sRNA STnc440 on virulence of Salmonella typhimurium

【目的】探究鼠伤寒沙门菌(STM)sRNA STnc440的分子特征及对细菌毒力的影响,为进一步揭示STnc440的调控机制奠定基础。【方法】克隆STnc440基因序列并进行分子特征分析,采用λ-Red重组技术构建了STnc440缺失株△STnc440和回补株△STnc440/STnc440,通过细胞侵染和小鼠感染试验研究STnc440对STM毒力的影响,预测分析STnc440调控的靶基因。【结果】sRNA STnc440基因大小为81 bp,上游序列含有-10 box和-35 box启动子核心区域,遗传进化分析表明该基因在沙门菌属中相对保守。与STM亲本株和回补株相比,缺失株△STnc440在生长特性上无明显差异,对巨噬细胞的粘附能力和侵袭力显著减弱,LD50显著升高,在小鼠肝脏和脾脏中的定殖能力均显著降低(P<0.05)。靶基因预测分析发现,STnc440的靶基因可能为SL1344_2880(Ⅰ型毒力岛蛋白)。【结论】sRNA STnc440对STM毒力有一定的调控作用,可能通过调控SL1344_2880来实现。

【Objective】This study explored molecular characteristics of Salmonella typhimurium(STM)sRNA STnc440 and its influence on bacterial virulence to lay foundation for further understanding of STnc440 regulatory mechanism.【Method】STnc440 gene sequence was cloned and molecular characteristics were analyzed.Then,gene mutant strain△STnc440 and complemented strain△STnc440/STnc440 were constructed byλ-Red recombination technology.The effects of STnc440 gene deletion on virulence of STM were explored through cell infection and mouse infection experiments.The target genes regulated by sRNA STnc440 were also analyzed.【Result】The sRNA STnc440 gene was 81 bp in length,containing-10 box and-35 box promoter regions in the upstream sequence.Phylogenetic analysis showed that the gene was relatively conservative in Salmonella.Compared with parental strains and complemented strain,there was no significant difference in growth in△STnc440.However,the adhesion and invasion ability of macrophages was significantly weakened in△STnc440 mutant strain.The LD50 of△STnc440 in mice was significantly increased and colonization ability of△STnc440 in liver and spleen of mice was significantly reduced(P<0.05).Target gene prediction analysis found that the target gene of STnc440 may be SL1344_2880(typeⅠvirulence island protein).【Conclusion】The sRNA STnc440 had regulatory effect on STM virulence likely by regulating SL1344_2880.