肺炎链球菌pcsB组成型表达的yycF缺陷菌株的构建及其致病能力

Construction of yycF-deficient mutant Streptococcus pneumoniae D39 with constitutive expression of pcsB and study on its pathogenicity

摘要:

【背景】YycFG双组分系统是肺炎链球菌(Streptococcus pneumoniae，S. pn)应对外界环境的重要信息传递系统，其中表达反应调节子YycF的编码基因是肺炎链球菌生长的必需基因，但其是否调控细菌毒力尚不清楚。【目的】构建肺炎链球菌pcsB组成型表达及yycF缺陷菌，分析YycF对肺炎链球菌生物学特征和毒力的影响。【方法】采用Janus cassette (JC)反选的方法构建pcsB组成型表达菌株(Pc-PcsB+)，从该菌株出发用替代失活的方法构建yycF缺陷菌株(Pc-PcsB+DyycF)，比较野生株D39rpsl41、pcsB组成型表达株及yycF缺陷株的生长特性、荚膜多糖(capsular polysaccharide，CPS)含量、粘附侵袭能力和致病性的差异。【结果】成功构建pcsB组成型表达的yycF缺陷菌株(Pc-PcsB+DyycF)；yycF缺陷导致细菌生长缓慢、分裂异常、胞内荚膜多糖和小分子荚膜多糖增多；体外实验结果显示，yycF缺陷菌株粘附能力较Pc-PcsB+菌株减弱(P=0.006)。体内毒力实验显示，感染野生菌的小鼠全部死亡，感染Pc-PcsB+和Pc-PcsB+DyycF菌株的小鼠死亡率分别为91.7%、75%，二者没有统计学差异(P=0.183)，但Pc-PcsB+DyycF菌株感染组有降低趋势；定殖结果显示，yycF缺陷菌株感染组的肺匀浆菌载量显著低于对照组(P=0.033)。【结论】成功构建yycF缺陷菌株，并初步证明yycF基因会影响肺炎链球菌的生物性状和致病能力，为后续探讨YycFG双组分系统对肺炎链球菌致病能力调控机制的研究奠定了基础。

Abstract:

[Background] The YycFG two-component regulatory system plays a critical role in the Streptococcus pneumoniae response to the external environment. The response regulator protein YycF(VicR) is essential for the growth of Streptococcus pneumoniae, but the function of YycF in regulating bacterial virulence is unclear. [Objective] To analyze the effect of the response regulator protein YycF on biological characteristics and pathogenicity, the pcsB constitutive-expressing, yycF-deficient mutant strain of Streptococcus pneumoniae was constructed and characterized. [Methods] First, the pcsB constitutive expression strain (Pc-PcsB+) was constructed by janus cassette (JC) counter selection, and the yycF gene in Pc-PcsB+ was replaced with an erythromycin resistance gene (erm). The growth characteristics, the contents of capsular polysaccharide, cell adhesion and invasion abilities, and pathogenicity of D39rpsl41, Pc-PcsB+ and Pc-PcsB+DyycF were assessed. [Results] The yycF-deficient mutant strain (Pc-PcsB+DyycF) was derived from Pc-PcsB+. Compared with Pc-PcsB+, we observed slower growth, abnormal division, increasing amount of capsular polysaccharide in intracellular and smaller molecule capsular polysaccharide in the Pc-PcsB+DyycF. In vitro studies showed that the adherence ability of Pc-PcsB+DyycF was significantly reduced than that of Pc-PcsB+ (P=0.006). The virulence test suggested that all mice infected with D39rpsl41 died, while the mortality rates of mice challenged with Pc-PcsB+, Pc-PcsB+DyycF were decreased to 91.7% and 75% respectively, though no statistical significance between them was observed (P=0.183). The colonization study revealed that the bacterial burden of Pc-PcsB+DyycF in the lung tissue was significantly lower than that of Pc-PcsB+ (P=0.033). [Conclusion] In this study, the yycF-deficient mutant Streptococcus pneumoniae D39 was constructed successfully, and the biological characteristics and pathogenicity of Pc-PcsB+DyycF were identified, which provide a theoretical basis for further study on the regulatory mechanism of YycFG on the pathogenicity of Streptococcus pneumoniae.