RfaH通过调控荚膜多糖合成介导肺炎克雷伯菌持留进化的作用研究

Microbial persistence is the leading cause of relapsing infections. Long-term antibiotic therapy can drive persistence evolution of pathogens, but the evolutionary mechanism is still unclear. RfaH is a transcriptional antiterminator, which is involved in the regulation of expression of certain extracytoplasmic macromolecules. The role of RfaH in the persistence evolution of pathogens has been barely revealed. In our previous study, we found an insertion mutation in the rfaH gene of high-persister mutant of K. pneumoniae. The complementary test showed that the persistence of the rfaH complemented strain was returned back to wild-type level, which suggested that rfaH played important roles during the persistence evolution of K. pneumoniae. In addition, we also found that the persistence evolution of K. pneumoniae was accompanied by the changes on colonial morphotypes, from an opaque form to a translucent phenotype. According to relevant research materials, we speculated that the changes of colonial morphotypes are closely related to the production of capsular polysaccharide. In this study, we aim to confirm that RfaH is a key factor of persistence evolution in K. pneumoniae, characterize the functions of RfaH on the production of capsular polysaccharide and reveal the regulatory network of RfaH on the persistence evolution of K. pneumoniae. Our project will help to lay a foundation for further elucidating the mechanism of bacterial persistence evolution, and provide new ideas and targets for clinical diagnosis and treatment of K. pneumoniae persistence infections.

微生物持留是导致临床复发感染的重要因素。长期药物治疗可驱动病原菌形成高水平持留，但该持留进化机制不详。RfaH是一种转录抗终止因子，参与调控细菌多种胞外组分的合成，但其在持留进化中的作用鲜有研究。我们的前期工作显示，肺炎克雷伯菌持留进化株的rfaH基因发生了插入突变。回补试验发现，rfaH的回补表达使得该进化株的持留能力几乎降至原始菌株水平，提示rfaH可能在肺炎克雷伯菌持留进化过程中发挥重要作用。前期研究还发现，该菌持留进化过程伴随着菌落形态由最初的不透明变成半透明。结合文献资料推测，这种表型改变与RfaH调控荚膜多糖合成有关。本项目拟证实RfaH是介导肺炎克雷伯菌持留进化的重要因子，明确RfaH对荚膜多糖合成的调控作用，鉴定RfaH对肺炎克雷伯菌持留进化的调控网络。本项目的开展有助于为深入阐明细菌持留进化机制奠定研究基础，并为肺炎克雷伯菌持留感染的临床诊治提供新思路和新靶点。

微生物持留是导致临床药物治疗失败和复发感染的重要原因，目前已成为全球关注的公共卫生问题，周期性抗生素治疗可导致病原菌群体中持留菌比例显著升高，即发生持留进化，但该进化机制还不很清楚。我课题组前期研究显示，rfaH可能参与了肺炎克雷伯菌的持留进化。于是，我课题组通过同源重组，成功构建了肺炎克雷伯菌rfaH的缺失株与回补株，但在探究rfaH对肺炎克雷伯菌持留进化和荚膜多糖合成的调控作用时，发现rfaH对荚膜多糖合成确有调控作用，但并不影响该菌的持留水平。我们进一步研究了rfaH对肺炎克雷伯菌毒力的调控作用，发现肺炎克雷伯菌rfaH参与调控了该菌荚膜的形成、菌毛的合成以及LPS的形成，该基因的缺失显著影响肺炎克雷伯菌细胞间黏性，使细菌抗血清杀伤能力显著降低，rfaH是肺炎克雷伯菌的一个重要的毒力调控因子。本研究为肺炎克雷伯菌感染的临床诊治提供了新的靶点。