质粒编码的PagO蛋白促进高毒力肺炎克雷伯菌致病性的分子机制

Hypervirulent Klebsiella pneumoniae (hvKp) possesses the ability to cause serious infections, such as community acquired liver abscess in healthy hosts, which is associated with high mortality rate, while its pathogenesis mechanisms still remain elucidated. Previous investigations revealed that the virulence plasmid universally carried by hvKp encoded a new kind of transmembrane protein, PagO, which acted as the secondary active transporter for amino acids and played a key role in host innate immunity resistance. To elucidate the molecular mechanisms of plasmid-encoded PagO for enhancing virulence in hvKp, its secretion of cysteine was analyzed based on the previous construction of pagO knock-out and complementation strains. Hydrogen peroxide sensitivity assay and phagocytosis analysis will be employed to determine the role of PagO in cysteine-cystine transmembrane shuttle system, and to elucidate the molecular mechanisms for PagO in defending host oxidative stress and enhancing hvKp virulence. The cofactors of PagO will also be screened and their biological functions will be determined. This study also aims to investigate the regulation mechanisms of pagO gene expression exerted by the PhoP/Q two-component regulatory system and global regulators. The whole regulation pathway, from stimulus sensing of PhoP/Q to pagO gene activation, will also be determined. This research focuses on the interaction between chromosome and virulence plasmids to investigate the mechanisms of the development of pathogenicity in hvKp, and the findings will provide new ideas for the treatment of hvKp infections.

高毒力肺炎克雷伯菌（hvKp）可引起肝脓肿等严重感染，病死率高，其高毒力形成及致病机制仍有待阐明。申请者前期研究发现hvKp携带的毒力质粒中编码PagO蛋白，该蛋白为氨基酸次级主动转运体，在抵抗宿主固有免疫防御和促进hvKp致病性中扮演重要角色。为阐明PagO蛋白促进hvKp致病性的分子机制，本项目拟在已获得pagO基因敲除和回补株基础上，探讨其对半胱氨酸的分泌作用，构建过氧化氢应激及免疫细胞吞噬模型，阐明该蛋白参与跨膜的半胱氨酸-胱氨酸循环，增强细菌抗氧化应激损伤，抵抗免疫细胞吞噬和清除，进而促进体内侵袭性感染能力，并对其辅助蛋白进行初步筛选和功能分析。同时探讨PhoP/Q双组分调控系统和全局调控子对pagO基因表达的调控作用，明确PhoP/Q从应激物感知至pagO基因活化的整体分子调控通路。本项目拟从染色体和质粒相互作用的角度探讨hvKp高毒力的形成机制，为临床抗感染治疗提供新思路。

高毒力肺炎克雷伯菌（hvKp）可引起肝脓肿等严重感染，病死率高，其高毒力形成及致病机制仍有待阐明。项目负责人前期研究发现，hvKp携带的毒力质粒中编码PagO蛋白，该蛋白为氨基酸次级主动转运体，在抵抗宿主固有免疫防御和促进hvKp致病性中扮演重要角色。本项目拟在已获得pagO基因敲除和回补株基础上，探讨PagO蛋白促进hvKp致病性的分子机制及pagO基因表达调控的分子机制。研究发现，PagO蛋白为半胱氨酸转运体，通过与胱氨酸转运体Fliy协同作用，参与跨膜的胱氨酸-半胱氨酸循环，共同对抗外源性活性氧介导的氧化应激损伤。且PhoP/Q双组份调控系统及全局调控子并未参与对pagO基因的调控作用，表明该基因的表达具有相对的独立性。研究成果补充和完善了hvKp相关的致病因子及调控网络，可为针对此类细菌感染的治疗提供新思路，同时为设计新的抗感染药物或替代性治疗策略提供潜在的药物靶标和实验依据。

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