质粒编码IV型分泌系统Hfc蛋白促使pLVPK-like毒力质粒在肺炎克雷伯菌中接合转移机制研究

高毒力肺炎克雷伯菌常导致肝脓肿、脓毒血症及迁徙性感染而严重危害人类健康。pLVPK-like毒力质粒接合转移导致肺炎克雷伯菌高毒力形成，但机制未明。申请人前期研究发现pLVPK-like毒力质粒可获取F型T4SS而促使接合转移，T4SS中Hfc蛋白具备DNA结合区域，可调控F菌毛蛋白合成。据此推测，质粒编码F型T4SS的Hfc蛋白调控F菌毛形成而影响pLVPK-like毒力质粒接合转移。本项目拟从质粒编码F型T4SS及Hfc与pLVPK-like毒力质粒流行相关性入手；通过基因定点突变、缺陷及回补技术明确Hfc对pLVPK-like毒力质粒接合转移的影响及作用方式；EMSA、DNase I footprinting鉴定与Hfc蛋白互作的DNA靶序列及结合位点，阐述其对F菌毛形成的调控，揭示Hfc调控pLVPK-like毒力质粒接合转移分子机制，有望为控制高毒力肺炎克雷伯菌传播提供新靶点。

Hypervirulent Klebsiella pneumobniae, which often causes liver abscess, sepsis and metastatic infections, become a serious threat to human health. It suggests that hypervirulence could be directly caused by conjugational transfer of pLVPK-like plasmids in Klebsiella pneumoniae. However the mechanism was still unclear. In our previous studies, we found that pLVPK-like virulence plasmid carried a plasmid-encoding F-type T4SS for conjugational transfer. Among the T4SS, Hfc protein has a DNA binding region, which can regulate the synthesis of F pili-associated protein. We presume that Hfc protein in the plasmid-encoded F-type T4SS could mediate F-pili formation and regulate pLVPK-like virulence plasmid conjugational transfer which promoted the hypervirulence of Klebsiella pneumoniae. This project plan to clarify the closely relationship between Hfc in the plasmid encoding F-type T4SS and the epidemic pLVPK-like virulence plasmids by using an epidemiological perspective. Then we will investigate the mechanisms that Hfc influences conjugational transfer of pLVPK-like plasmids by site directed mutagenesis, constructing series of hfc-deletion mutant and a complementation study. EMSA and DNase I footprinting are used to identify the DNA target sequence and binding region to Hfc protein, elaborate the molecular mechanism of Hfc to regulate the F pili formation, and reveal the mechanism of Hfc to regulate pLVPK-like virulence plasmid conjugational transfer. Our study will provide a new target for controlling the transmission of hypervirulent Klebsiella pneumoniae.