问号钩端螺旋体（简称钩体）能迅速侵入人体及血流、广泛体内播散和尿液排菌，仅我们报道的胶原酶难以解释其强大侵袭力。近年发现细菌可产生胞外金属蛋白酶eMPs并有侵袭作用。我们发现钩体有十个M14/16/20/23家族分泌型S-eMPs或膜型M-eMPs基因，钩体感染细胞时pH下降但ROS和eMPs-mRNAs升高。近年发现细菌KdpDE信号系统（TCS）感受pH、氧分压或渗透压上调胞外酶表达并经AcrAB-TolC和Sec-Gsp分泌器分泌。本项目拟用kdpDE敲除突变株及细胞感染模型，确定钩体eMPs水解胞外基质分子活性与机制差异、KdpDE-TCS感受低pH和高ROS或渗透压信号上调eMPs基因表达增强侵袭力、S-eMPs经AcrAB-TolC和Sec-Gsp分泌及M-eMPs钩体表面定位发挥作用，然后用金地鼠感染模型验证上述体外结果，进一步阐明钩体侵袭力分子机制，有较高创新性和医学意义。

Leptospira interrogans can rapidly invade into human body and blood stream, extensive diffusion in vivo and discharge of leptospires from urine, but a collagenase reported by us is difficulty to explain its powerful invasiveness. We found that L.interrogans possesses ten extracelluar metalloprotease (eMPs) encoding genes belonging to M14/16/20/23 families and their products are predicted as secretion or membrane type eMPs (S-eMPs and M-eMPs) as well as the decrease of pH values and increase of ROS and eMPs-mRNAs during leptospiral infection of cells. The recent studies found that bacterial KdpDE two-component signaling system (KdpDE-TCS) could sense pH, oxygen partial pressure and osmotic pressure to up-regulate the expression of their extracellular enzymes that secreted through AcrAB-TolC and Sec-Gsp secretors. In the present study, we plan to use kdpDE-knockout mutant and Leptospira-infected cell models to determine the activity of leptospiral eMPs hydrolyzing molecules in extracellular matrix (ECM) and diversity of hydrolytic mechanisms, the KdpDE-TCS up-regulating the expression of eMPs to cause invasiveness enhancement after sensing low pH and high ROS or osmotic pressure, and the roels of S-eMPs secreted through AcrAB-TolC and Sec-Gsp secretors and M-eMPs located on leptospiral surface. All the results in vitro are verified using Leptospira-infected Syrian hamsters. The results of this study will further elucidate the powerful invasiveness of L. interrogans, which has higher creativeness and medical significance.