钩端螺旋体KdpDE-TCS调控表达的胞外金属蛋白酶侵袭作用和分泌机制研究

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.

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。

钩体病是全球流行的自然疫源性人兽共患传染病，也是我国重点监控及自然灾害时重点防疫的传染病之一。致病性钩体能迅速通过黏膜或破损皮肤侵入人体内并进入血流引起中毒性败血症、约一周后血流中钩体穿越小血管播散至多种内脏及脑脊液而导致疾病显著加重和较高病死率、部分钩体病人出现恢复期尿液排菌以及感染动物长期尿液排菌，均提示致病性钩体强大的侵袭力与其致病机制甚至传染源（疫水）形成密切相关。我们通过基因克隆和重组表达、基因敲除、激光共聚焦显微镜、实时荧光定量RT-PCR、Western Blot检测、Transwell法等技术明确了问号钩体KdpDE-TCS感知感染微环境信号（低pH、高ROS或渗透压等）后磷酸化激活上调胞外金属蛋白酶（eMPs）基因表达而使侵袭力显著增强、分泌型或膜表面型eMPs（S-eMPs或M-eMPs）水解宿主细胞胞外基质ECM分子（FN、LN、COL和ELN）以及不同eMPs协同发挥侵袭作用的分子机制，并采用金地鼠感染模型验证，阐明钩体侵袭力分子机制。揭示了致病性钩体具有迅速侵入宿主、体内广泛播散、尿液排菌强大侵袭力的物质基础。