Role and mechanism of Leptospira interrogans vWF-A gene products binding to human collagen proteins / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To investigate the role and mechanism of Leptospira interrogans ( L. interrogans) vWF-A gene products binding to human collagen proteins. Methods:Bioinformatic software was used to analyze the structure and function of the vWF-A genes (LA_0012, LA_0697 and LA_4207) of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai. Prokaryotic expression systems for the vWF-A domain segments in the vWF-A genes were generated. The target recombinant proteins, rLep0012, rLep0697 and rLep4207, were purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE. ELISA and surface plasmon resonance (SPR) were performed to detect the binding ability of the target recombinant proteins to humanⅠ, Ⅲ, Ⅳ and Ⅵ types of collagen proteins (hCOL1/3/4/6). Expression of the vWF-A genes at mRNA and protein levels were detected by real-time fluorescent quantitative RT-PCT and Western blot during infection of human umbilical vein endothelial cells (HUVEC) and mouse hemangioendothelioma endothelial cells (EOMA). Results:The products of vWF-A genes were vWF-A superfamily domain-containing surface or transmembrane proteins, but LA_0697 and LA_4207 genes also contained metal ion-dependent adhesion sites (MIDAS). The established prokaryotic expression systems efficiently expressed the target recombinant proteins and each of the proteins extracted by Ni-NTA affinity chromatography showed a single band in SDS-PAGE. ELISA results showed the strong binding of rLep0697 to hCOL3/6 and rLep4207 to hCOL1/4. SPR results showed the rapid binding and dissociation of rLep0697 with hCOL3/6 ( KD values=5.71×10 -8 and 5.89×10 -8 mol/L) and the rapid and stable biding of rLep4207 with hCOL1/4 ( KD values=6.4×10 -9 and 3.2×10 -9 mol/L). Expression of the vWF-A genes at both mRNA and protein levels were significantly elevated ( P<0.05) during infection of HUVEC and EOMA cells. Conclusions:The products of LA_0697 and LA_4207 genes could act as the adherence factors of L. interrogans during infection.