Role And Mechanism And Pathogenic Significance Of Leptospira Interrogans VWF-A Gene Products Binding To Human Collagen Proteins

Background:Leptospirosis caused by pathogenic Leptospira soecies is a worldwide zoonotic infectious disease.The disease is also one of the most important infectious diseases for prevention and monitoring in China.After invading into human body through the mucosa or damaged skin,pathogenic Leptospira species rapidly enter the bloodstream to cause toxic septicemia.High fever,myalgia and superficial lymphadenectasis are the common clinical sings and symptoms of leptospirosis patients.Subsequently,the leptospires in bloodstream pass through small vessels to diffuse into many internal organs such as lungs,liver and kidneys to cause aggravation of disease.Adhesion to cells and invasion into hosts are the prerequisites for pathogenic microbes causing diseases.However,the adhesion factors and their action mechanisms of pathogenic Leptospira species remain poorly understood.von Willebtand factor（v WF）is a key blood coagulation factor and it plays the role in blood coagulation through its region A（v WF-A）to bind to theα-subunit of cytomembrane glucoprotein-Ib（GPIbα）on platelets and exposed collagens（COL）that cause the aggregation of platelets on the exposed COLs in blood vessel basement membrane of damaged vascular endothelium.We found that the products of LA＿0012,LA＿0697 and LA＿4207genes of serogroup Icterohaemorrhagiae serovar Lai strain Lai of pathogenic L.interrogans contain v WF-A superfamily domains,but have no GPIbα-bindng sites.Until now,the pathogenic role and mechanism of the products of these genes have not been reported.We presumed that the products of leptospital v WF-A genes can adhere to the COLs in extracellular matrix（ECM）,intercellular space and blood vessel basement membrane to play roles in invasion into hosts and diffusion in vivo of L.interrogans.Methods:The structure and function of the v WF-A genes（LA＿0012,LA＿0697and LA＿4207）of L.interrogans strain Lai were analyzed using bioinformatic softwares.The prokaryotic expression systems of the v WF-A domain segments in the v WF-A genes were generated and the target recombinant proteins,r Lep0012,r Lep0697 and r Lep4207,were extracted by Ni-NTA affinity chromatography.The expression and purification of r Lep0012,r Lep0697 and r Lep4207 were examined by SDS-PAGE.Rabbits were immunized with the purified r Lep0012,r Lep0697 and r Lep4207 to obtain antisera and r Lep0012-Ig G,r Lep0697-Ig G and r Lep4207-Ig G in the antisera were isolated by saturated ammonium sulfate precipitation and DEAE-52 column chromatography.ELISA and surface plasmon resonance（SPR）were applied to detect the binding ability of r Lep0012,r Lep0697 and r Lep4207 with type I,III,IV and VI types of human collagen proteins（COL1/3/4/6）.The transcription and expression levels of the v WF-A genes of L.interrogans strain Lai during infection of human and mouse vascular endothelial cells（HUVEC and EOMA）were detected by real-time fluorescent quantitative RT-PCT and Western Blot assay.Results:The products of L.interrogans strain Lai v WF-A gens（LA＿0012,LA＿0697和LA＿4207）were predicted as v WF-A superfamily domain-containing surface or transmembrane proteins,but LA＿0697 and LA＿4207 genes also contains metal ion-dependent adhesion sites（MIDAS）.The generated prokaryotic expression systems efficiently expressed the target recombinant proteins（r Lep0012,r Lep0697 and r Lep4207）and each of the proteins extracted by Ni-NTA affinity chromatography showed a single band in gel after SDS-PAGE.The ELISA showed the strong binding to COL3/6 and weak binding to COL1/4 of r Lep0697,strong binding to COL1/4 and weak binding to COL3/6 of r Lep0697,and weak binding to COL1/3/4/6 of r Lep r Lep0012.The SPR showed the rapid binding and dissociation of r Lep0697 with COL3/6(K_Dvalues=5.71×10^-8 and 5.89×10^-8M)and the rapid and stable biding of r Lep4207 with COL1/4(K_Dvalues=6.4×10^-9 and 3.2×10^-9M),but no binding of r Lep0012 with COL1/3/4/6.Both the transcription and expression levels of the v WF-A genes of L.interrogans strain Lai were significantly elevated（p<0.05）during infection of HUVEC and EOMA cells.Conclusion:The products of LA＿0697 and LA＿4207 genes can act as the adherence factors of L.interrogans to bind to COLs in ECM,intercellular space and blood vessel basement membrane in hosts and play important roles in invasion into hosts and diffusion in vivo of L.interrogans.