Study On PAF-AH And PLA₂ Activities Of Leptospira Interrogans LA 2144 Gene Product

Background:Leptospirosis caused by pathogenic Leptospira genospecies is a world-spreading zoonotic infectious disease.Our country,China,is also one of the epidemic areas of the disease.Hemorrhage and jaundice are the typical pathological features of leptospirosis in human,in which the mortality of patients with pulmonary diffuse hemorrhage?PDH?is over 50%.We recently found that the pathogenic Leptospira interrogans can cause hemorrhage by competitive inhibition of vWF-mediated platelet aggregation,but the mechanism of pulmonary hemorrhage in leptospirosis remains little understood.L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai LA₂144 gene product has been annotated as SGNH hydrolase.SGNH hydrolase superfamily is known to contain platelet activating factor acetylhydrolase?PAF-AH?and phosphatidase A2?PLA2?.However,the roles and mechanisms of leptospiral LA 2144 gene product in pulmonary hemorrhage and inflammatory reaction in leptospirosis have been poorly understood or not been reported.Methods:Bioinformatic softwares were used to predict transmembrane regions,signal peptide and domains in LA₂144 gene of L.interrogans strain Lai.A prokaryotic expression system of signal peptide-free LA₂144 gene was generated and the target recombinant protein rLep2144 was extracted by Ni-NTA affinity chromatography.SDS-PAGE was used to detect the effect of rLep2144 expression and extraction.The purified rLep2144 was renatured by surfactant and highly polymerized cycloamylose method.New Zealand rabbits were immunized with the purified rLep2144 to obtain antiserum and rLep2144-IgG in the antiserum was isolated by saturated ammonium sulfate precipitation and DEAE-52 column chromatography.Spectrometry was applied to detect the activity of rLep2144 hydrolyzing PAF-AH substrate 2-thio PAF and its Km and Kcat values as well as the activity of rLep2144 hydrolyzing PLA2 substrate arachidonoyl 2-thio PC.Real-time fluorescence quantitative RT-PCR and Western Blot assay were performed to detect the transcription,protein expression and secretion of LA 2144 gene during infection of human and mouse vascular endothelial cells?HUVEC and EOMA?with the spirochete.Results:LA 2144 gene contains a signal peptide and a domain belonging to SGNH hydrolase superfamily but has no transmembrane region.The generated prokaryotic expression system of signal peptide-free LA₂144 gene could efficiently express rLep2144 and the extracted rLep2144 by affinity chromatography was shown as a single protein fragment in gel of SDS-PAGE.The purified rLep2144 was efficiently renatured.The rLep2144 presented a powerful PAF-AH activity with the Km and Kcat values of 688.235 ?moL/L and 0.976 s-1 in hydrolysis of 2-thio PAF substrate,but its PLA2 activity was relatively low in hydrolysis of arachidonoyl 2-thio PC substrate.The mRNA transcription and protein expression levels of L.interrogan strain Lai LA 2144 gene were rapidly increased during infection of HUVEC and EOMA cells?p<0.05?and the secretion of LA 2144 gene product could be found.Conclusion:LA₂144 gene product of L.interrogans has a powerful PAF-AH and a lower PLA2 activity that may play roles in pulmonary hemorrhage and lipid inflammatory reaction in leptospirosis.