Diversity of outer membrane protein expression of Leptospira interrogans during infection of human THP-1 monocytes

Abstract

Objective To determine the diversity of outer membrane protein expression of Leptospira interrogans during infection of human THP-1 monocytes,and protein antigen candidates for developing leptospiral genetically engineering vaccines.Methods The outer membrane proteins(OMPs) of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai before or after infection of human THP-1 monocyte line were extracted using Triton X-114 method.The OMPs extracts were separated by two-dimensional gel electrophoresis(2-DGE) and then detected using silver staining method.Four up-regulated and four down-regulated OMPs were selected for hydrolysis by trypsin and then identified by using liquid chromatography-tandem mass(LC-MS/MS) method.The transmembrane regions and signal peptides in the eight target OMPs were analyzed using bioinformatic software.Several real-time fluorescent quantitative RT-PCRs were performed to determine the mRNA level changes of the eight target genes of L.interrogans strain Lai before or after infection of THP-1 cells.The prokaryotic expression systems of target genes were generated and the immunoprutection of target recombinant proteins was determined using leptospire-infected guinea pigs model.Results After a 60 min infection of THP-1 cells,four OMPs( Loa22,GroEL,F0F1 ATP synthase alpha- and beta-subunits) of L interrogans strain Lai were noticeably up-regulated ( P＜0.05 ),while the other four OMPs( FlaB2,LigB,OmpA family protein and OmpA) were significantly down-regulated (P＜0.05).The results of real-time fluorescent quantitative RT-PCRs were coincident with those confirmed by the 2-DGE phus silver staining.The bioinformatic analysis indicated that among the eight OMPs,OmpA and OmpA family protein belonged transmembrane proteins,while the rest had no membrane structure.Loa22,LigB and OmpA family protein contained a signal peptide in their sequences.200 μg of the recombinant proteins rLoa22 and rGroEL presented 75.0％ immunoprotective rates in the infected guinea pigs.Conclusion An obvious change of the OMP expression map of L.interrogans strain Lai occurs during infection of host cells.The up-regulated leptospiral OM Ps during infection,especially for the GroEL and Loa22 proteins,can be used as the immunogen candidates for developing leptospiral genetically engineering vaccines. Key words: Leptospira interrogans; Outer membrane proteins; Two-dimensional gel electrophores