Diversity Of Outer Membrane Protein Expression Of Leptospira Interrogans During Infection Of Human THP-1Monocytes

Objective To determine the diversity of outer membrane proteins expressed by Leptospira interrogans during infection of human THP-1monocytes and screen 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-1monocyte line were extracted using Triton X-114method and were detected by Western blot using the corresponding antibodies. 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 followed by identification by using liquid chromatography-tandem mass (LC-MS/MS) method. The transmembrane regions and signal peptides in the target proteins were analyzed using bioinformatica software. Several real-time fluorescent quantitative RT-PCRs were performed to determine the mRNA level changes of the target genes of L. interrogans strain Lai before or after infection of THP-1cells.Results After a60min infection of THP-1cells, four OMPs (Loa22, GroEL, F0F1ATP synthase alpha-and beta-subunit) 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 the2-DGE plus silver staining (P<0.05). The bioinformatica analysis indicated that there was a transmembrane region in OmpA and OmpA family protein sequences, and a signal peptide in Loa22, LigB and OmpA family protein sequences. Western blot results showed that the signal of Loa22in leptospirosis OMPs infection with THP-1cells reacted with rabbit anti-serum of Loa22was stronger than that before non-infection with THP-1cells.Conclusion An obvious change of leptospiral OMPs occurs during infection of host cells. The up-regulated OMPs after infection, especially for the GroEL and Loa22proteins, can be used as the immunogen candidates for developing leptospiral genetically engineering vaccines.