Function Identification Of Adhesion And Invasion Associated Mce Gene From Leptospira Interrogans

Background and Objective:Leptospirosis caused by spirochetes of the genus Leptospira is a zoonotic spirochetal disease of global importance.It has been estimated that there were more than 500,000 people infected by pathogenic leptospiral species every year in the world wide and the death rate was 5～20%.However,there is still limited knowledge about the physiopathology of leptospirosis.As a result,we can't control leptospirosis effectively.Leptospira includes classically two species: Leptospira interrogans,pathogenic for humans and animals,and Leptospira biflexa,a saprophyte found in surface waters and soils.Adhesion to host cell surface,invasion and survival within the macrophages seem to be important properties of virulent leptospires in pathogenesis.Virulent leptospires which survive by evading phagocytosis can be isolated ordinarily from blood in the first week of the disease course,but late isolation of leptospires in blood or in cerebrospinal fluid has also been reported.A latest study on the interaction between macrophages and leptospires suggested that the virulent strain demonstrated an ability to actively invade the monocyte-macrophage-like J774A.1 cells during the early stages of contact.However, the molecular mechanisms used by Leptospira to adhere and entry macrophages are not elucidated.The putative mce gene presented in L.interrogans was supposed to be related to adhesion and invasion to the macrophages.However,the function of mce gene hasn't been verified.The aim of this study is not only to determine the mce gene carrying frequency in Leptospira interrogans and investigate the gene transcription level alteration of L.interrogans before and after infecting cells,but also to confirm the function of mce by studying the adherence and invasiveness of L.interrogans mce mutant.Further characterization of the gene product may improve our understanding of its role in the pathogenesis of leptospiral disease.Methods:The segments of entire mce genes from thirteen L.interrogans strains and one L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. By using genetic engineering technique,a prokaryotic expression system of mce gene was constructed.SDS-PAGE and Bio-Rad Agarose Image Analyzer was applied to examine the expression and output of the target recombinant protein rMce Rabbits were intradermally immunized with rMce to prepare the antiserum and Western Blot assay was performed to identify the expressed rMce.The alteration of mce gene transcription levels of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain Lai before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR.A mce mutant was constructed by allelic exchange in L.interrogans strain Lai.Fontana silver staining and double-fluorescence staining detection was used to study the change of adherence and invasiveness of L.interrogans mce mutant strain Lai.Results:All the tested L.interrogans strains was carrying mce gene but L.biflexa serogroup was not.The similarities of nucleotide and putative amino acid sequences of the cloned mce genes compared to the reported sequences(GenBank accession No.:NP₇12236) were 99.0%～100%and 97.9%～100%,respectively.The constructed prokaryotic expression system of mce gene could express rMce and the output of rMce was about 5%of the total bacterial proteins.The antiserum against the whole cell of L.interrogans strain Lai and rMce could efficiently recognize rMce.After infecting J774A.1 cells,the mce gene transcription level of L.interrogans strain Lai was remarkably up-regulated.This increase being 6.10 fold over control at most,after 30,60,90 and 120rain of treatment,respectively(P＜0.05).A mce mutant was constructed by allelic exchange in L.interrogans strain Lai.Through fontana silver staining and fluorescence staining detection,we found that adherence ratios and invasiveness of mce mutant L.interrogans strain Lai was inhibited by 59.8%and 72.7%,respectively(P＜0.05).Conclusion:mce gene is only existed in pathogenic L.interrogans strains.The product of mce gene is a sequence-conserved outer membrane protein.The constructed prokaryotic expression system of mce gene as well as the prepared antiserum against rMce offer useful tools to further study the function of the gene.Transcription level of the gene displays a pattem of cell-touch up-regulation.Adherence ratios and invasiveness of race mutant L.interrogans strain Lai decreased remarkblely,indicating a close correlation between the gene and pathogenesis of L.interrogans.