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Mitochondria-Related Pathway Involved in Leptospira Interrogans- Induced Macrophage Apoptosis and Characterization of OmpL1 of Pathogenic Leptospira Species

Author: DongHaiYan
Tutor: YanJie
School: Zhejiang University
Course: Microbiology
Keywords: Apoptosis Leptospira interrogans mitochondrial-related pathway AIF EndoG caspase-independent pathway Bcl-2 family ompL1 cross-immunogenicity cross-immunoprotection
CLC: R514
Type: PhD thesis
Year: 2009
Downloads: 107
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AIM AND GROUNDOur previous study found that Leptospira infection could induce monocyte-macrophage-like cells(J774A.1) apoptosis,but the specific mechanism of apoptosis has not been clarified.The purpose of this study is to investigate mitochondria-related pathway of L.interrogans-induced macrophages apoptosis.MATERIAL AND METHODS(1) Establish pathogenic leptospires infected macrophages apoptosis model in vitro: murine peritoneal macrophages(MPMs),murine monocytic macrophage-like cells (J774A.1) and human monocyte-like cells(THP-1) were infected with L.interrogans serovar Lai strain Lai with different MOIs for different times,respectively.Detect apoptosis of the macrophages by Armexin V-FITC/PI double staining method;analyze apoptosis rates by flow cytometry(2) Detect morphological change and the mitochondria impairment by transmission electron microscopy of leptospiral induced apoptotic macrophages.(3) Caspase-8 and caspase-9 activity was measured with assay kits.To detect mitochondrial potential with JC-1 staining by flow cytometry and fluorescence microscopy,simultaneously detect cells of reactive oxygen species(ROS) levels by DCFH-DA fluorescent probe.(4) Expression of cytochrome C,Smac,EndoG and AIF in macrophages were determined in the mitochondrial and cytosol component after infection by Western Blot,and translocation of AIF and EndoG were examined by immunofluorescence staining.(5) Apoptosis-related Bcl-2 family mRNA expression levels was detected by microarray and confirmed by real-time PCR.RESULTS(1) Pathogenic L.interrogans serogroup Lai strain Lai could induce apoptosis of MPMs,J774A.1 and THP-1 cells with a time-and dose-dependent manner.However, with the increase of MOI,apoptosis ratio firstly rose,and then declined,while cell necrosis ratio increased.(2) Transmission Electron Microscopy results showed that all three types macrophages demonstrated apoptotic morphological changes,such as chromatin condensation and marginalization and cell vacuolization,mitochondria swelling and disappearance of mitochondrial ridges.(3) The activity of caspase-8 of the L.interrogans strain Lai-infected macrophages increased,but activity of caspase-9 did not increase obviously.However,pan-caspase inhibitors could not completely block macrophages apoptosis.Mitochondrial membrane potential(MPT) in infected macrophages declined gradually as infected time;while the cells showed a visible color change under the fluorescence microscope.At the same time,reactive oxygen species (ROS) levels increased in the infected macrophages.(4) The results of Western Blot showed that it was not detectable of cytochrome C and Smac release from mitochondria to cytoplasm in the L.interrogans strain Lai induced-apoptotic macrophages.However, pro-apoptotic proteins,AIF and EndoG,releasing from mitchondria to cytosol was detectable in the infected MPMs and J774A.1 cells,and AIF releasing was detectable in THP-1 cells.Results of immunofluorescent staining confirmed that AIF and/or EndoG translocation from mitochondria to the nucleus,thereby inducing cell apoptosis.MPT blocker could completely block the release of AIF or EndoG,and partly blocked the macrophages apoptosis.(5) The results of microarray and real-time PCR showed that Bcl-2 family mRNA levels changed in the L.interrogans strain Lai induced apoptotic macrophages.Pro-apoptotic genes(Bid,Bik1,Bnip3,Bcor,Bcl-211 and Bclafl) were significantly up-regulated in the infected MPMs and J774A.1 cells,pro-apoptotic genes (Bid,Bik1,Bnip3,Bcl-211 and Bclafl) were lightly up-regulated in the infected THP-1 cells,other proapoptotic genes were unchanged.CONCLUSIONS (1) L.interrogans serovar Lai strain Lai induced apoptosis and necrosis in macrophages in vitro;apoptosis rates in primary macrophages was higher than that of continuous cell lines,apoptosis rates in murine macrophage was higher than people originate macrophages.The leptospires induced apoptosis in a certain range was time-and dose-dependent.(2) L.interrogans strain Lai-induced macrophages apoptosis via caspase-8 dependent pathway and mitochondrial related caspase-independent pathway.(3) In the process of Leptospiral stain Lai-induced macrophages apoptosis,mRNA expression level of some pro-apoptotic genes of Bcl-2 family were up-regulated,which may be involved in the process of apoptosis. AIM AND GROUNDThe usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp.Identification of genus-specific protein antigens(GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1,a transmembrane porin of pathogenic leptospires,was identified as a possible GP-Ag,but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.MATERIAL AND METHODS(1) Amplification and sequencing the ompL1 gene in all 15 official Chinese standard strains and 163 clinical isolated strains of pathogenic leptospires.(2) Molecular phylogeny analysis and gene-typing of ompL1 genes,phylogenetic analysis and secondary structure prediction of OmpL1 protein.(3) Prokaryotic expression and identification of recombinant OmpL1(rOmpL1) proteins,then preparation of rabbit antisera against rOmpL1 proteins.(4) Detect cross-immunoagglutination among the antisera against(rOmpL1) and leptospiral strains belonging to different ompL1 gene types by microscopic agglutination test(MAT).Then detect cross-immunoreactions by using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients by ELISAs.(5) The effects of the antisera against rOmpL1 proteins to inhibit L.interrogans strain Lai from adhering to J774A.1 cells.(6) Detect cross-immunoprotection of each rOmpL1 by challenging immunized guinea pigs with lethal dose leptospires from different ompL1 gene types.RESULTSPCR analysis demonstrated that the ompL1 gene is expressed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China.In the standard strains,the ompL1 gene could be divided into three groups (ompL1/1,ompL1/2 and ompL1/3) according to their sequence identities.Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires.The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1(rOmpL1) and leptospiral strains belonging to different ompL1 gene types.ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients further verified these cross-immunoreactions.All the antisera against rOmpL1 proteins could inhibit L.interrogans strain Lai from adhering to J774A.1 cells.Furthermore,immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types.CONCLUSIONThree types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.

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