Pathogenic Role And Mechanism Of Zn²⁺-dependent Matrix Metalloproteinases Of Leptospira Interrogans

Background:Leptospirosis, caused by pathogenic Leptospira, is a worldwide prevalent zoonotic infectious disease. The disease is one of the important infectious diseases that has been nationally prevented and monitored by Chinese government. Pathogenic Leptospira contains seven genospecies, but Leptospira interrogans is the predominant genospecies in the most of countries and areas including China. In China, about 70% of leptospirosis patients are due to the infection of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. After infection with L. interrogans, the animals are usually symptomless or have mild symptoms, but can persistently discharge leptospires from urine to contaminate natural bodies of water to form infection sources. Human individuals are infected with the spirochete by contact with the leptospire-contaminated water. L. interrogans possesses a powerful invasiveness which enables the spirochete to rapidly invade into human body through mucosa or damaged skin and then enter blood stream mucous to cause grippotyphosa type leptospirosis. Subsequently, the spirochete passes through small blood vessels to disseminate into lungs, liver, kidneys and erebrospinal fluid to cause pulmonary hemorrhage or pulmonary diffuse hemorrhage, icterohemorrhage, nephropathy or meningoencephalitis type leptospirosis. The fatality rate of severe leptospirosis patients is as high as 30-50%. It is well known that invasive enzymes are the virulence factors to determine invasive ability of bacterial pathogens. In the genome of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai (No. in China:56601), the products of LA0948, LA3400 and LA3401 genes are annotated as Zn-dependent peptidase. However, the function and pathogenic role of the three genes’ products have not been reported yet. Metalloprotease (MP) is also a large group of Zn2+-dependent proteinases and molecules in extracellular matrix (ECM) are their major hydrolytic substrates. Therefore, determination of MMP function and role in infection of LA0948, LA3400 and LA3401 genes’ products of L. interrogans strain 56601 is significant to understand the invasive substances and effective mechanism of L. interrogans.Methods:Bioinformatic professional softwares were used to analyze the structure and domains in the sequences of LA0948, LA3400 and LA3401 genes of L. interrogans strain 56601. The prokaryotic expression systems of the three leptospiral genes were constructed. The expressed target recombinant proteins, rLA0948, rLA3400 and rLA3401, were extracted by Ni-NTA affinity chromatography. SDS-PAGE plus Bio-Rad image analysor was used to detect the expression and extraction effects of the target recombinant proteins. The extracted rLA0948, rLA3400 or rLA3401 protein was intracutaneously injected into rabbits to obtain antiserum. The total IgG in each of the antisera was separated with ammonium sulfate precipitation plus DEAE-52 ion exchange chromatography. Azo-substrate spectrophotometry was used to determine the enzymatic activity, Km and Kcat values, and optimal pH, temperature and metal ion types of rLA0948, rLA3400 and rLA3401 proteins during hydrolysis of Azocasein substrate. Moreover, the activities of rLA0948, rLA3400 and rLA3401 proteins to hydrolyze several ECM molecles were detected using SDS-PAGE. On the other hand, several protease inhibitors and metal ion chelators were applied to further confirm the hydrolytic activities of rLA0948, rLA3400 and rLA3401 proteins by Azo-substrate spectrophotometry as described above. Real-time fluorescence quantitative RT-PCR and Western blotting assay were used to detect the mRNA levels and protein external secretion of LA0948, LA340 and LA3401 genes of L. interrogans strain 56601 during infection of human umbilical vein endothelial cell line (HUVEC) or human embryo kidney epithelial cell line (HEK293). The LA0948, LA3400 or LA3400 gene-knockout mutant (ALA0948, ΔLA3400 or ΔLA3401) of L. interrogans strain 56601 was generated. Compared to the wild-type strain, the ability to pass through HUVEC or HEK293 monolayers of ΔLA0948, ΔLA3400 and ALA3401 mutants by Transwell assay. L. interrogans-infected Syrian hamster model was used to detect the diffusion into internal organs, leptospiral discharge form urine and histopathological changes of ΔLA0948, ΔLA3400 andΔALA3401 mutants, which compared to that of the wild-type strain.Results:The result of bioinformatic analysis showed that LA0948, LA3400 and LA3401 genes of L. interrogans strain 56601 contain M16 family enzymatic domains, but only the the LA0948 or LA3401 gene has a positive MMP motif HXXEH, HFLEX or HLLEH. The constructed prokaryotic expression systems of the LA0948, LA3400 and LA3401 genes could efficiently express the target recombinant proteins, rLA0948, rLA3400 and rLA3401. Each of the rLA0948, rLA3400 and rLA3401 proteins extracted by Ni-NTA affinity chromatography showed a single protein fragment in gel after SDS-PAGE. rLA0948, rLA3400 or rLA3401 protein could hydrolyze Azocasein as well as ECM molecles I/III/IV/V type collagens, elastin, fibronectin, laminin and decorin. When hydrolzing Azo-casein substrate, the Km and Kcat values of rLA0948, rLA3400 or rLA3401 proteins were 123.1785 μmol·L-1 and 5.987 s-1,742.7561 μmol·L-1 and 24.113 s-1 or 340.1358 μmol·L-1 and 17.676 s-1, and the optimal pH and temperature were 7.4 and 37℃. Among the six tested metal ions, Zn2+ and then Mg2+ presented the role to increase Azocasein-hydrolytic activitities of the rLA0948, rLA3400 and rLA3401. All the protease inhibitors and metal ion chelators used could inhibit the proteolytic activities of rLA0948, rLA3400 and rLA3401 proteins, but the protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), presented the most powerful inhibitory effect. Thus, rLA0948, rLA3400 and rLA3401 proteins could be considered as MPs that named L-MMP1, L-MMP2, and L-MMP3. After HUVEC or HEK293 were infected with L. interrogans strain 56601, L-MMP1-mRNA, L-MMP2-mRNA and L-MMP3-mRNA levels were significantly increased (p<0.05) and all the L-MMPs were secreted. The abilities of ΔLA0948 and ΔLA3401 mutants passing through HUVEC or HEK293 cell monolayers were significantly lower than the wild-type strain (p<0.05). However, The abilities of ΔLA3400 mutant passing through HUVEC or HEK293 cell monolayers were lower than the wild-type strain, but there was no significant difference (p>0.05). The leptospiral numbers in lungs, liver, kidneys and urine of ΔLA0948, ΔLA3400 or ΔLA3401 mutant-infected hamsters were significantly less than the wild-type strain (p<0.05), and the histopathological changes were also attenuated compared to the wild-type strain.Conclusions:The products of LA0948, LA3400 and LA3401 genes of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain 56601 are the Zn2+-dependent matrix metalloproteinases (MMP) belonging to M16 family (L-MMP1, L-MMP2 and L-MMP3). L-MMP1, L-MMP2 and L-MMP3 play important roles in the diffusion into internal organs, discharge leptospires from urine and histopathological changes caused by L. interrogans strain 56601, indicating the L-MMPs act as the important virulence factors of L. interrogans.