Collagenase A:a Critical Invasive Factor In Virulence Of Pathogenic Leptospira Secies

General AbstractBackground:All leptospires in the genus of Leptospira are classified into two groups:pathogenic and non-pathogenic. Non-pathogenic leptospires exist in surface waters and soil and never cause disease in humans or animals. At least seven genospecies of pathogenic Leptospira species have been identified such as L. interrogans, L. borgpetersenii, L. kirschneri and L. weilii. The species L. interrogans comprises at least250antigenically distinct variants known as serovars belonging to24serogroups. Leptospirosis caused by pathogenic species of the genus Leptospira is an important zoonotic disease that occurs worldwide and is most commonly found in tropical or subtropical countries. Leptospirosis is also prevalent in P.R.China. The symptoms of human leptospirosis vary from mild to rapidly fatal. Clinical manifestations of the disease range from mild "flu-like" symptoms, moderate dengue-like symptoms such as high fever and severe headache as well as muscle pain to very severe symptoms such as pulmonary hemorrhage, jaundice and meningitis which frequently lead to respiratory failure and renal failure with high mortality rates. However, the pathogenetic mechanism is still unkown or less known. Thus bacterial strategies based on attacking the hosts are important for virulence. However, any report was found in the literature about collagenase produced by Leptospira species. Collagenase is an enzyme that is highly specific for both native and denatured collagen, in other words, a protease capable of causing directly hydrolytic cleavage of the peptide bonds in the triple-helical region of collagen molecules with unique specificity and shows high substrate specificity. Since, collagenases produced by bacteria are well documented for a long time. Only a few studies on collagenolytic proteases from bacteria have included elaborate molecular analyses. Many reports talked about enzymatic properties, general features and collagenolytic activity of various pathogenic bacteria such as Borrelia burgdorferi, Peptostreptoccoccus magnus and Clostridium spp. But many of these works have lacked detailed investigations. Moreover, bacterial virulence factors such as exotoxin, endotoxin and invasive enzyme are responsible for pathogenicity, promoting invasion of pathogenic bacteria into their hosts and cause tissue injury. However, apart from several hemolysins, pathogenic Leptospira species have not been shown to possess any exotoxins. In addition, virulence factors by themselves are not sufficient for successful infection. Moreover, collagenolytic activity was included as an activity within other proteolytic activities and there were few separate reports about collagenase as an essential factor in pathogenesis. Collagenolytic enzymes of some pathogens are considered to be virulence factor, as the can efficiently degrade collagen in connective tissue. In addition tissue putrefaction was attribuated to Clostridium collagenases of proteolytic clostridia. Collagenase from many pathogenic bacteria was related to pathogenicity as from Aeromonas veronii, involved in the pathogenesis of this pathogenic bacteria during infection of goldfish and mice. L. interrogans possesses a powerful invasiveness action by penetrating skin and mucous membranes and disseminate rapidly to other tissues shortly after infection, having the capacity to survive and grow in tissue by escaping natural defense mechanism. However the nature of the collagenase of L. interrogans species and its genetics are unknown.Aim:First of all, this study is to understand the whole collagenase enzyme ability and specificity hydrolyzing different collagen substrates, with different pH, metal ions concentration and buffer systems and the kinetic characteristic of the enzyme. Moreover, measure the rColA protein expression patterns during infection with human umbilical veins and nephric epithelial cells and examinate whenever Leptospira ColA protein is the secreted type or not. Finally, generate knocked-out colA mutant leptospires to further determine the function and the pathogenic significance for collagenase enzyme from this leptospiral colA gene through out histopathological damages in lung, liver and kidney tissues of infected Golden Syrian hamsters.Result:colA gene from Leptospira interrogans serovar strain Lai cloned in pMD18-T, sequenced, expressed in E-coli BL21DE3was highly conserved in protein sequence. The recombinant collagenase protein, purified to homogeneity by Ni-NTA affinity chromatographic column had a collagenolytic activity in50mM Tris-HC1Buffer with0.4mM CaCl2, pH7.4CCB at37℃and showed broad substrate specificity by digesting Azocoll, both water-insoluble native and water-soluble denatured type Ⅰ, Ⅱ, Ⅲ and Ⅳ collagens. Then, confirmed that the leptospiral rColA protein is a collagenase which preferentially hydrolyzes type Ⅲ collagen. The corresponding values of the apparent Michaelis-Menten constant (Km) and the substrate turnover number(Vmax) of type Ⅲ collagen were respectively6.67mM and1818.18U approximatively. Thus this collagenolytic enzyme is not only a collagenolytic protease, but it's also an amino peptidase, which recognize and digest collagen-specific sequences of the synthetic substrate Pz-peptide (but with low efficiency). In addition, Pathogenic Leptospira species was confirmed to possess a powerful invasiveness action by penetrating skin and mucous membranes, disseminate rapidly to other tissues shortly after infection. Infection of virulent wild-type L. interrogans with HUVEC or HEK293cells at different incubation times revealed by qPCR an augmentation of the production of the mRNA level with a peak after6hours of infection time compared to uninfected leptospires incubated in the same conditions. These results were confirmed by Western-Blot analysis where the concentration of the collagenase produced was increased at a maximum after6h of infection. Moreover, whenumbilical veins and nephric epithelial cells were infected with leptospires in the DMEM medium and incubated in the same condition, Western-Blot analysis of the supernatant revealed only the presence of rColA protein in the supernatant not in the wild-type leptospires in DMEM medium. This suggested that the colA gene is required by the spirochete during infection of host cells and is a secreated-type and confirmed the presence of the signal peptide in the colA gene sequence. Finally, the colA-mutant leptospires (with a333-bp amplicon between1388-bp andl721-bp of the colA coding region knocked-out and knocked-in in the same position with a1065-bp kan cassette) presented an attenuated virulence in hamsters. The50%lethal dose (LD50) in hamsters within14days after challenge was approximately1×10leptospires for the colA-mutant and1×10for the wild-type. In addition, colA-mutant leptospires caused mild histopathological damages in kidney, liver and lung compared to the wild-type strain. Results convincingly demonstrated that the rColA protein is a collagenase enzyme and is a pivotal virulence factor in pathogenic Leptospira species. Methods:To amplify and sequencing of colA gene of pathogenic L. interrogans strain Lai, the genomic DNA was prepared and PCR technique was realized. Procaryotic expression and purification of the recombinant ColA protein were obatained by expressing the E. coli BL21DE3PET42a-coiA with0.5mM IPTG, growth at28℃for8hours and using a Ni-NTA affinity chromatographic column protocol respectively.12%SDS-PAGE was performed for the determination of molecular mass of the rColA. Collagenase enzymatic assay against collagens, Azocoll and Pz-peptide was used to determine the enzymatic characteristics. The apparent Michaelis-Menten constant (Km) and the substrate turnover number (Vmax) of the leptospiral collagenase were determined by a least-squares analysis from the x-and y-intercepts, from Lineweaver-Burk plot with collagen type Ⅲ as the substrate. To measure the expression patterns at the transcriptional level of colA gene during infection, Real Time RT-PCR was performed after cells infection. Western-Blot analysis was performed to confirm the previous results. Then the leptospiral rColA protein expression and its secretion patterns were examinated by12%SDS-PAGE technique of the all proteins collected in the supernatant after infection and followed by Western-Blot analysis. To determine the function of leptospiral colA gene as a pivotal factor virulence of Leptospira species in hamster, a colA mutant from wild-type was constructed by knocked-out a333-bp amplicon between1388-bp and1721-bp of the colA coding region and knock-in of a1065-bp kan cassette in the same position. This was followed by the electro-transformation of recombinant suicide plasmid pUC19kan-colA-in the wild-type Leptospira interrogans. The method of the LD50calcul was applied to detect the attenuated virulence of the colA-mutant in the hamsters. Finally, Silver Staining and Hematoxylin&Eosin (HE) staining methods were performed to observe leptospires in the tissues and histopathological damages repectively in the lung, liver and kidney tissues of the hamsters. Conclusion:We expressed and purified a new collagenolytic protease, which possesses high specific activity with type Ⅲ collagen. This pathogenic leptospiral rColA did not express in media but during the infection of the host cells, meaning that this collagenase A is required by the spirochete during infection and is a secreted-type. Moreover, it presented a high virulence in hamsters, causing histopathological damage in lung, kidney and liver. These progresses make lpetospiral collagenase have a special interest. Future work will investigate more it functions in pathogenicity to have greater vaccine applications in the future.