Abstract
Background: Leptospirosis is a global zoonotic infectious disease. Leptospira interrogans, the major pathogen of leptospirosis, can rapidly invade into hosts and diffuse from the bloodstream into many internal organs, and excrete leptospires from the urine to form sources of infection in the environment. However, the molecular basis of the powerful invasiveness of L. interrogans remains poorly understood. Methods: M16-type metallopeptidase-encoding genes in different serogroups and serovars of L. interrogans were detected by PCR and sequencing. Proteolytic activity of native/recombinant M16-type metallopeptidases (n/rLep-MP1/2/3) from L. interrogans strain Lai was determined by casein/azocasein and extracellular matrix (ECM) protein hydrolytic tests. Expression and secretion of Lep-MP1/2/3 during infection of vascular endothelial, enteric and renal epithelial cells (FHC, HUVEC and Hek293) were detected by qRT-PCR, Western Blot assay and confocal microscopy. Lep-MP1/2/3-encoding gene-deleted and complemented mutants (ΔMP1/2/3 and CΔMP1/2/3) were generated to determine the roles of Lep-MP1/2/3 in invasiveness by transwell test and virulence in hamsters. Findings: All the tested L. interrogans but not saprophytic L. biflexa strains possess the sequence-conserved M16-type MP-encoding genes. rLep-MP1/3 rather displayed stronger hydrolysis of casein and azocasein than rLep-MP2, with Km of 123.179/340.136 µmol•L-1 and Kcat of 0.154/0.159 s-1 but all the three rLep-MPs hydrolyzed ECM proteins. The expression, secretion or membrane translocation of Lep-MP1/2/3 during infection of different cells were notably increased. The ΔMP1/3 mutant, but not ΔMP2 mutant, presented significantly attenuated transmigration through different cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys and urine, and 10/13-fold-decreased LD50 and milder histopathologic injury in hamsters. Interpretation: M16-type MP1/3 are virulence factors of L. interrogans in invasion into hosts, leptospiral diffusion in vivo, and transmission of leptospirosis. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (81802021, 81671974, 81760366 and 81971951), a grant from the Zhejiang Medical and Health Science and Technology Project of China (2019KY013) and a grant from the Research Initiation Fund of Zhejiang People’s Hospital in China (ZRY2018B004). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The animal experimental protocol was approved by the Ethics Committee for Animal Experiment of Zhejiang University.
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