Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity

Abstract

Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl–fluoride inhibitor of serine peptidases

“…Finally, another lower pappalysin, leptolysin, was also insensitive to PMSF but AEBSF was not tested. 46…”

A New Recombinant Multiepitope Chimeric Protein of Leptospira interrogans Is a Promising Marker for the Serodiagnosis of Leptospirosis

“…LIC10713 protein, namely LruB, has been shown to react with antibodies in sera from humans who have leptospiral uveitis [46]. LIC13434 protein has recently been characterized as a metalloprotease of the pappalysin family, capable of degrading host proteins and possibly related to leptospiral invasiveness [47].…”

Human leptospirosis: In search for a better vaccine

“…Pathogenic leptospires have developed several strategies to evade the immune system, thus allowing their survival in the host, causing several complications. The main mechanisms of evasion are recruitment of host regulatory proteins of the complement system 33,42,[67][68][69][70][71][72] ; secretion of metalloproteinases, 28,31,73 biofilm formation, 74 changes in LPS composition and expression of catalase. 75 Pathogenic Leptospira also binds in vitro to plasminogen, laminin, fibronectin, thrombin, fibrinogen, extracellular matrix components and certain proteins of the Coagulation System.…”