Expression and functional characterisation of the zinc-dependent metalloprotease LIC11568 from Leptospira interrogans reveals collagenolytic activity.

backgroundMetalloproteases are important virulence factors that facilitate bacterial dissemination through degradation of host extracellular matrix proteins. While proteolytic activities have been reported in pathogenic Leptospira, the biochemical characterisation of individual metalloproteases remains limited. METHODS AND

resultsIn this study, the putative M23 metalloprotease LIC11568 from Leptospira interrogans was characterised using bioinformatic analysis, recombinant protein production by cell-free protein synthesis, affinity purification, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) peptide mass fingerprinting, and functional enzymatic assays. Bioinformatic analysis supported the previously reported LysM and M23 metallopeptidase domain architecture of LIC11568 and additionally predicted an N-terminal signal peptide. Recombinant LIC11568 was successfully produced using cell-free protein synthesis after recombinant protein production was not detectable by SDS-PAGE in Escherichia coli under the conditions tested, and its identity was supported by MALDI-TOF peptide mass fingerprinting. Functional assays demonstrated that LIC11568 degraded Type I collagen in a time-dependent manner, with maximum activity observed after 5 h at pH 8.0 and 37 °C. Collagenolytic activity increased with enzyme concentration and was strongly inhibited by 1,10-phenanthroline. Activity was restored by Zn²⁺, whereas Ca²⁺ alone failed to recover activity, supporting the zinc dependence of LIC11568 under the tested conditions.

conclusionThis study provides the first biochemical characterisation of LIC11568 as a functional M23 collagenolytic metalloprotease from L. interrogans. Its ability to degrade Type I collagen in vitro identifies collagen as a substrate under the conditions tested and provides a basis for further investigation of its substrate specificity and potential role in extracellular matrix interactions during leptospiral infection.