Metalloproteases have long been recognised as key virulence factors in bacterial pathogens. By cleaving the protein scaffolds that hold tissues together, these enzymes help bacteria breach epithelial barriers, disseminate through the bloodstream and colonise new niches within the host. Proteolytic activity has been detected in pathogenic Leptospira before: secreted proteases from L. interrogans have been shown to degrade extracellular matrix and plasma proteins, and a metalloprotease called Leptolysin, a member of the pappalysin family, was recently described with broad-spectrum activity against host targets. Other Leptospira proteases have been implicated in cleaving complement proteins, a strategy that helps the spirochaetes evade the innate immune response. What has been missing, however, is detailed biochemical information about individual enzymes, particularly members of the M23 family, which in most bacteria are known primarily as peptidoglycan hydrolases involved in cell wall remodelling.

LIC11568 caught the researchers’ attention because of its unusual domain architecture. Bioinformatic analysis using resources such as InterPro and Pfam confirmed the previously reported presence of an M23 metallopeptidase domain alongside a LysM motif, a widely distributed module that binds peptidoglycan and related glycans. The team’s predictions went a step further, identifying an N-terminal signal peptide, the molecular postal code that directs proteins to the secretory pathway. Taken together, these features suggested a protein that could be exported to the bacterial surface or beyond, where it might encounter host tissue components. The M23 fold is a striking example of structural economy: a single protein fold that has been recruited for functions ranging from bacterial cell wall turnover to, as this study now suggests, the degradation of mammalian collagen.

Getting enough of the protein to study experimentally proved to be the first major hurdle. The researchers initially attempted to produce recombinant LIC11568 in Escherichia coli, the workhorse of protein expression laboratories worldwide. Under the conditions tested, however, no detectable protein appeared on SDS-PAGE gels, a frustrating but familiar outcome for proteins that are toxic to their bacterial hosts, difficult to fold, or prone to degradation. Rather than abandon the project, the team turned to cell-free protein synthesis, a technique in which transcription and translation are carried out in a test tube using cellular extracts stripped of their own genomes. Because the method bypasses living cells entirely, it sidesteps problems of toxicity and allows direct control over the reaction environment. Cell-free systems have matured considerably since their early days and are now routinely used for proteins that resist conventional expression, and LIC11568 proved to be a textbook case.

With soluble recombinant protein in hand, the researchers confirmed its identity using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, or MALDI-TOF peptide mass fingerprinting. This technique digests a protein into peptides and measures the precise masses of the resulting fragments, generating a fingerprint that can be matched against theoretical digests of known sequences. The match supported that the product of the cell-free reaction was indeed LIC11568, clearing the way for the functional experiments that form the heart of the study.

Those experiments delivered the study’s headline finding: LIC11568 degrades Type I collagen in a time-dependent manner. Collagen is not an easy substrate. Its triple-helical structure, stabilised by tight packing of glycine-rich chains, resists most proteases and gives skin, tendon, bone and connective tissue their remarkable tensile strength. Enzymes capable of cleaving it, collectively known as collagenolytic proteases, are relatively rare among bacteria and are strongly associated with tissue-invasive pathogens such as Vibrio species. The Leptospira enzyme showed maximum activity after five hours of incubation at pH 8.0 and 37 degrees Celsius, conditions that mirror the neutral to slightly alkaline environment of mammalian body fluids at physiological temperature. Activity also increased with enzyme concentration, consistent with a genuine enzymatic reaction rather than a contaminating artefact.

The clearest evidence for the enzyme’s mechanistic class came from inhibitor and rescue experiments. The chelating agent 1,10-phenanthroline, which sequesters divalent metal ions from the active site of metalloproteases, strongly inhibited collagen degradation. When the researchers added zinc ions back to the inhibited reaction, activity was restored. Calcium ions, by contrast, failed to rescue activity on their own, indicating that the catalytic metal is specifically zinc rather than any available divalent cation. This pattern of inhibition and metal-dependent rescue is a classic signature of zinc-dependent metalloproteases and firmly places LIC11568’s collagenolytic behaviour within that mechanistic family under the conditions tested.

The significance of the finding lies in what it suggests about leptospiral pathogenesis. During infection, L. interrogans must cross tissue barriers, survive in the bloodstream and ultimately colonise organs such as the kidney and liver. An enzyme that can digest Type I collagen would, in principle, help the spirochaete tunnel through the dense connective tissue matrices that stand in its way. The combination of a signal peptide, a LysM binding module and a catalytic M23 domain paints a coherent picture of a protein positioned at the interface between the bacterium and its host environment. The authors are careful, appropriately, to frame the result as identifying collagen as a substrate in vitro, and they note that the work provides a basis for further investigation of the enzyme’s substrate specificity and its potential role in extracellular matrix interactions during infection. Whether LIC11568 is secreted in vivo, how much it contributes to virulence relative to other Leptospira proteases, and whether it can serve as a vaccine or drug target are questions that remain open.

Methodologically, the study also offers a useful lesson for the broader protease research community. The successful use of cell-free protein synthesis after conventional expression failed demonstrates a practical route to characterising difficult Leptospira proteins, many of which may share the expression problems that plagued LIC11568 in E. coli. As the leptospirosis research field works to fill the gaps in its virulence atlas, an area that reviewers have repeatedly described as underexplored compared with other bacterial pathogens, approaches that decouple protein production from living cells are likely to become increasingly valuable. Each newly characterised enzyme, in turn, narrows the search space for interventions. If LIC11568 or its relatives prove essential for tissue invasion, inhibitors modelled on 1,10-phenanthroline’s mechanism of metal chelation, or antibodies raised against the enzyme, could one day complement the vaccines and antibiotics that currently form the limited arsenal against this globally distributed disease.

Subject of Research: Biochemical characterisation of the zinc-dependent M23 metalloprotease LIC11568 from Leptospira interrogans and its collagenolytic activity

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

Article References: Merrin, S. B., & Ramya, M. (2026). Expression and functional characterisation of the zinc-dependent metalloprotease LIC11568 from Leptospira interrogans reveals collagenolytic activity. Molecular Biology Reports, 53(1), Article 1685. https://doi.org/10.1007/s11033-026-12887-4

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12887-4

Keywords: Leptospira interrogans, leptospirosis, metalloprotease, M23 metallopeptidase, collagenolysis, Type I collagen, zinc-dependent enzyme, extracellular matrix, virulence factor, cell-free protein synthesis, MALDI-TOF, signal peptide