Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae
Michelle Lopes Ribeiro-Guimarães1, Eliana Blini Marengo, Antonio Jorge Tempone
1Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, Brasil.
Abstract:
Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40 degrees C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.
Insights
Mycobacterium leprae possesses an HtrA-like protease, ML0176, crucial for bacterial pathogenesis. This enzyme shows high activity at elevated temperatures and could be a new therapeutic target for leprosy.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- High temperature requirement A (HtrA) proteases are implicated in bacterial pathogenesis.
- The Mycobacterium leprae gene ML0176 encodes a conserved HtrA-like protease found in human leprosy lesions.
Purpose of the Study:
- To characterize the enzymatic properties of the recombinant ML0176 protein from Mycobacterium leprae.
- To investigate the potential of ML0176 as a therapeutic target for mycobacterial infections.
Main Methods:
- Production of recombinant ML0176 protein.
- Assay of proteolytic activity against synthetic and natural peptides.
- Determination of optimal temperature for enzyme activity.
- Inhibition studies using aprotinin.
- Analysis of enzyme specificity.
Main Results:
- Recombinant ML0176 effectively hydrolysed various peptides.
- Optimal proteolytic activity was observed above 40°C.
- The enzyme was inactivated by the serine protease inhibitor aprotinin.
- ML0176 exhibited broader cleavage specificity compared to other HtrA homologues.
Conclusions:
- ML0176 is a functional HtrA-like serine protease in Mycobacterium leprae.
- Its enzymatic properties suggest a role in bacterial pathogenesis.
- ML0176 represents a potential novel target for anti-mycobacterial therapies.
