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Characterization and pathogenicity of extracellular serine protease MAP3292c from Mycobacterium avium subsp.
Hongxiu Liu1, Guanghui Dang1, Xinxin Zang1
1State Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Street, Harbin, 150069, PR China.
Abstract:
Serine protease is the virulence factor of many pathogens. However, there are no prevailing data available for serine protease as a virulence factor derived from Mycobacterium avium subsp. paratuberculosis (MAP). The MAP3292c gene from MAP, the predicted serine protease, was expressed in Escherichia coli and characterized by biochemical methods. MAP3292c protein efficiently hydrolyzed casein at optimal temperature and pH of 41 °C and 9.0, respectively. Furthermore, divalent metal ions of Ca2+ significantly promoted the protease activity of MAP3292c, and MAP3292c had autocleavage activity between serine 86 and asparagine 87. Site-directed mutagenesis studies showed that the serine 238 residue had catalytic roles in MAP3292c. Furthermore, a BALB/c mouse model confirmed that MAP3292c significantly promoted the survival of Mycobacterium smegmatis in vivo; caused damage to the liver, spleen, and lung; and promoted the release of inflammatory cytokines IL-1β, IL-6, and TNF-α in mice. Finally, we confirmed that MAP3292c was relevant to mycobacterial pathogenicity.
Insights
Mycobacterium avium subsp. paratuberculosis (MAP) serine protease MAP3292c promotes pathogen survival and causes organ damage. This study confirms MAP3292c
Area of Science:
- Microbiology
- Biochemistry
- Immunology
Background:
- Serine proteases are key virulence factors in many pathogens.
- Data on serine proteases as virulence factors in Mycobacterium avium subsp. paratuberculosis (MAP) are limited.
Purpose of the Study:
- To investigate the role of the predicted serine protease MAP3292c from MAP as a virulence factor.
- To characterize the biochemical properties and pathogenicity of MAP3292c.
Main Methods:
- Expression and biochemical characterization of MAP3292c in Escherichia coli.
- Site-directed mutagenesis to identify catalytic residues.
- In vivo studies using a BALB/c mouse model with Mycobacterium smegmatis.
Main Results:
- MAP3292c demonstrated caseinolytic activity with optimal conditions at 41°C and pH 9.0.
- Calcium ions (Ca2+) enhanced MAP3292c activity; autocleavage occurred between Ser86 and Asn87.
- Serine 238 was identified as a catalytic residue.
- MAP3292c promoted Mycobacterium smegmatis survival in vivo, causing liver, spleen, and lung damage.
- MAP3292c induced the release of inflammatory cytokines (IL-1β, IL-6, TNF-α).
Conclusions:
- MAP3292c functions as a serine protease with significant implications for mycobacterial pathogenicity.
- MAP3292c contributes to virulence by promoting pathogen survival and inducing host inflammatory responses and tissue damage.
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