Identification of secreted metalloprotease from the culture supernatant of Mycobacterium bovis BCG

Abstract

Tuberculosis is considered one of the most prevalent diseases worldwide, claiming the lives of approximately 1.2 to 1.5 million people each year. It is caused by a member of a class of bacteria called Mycobacterium (M. tuberculosis), which includes saprophytic and pathogenic species. M. bovis causes similar disease in cattle. This latter species is used for the production of all currently available tuberculosis Bacillus Calmette-Guérin (BCG) vaccines. There has been progress made in the past 20 years in understanding the pathogenesis of Tuberculosis but much more research is needed to fully understand the mechanisms that mediate the survival of the pathogen in phagocytic cells. Metalloproteases are a subfamily of proteases that utilize metals, mostly zinc, for their catalytic activities. They are involved in virulence, cell wall processes and intermediary metabolism in bacteria. Metalloprotease activity in the culture supernatant of M. tuberculosis has been detected previously in other laboratories and we are able to detect this activity in the culture supernatant of M. bovis BCG. Bioinformatics analysis of the proteome of M. tuberculosis reveals a large number of putative proteases (66 in total) with 23 having the zinc-binding motif (PROSITE PDOC00129). The goal of this study was the direct identification of the protein responsible for the protease activity in the culture supernatant of M. bovis BCG. The protease in the supernatant was identified by zymography, with partial purification of the protease activity by ion exchange chromatography. A band on electrophoresis gels associated with the protease activity was then subjected to N-terminal sequencing of the purified protein, which identified hexapeptide sequence VTGGGA. Using a bioinformatics approach, the best matching protein with this sequence was identified as PstS, a phosphate binding protein normally considered a component of the phosphate transport system.