Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Maria Florencia Del Papa, Lynn E. Hancock, Vinai C. Thomas, Marta Perego

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and 1305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.

Original languageEnglish (US)
Pages (from-to)8835-8843
Number of pages9
JournalJournal of bacteriology
Volume189
Issue number24
DOIs
StatePublished - Dec 2007
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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