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

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 I305 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.

FIG. 1.

Amino acid sequence alignment of members of the M4 family of bacterial metalloproteases. The amino acid sequences of aureolysin of S. aureus, thermolysin of B. thermoproteolyticus, and gelatinase of E. faecalis were aligned using the ClustalW program. The active site residues are indicated by the gray boxes. Symbols: asterisks, identical residues in all sequences; colons, conserved substitutions; periods, semiconserved substitutions. The sequence of the C-terminal 18 amino acids is shown at the bottom along with the calculated molecular masses of GelE mutant proteins lacking the last 5 (Δ5), 6 (Δ6), 12 (Δ12), 13 (Δ13), 14 (Δ14), and 15 (Δ15) amino acids. The predicted molecular masses (mw) were obtained with the ExPASy pI-M_w tool. The predicted molecular masses of the GelE Q306P mutant proteins lacking the last five or six amino acids are indicated in parentheses. D, daltons.

FIG. 2.

Schematic representation of gelatinase proteolytic processing. The gelE gene encodes a 509-amino-acid protein with a probable signal peptide (S.P.) that is 29 amino acids long. The propeptide sequence extends from residue 30 to residue 191, and the mature gelatinase starts at residue V192. The predicted masses of the mature protein and C-terminal peptide were determined with the ExPASy pI-M_w tool. The molecular weight of the mature protein purified from culture supernatants was determined by mass spectrometry.

FIG. 3.

Analysis of the protein profiles for the culture supernatants of GelE derivatives. The supernatants were concentrated 20-fold and loaded on a 12% polyacrylamide gel for Coomassie blue staining (A) or for Western blot analysis using anti-C-terminal His tag antibody (B). (C) Protein profile of the same supernatants after overnight incubation al 37°C.

FIG. 4.

Analysis of the electrophoretic mobility of GelE proline substitution mutant proteins. Gelatinases purified from culture supernatants of strain FA2-2 expressing the wild-type protein (GelE wt) and the E303P, D304P, I305P, and Q306P mutant proteins were run on a 12% SDS-PAGE gel and stained with Coomassie blue. The broad range protein marker (New England Biolabs) (lanes MW) was included in the analysis. The molecular weights of the five proteins were determined by matrix-assisted laser desorption ionization—time of flight mass spectrometry. MW, molecular weight.

FIG. 5.

Hydrolysis of azocoll by GelE and site-specific mutants as a function of time measured by the absorbance of released azocoll dye. The assay was carried out with FA2-2 strains carrying plasmids pML28 (vector alone) (♦), pML29 (GelE wild type) (▪), pML38 (GelE E303P) (▵), pML39 (GelE D304P) (○), pML40 (GelE I305P) (⋄), and pML41 (GelE Q306P) (•). The data are the averages of two independent experiments; the error bars indicate the standard deviations. Each data point is the average of triplicate measurements. OD₅₅₀, optical density at 550 nm.

FIG. 6.

Autolysis assay with FA2-2 strains expressing gelatinase. The autolysis assay was carried out with FA2-2 strains carrying plasmids pML28 (vector alone) (•), pML29 (GelE wild type) (▴), pML34 (GelE-CT14aa) (▪), and pML41 (GelE Q306P) (♦). Samples were analyzed at 30-min intervals as described in Materials and Methods. OD₆₀₀, optical density at 550 nm; T0, time zero.

FIG. 7.

Analysis of GelE E137Q protein secretion and biofilm formation. The culture supernatants were concentrated 20-fold and loaded on a 12% polyacrylamide gel for Coomassie blue staining (A) or for Western blot analysis using anti-C-terminal His tag antibody (B). The molecular weight standards were the New England Biolabs broad range protein marker (A) and the broad range prestained protein marker (B). The arrowhead indicates the full-length GelE protein band. (C) Analysis of biofilm formation by FA2-2 strains carrying plasmids expressing wild-type GelE (pML29), GelE E137Q (pML44), and His-tagged GelE E137Q (pML45). The control strain carried only the vector pML28. The error bars indicate the standard deviations. OD₅₅₀, optical density at 550 nm.