The zinc metalloprotease secreted by E. faecalis
, gelatinase, is one of the few characterized virulence factors of this organism. Gelatinase has been shown to contribute to disease pathogenesis in a number of model systems, including peritonitis in mice (34
), endophthalmitis in rabbits (7
), and nematode killing (34
). In addition to gelatin, only a few substrates are known for this protease. The list of known substrates includes the enterococcal conjugative sex pheromones and a number of host-derived in vitro substrates, including the insulin B chain, endothelin, hemoglobin, fibrinogen, fibronectin, collagen, and laminin (18
). In addition, gelatinase has been shown to interfere with innate immune defenses through inactivation of the antibacterial peptides LL-37 and α-defensin (31
). Recently, gelatinase has been shown to be relevant for in vitro translocation of E. faecalis
across polarized human enterocyte-like T84 cells (42
As a member of the M4 family of zinc metalloproteases, gelatinase possesses a conserved catalytic domain typified by the primary-sequence HEXXH motif responsible for coordinating zinc in the active site (13
). In addition, these zinc metalloproteases bind three or four calcium atoms, which are thought to stabilize the overall structure; the more thermal stable proteases bind four calcium atoms.
Members of the M4 family are secreted bacterial enzymes that are synthesized as preproenzymes. The presequence, or signal peptide, targets the proenzyme to the secretion apparatus of either gram-negative or gram-positive cells. Maturation of the secreted proenzyme to its active mature form is thought to proceed by autocatalytic processing and transient association of the propeptide or prosequence with the active form (19
). The primary purpose of the propeptide is to function as an intramolecular chaperone facilitating the proper folding of the active protease (20
Here we showed that an active gelatinase is required for processing of the propeptide at the amino terminus and cleavage of the C-terminal tail. Mutation of a critical residue in the GelE active site (E137) resulted in secretion of a protein whose molecular weight approximated the molecular weight of the gelatinase proenzyme and that retained the carboxy-terminal histidine tag. This is reminiscent of the previously reported autocatalytic processing of thermolysin (19
). However, autoprocessing of GelE may not necessarily be an intramolecular event, as reported for thermolysin (19
). In supernatants of strain FA2-2 expressing the GelE E137Q His-tagged protein, we observed disappearance of the histidine tag upon addition of purified wild-type GelE, as determined by Western blot analysis with an anti-C-terminal His tag antibody (see Fig. S4 in the supplemental material). This suggested that at least the C-terminal processing event may not be an intramolecular event.
In addition to the amino-terminal processing, some M4 family members from bacteria belonging to the family Vibrionaceae
also require processing at the carboxy terminus to become fully active (15
). Here we show that the E. faecalis
gelatinase also requires proteolytic processing at the carboxy terminus, which makes it unique among members of the M4 family from gram-positive bacteria. Determination of the precise molecular mass of GelE purified from E. faecalis
culture supernatants resulted in a value (33,030 Da) in good agreement with the original estimates of Mäkinen et al. obtained by using SDS-PAGE (33,000 Da), sizing column analysis (32,000 Da), or fast protein liquid chromatography (31,500 Da) (18
Our investigation of the reason for the approximately 1,500-Da discrepancy with the predicted molecular mass of gelatinase indicated that a processing event takes place at the carboxy-terminal end, most likely between the D304 and I305 residues, and this requires the Q306 residue. As mentioned above, evidence suggested that GelE itself may be involved in the processing. An isoleucine residue is not as common as leucine on the imino side of the scissile bond of known substrates, but such a residue was found for angiotensin and neurotensin, providing further support for the autocatalytic hypothesis (18
). Additionally, a hydrophobic amino acid residue has often been found at this position. Furthermore, the molecular weight of the I305P mutant protein suggests that there is an alternative processing event, possibly between Q306 and V307; this could also be a gelatinase processing site as glutamine at the carboxylic side and leucine at the imino side of the bond were found in the gelatinase processing site of the insulin B chain (18
Nevertheless, additional proteases, such as the coregulated enzyme encoded by sprE
, may contribute to the C-terminal processing, as suggested by the molecular weight of the Q306P mutant protein, for which cleavage between the E312 and S313 or S313 and V314 residues must have occurred to explain the molecular weight obtained by mass spectrometry. Singh et al. recently showed that in the absence of a functional fsr
system, a basal level of GelE and SprE is produced; thus, we cannot rule out the possibility that a basal level of SprE contributes to GelE maturation in strain FA2-2 (35
The role of the Q306 residue in the processing of the C-terminal end of GelE is clearly critical for efficient cleavage, as observed with the wild-type protein; in the purification conditions used in this study, this protein is found essentially fully processed, while the Q306P mutant protein was processed at an alternative site, resulting in a protein with a slightly higher molecular weight than the wild-type protein (Fig. ). Nevertheless, the GelE-CT14 His-tagged variant seemed to be processed to a molecular weight essentially identical to that of the wild-type protein (Fig. ) despite the lack of the Q306 and I305 residues, which were replaced by Glu and Leu residues (introduced by the XhoI cloning site), respectively, that linked the protease to six histidine residues. However, this processing event was not highly efficient, and its efficiency seemed to increase when the I305 residue alone or the I305 and Q306 residues were still present (pML42 and pML43) (Fig. ), again suggesting a role for these residues in proper cleavage. Further studies of the biochemical properties of GelE are required to define the mechanism of this autocatalytic event.
The exact role of the GelE C-terminal processing event in the overall activity of the protease appears to be marginal because the Q306P mutant protein was only 25 to 30% less active than the wild-type proteins in the protease assay and it did not have a significant effect on biofilm formation. The C-terminal processing event, however, may delay maturation of the protein, and this may affect protease activity in some specific environmental conditions.
The observation that the Q306P mutant protein had a higher rate of autolysis (Fig. ) raised the possibility that this GelE variant may remain more tightly associated with the cell membrane or cell wall, perhaps due to the slightly hydrophobic nature of the 14-amino-acid C-terminal tail. This could favor processing and maturation of the autolysin substrate of GelE, muramidase-1 (33
), giving rise to the observed phenotype. Thus, processing of the C-terminal tail of gelatinase may have a regulatory role in cell division through the regulation of the autolysin's activity.
It is possible that the C-terminal 14 amino acids have a secondary signaling function. The role of the conjugative peptide pheromones in E. faecalis
, which arise from processing of a bacterial signal peptide sequence of surface lipoproteins, is well characterized and provides a precedent for secondary functions for processed peptide sequences. However, so far there is no evidence for such a role for the GelE C-terminal peptide (3
). There is also no evidence that the C-terminal 14 amino acids of GelE have a distinct function in the protein other than slightly inhibiting gelatinase activity, as reported for the Vibrio cholerae
non-O1 hemagglutinin/protease, whose processing of the C-terminal 2 kDa results in increased protease activity but decreased hemagglutinin activity (23
). Thus, the precise role of this domain in the biology of Enterococcus