Molecular Diversity of a Putative Virulence Factor: Purification and Characterization of Isoforms of an Extracellular Serine Glutamyl Endopeptidase of Enterococcus faecalis with Different Enzymatic Activities

Abstract

A previously identified gene sprE of Enterococcus faecalis strain OG1 was shown to encode an extracellular serine protease that appears to belong to the glutamyl endopeptidase I staphylococcal group. A single form of SprE with a molecular mass of 25 kDa and a pH optimum between 7.0 and 7.5 was isolated from culture supernatant of wild-type E. faecalis strain OG1RF (TX4002); this form was apparently generated by cleavage of the Ser ؊1 -Leu 1 and Arg 230 -Leu 231 peptide bonds of the secreted zymogen. In contrast, the culture supernatant of the gelatinase-null mutant, TX5264, with a nonpolar deletion of gelE which encodes the E. faecalis gelatinase, was found to contain several forms of SprE proteolytically processed on both the N and C termini; in addition to a full-length zymogen and a truncated zymogen, three mature forms of the SprE proteinase, Leu 1 -Ala 237 , Ser ؊1 -Glu 227 , and Leu 1 -Glu 227 , were identified. As with the V8 proteinase of Staphylococcus aureus, the closest homologue of SprE, all of the active forms cleaved specifically Glu-Xaa peptide bonds but with substantially different efficiencies, while none was able to hydrolyze peptide bonds with Asp in the P1 position. The most active of all these enzyme forms against several substrates, including human fibrinogen and ␤-chain insulin, was the Ser ؊1 -Glu 227 ( ؊1 S-SprE) isolated from TX5264; ؊1 S-SprE, in contrast to other forms of SprE, was unstable at 37°C, apparently due to autodegradation. In conclusion, our results demonstrate that sprE encodes a highly specific serine-type glutamyl endopeptidase, the maturation of which is dependent on the presence of gelatinase. In the absence of gelatinase activity, the aberrant processing of pro-SprE results in the appearance of a "superactive" form of the enzyme, ؊1 S-SprE.Enterococci, often viewed primarily as human commensals and even used as probiotics, have become problematic nosocomial pathogens, at least in part because of their increasing resistance to many antibiotics and their ability to infect the growing pool of severely debilitated and/or immunocompromised patients who undergo prolonged antibiotic therapy (27,(37)(38)(39). Several groups have recently undertaken a search for enteroccocal virulence factors in an effort to devise new solutions to the problems caused by these bacteria (20, 25). Included among these may be enterococcal proteinases, as enzymes of this class have been previously suggested to be important virulence factors for other bacterial pathogens. Examples include the V8 proteinase of Staphylococcus aureus involved in septicemia (2, 14, 44) and its homologue GluSE from S. epidermidis, found to be important for slime production and, consequently, biofilm formation by this bacterium in vitro (36,43). Furthermore, the cysteine endopeptidase SpeB of Streptococcus pyogenes (8,9,16,(29)(30)(31) and proteases of Porphyromonas gingivalis (3,4,24,42,45), Yersinia spp. (22,28,(54)(55)(56), and Pseudomonas aeruginosa (10, 17, 23) have all been implicated as virulence factors.Enterococ...

Gelatinase Is Important for Translocation of Enterococcus faecalis across Polarized Human Enterocyte-Like T84 Cells

“…Subsequent analysis of gelatinase activity, as measured by proteolysis of Azocoll, indicated a clear correlation between gelatinase activity of bacteria grown in tissue culture medium plus T84 cells and translocation: the gelE disruption mutant showed no detectable activity against Azocoll, the gelE deletion mutant showed a minimal amount of activity (OD 540 of 0.1 Ϯ 0.1 compared to 2.3 Ϯ 0.3 for OG1RF), and complementation fully restored activity (OD 540 of 2.7 Ϯ 0.1). The residual proteolytic activity shown by the gelE deletion mutant is likely due to production of a superactive serine protease in this mutant (6), the activity of which is different from that of SprE in wild-type OG1RF due to different processing (6).…”

Fsr-Independent Production of Protease(s) May Explain the Lack of Attenuation of an Enterococcus faecalis fsr Mutant Versus a gelE - sprE Mutant in Induction of Endocarditis

“…Bacteria used (Table 1) include wild-type OG1RF (Gel ϩ , Spr ϩ ) (13), the fsrB deletion mutant TX5266, and the gelE insertion disruption mutant TX5128 (10,21); the latter two are Gel Ϫ using a standard plate assay, (20,21) and by Northern blot analysis we had previously shown that gelE and sprE are cotranscribed in OG1RF and no sprE mRNA is detected in TX5128 (21). TX5283 (20) is the fsrB deletion mutant TX5266 containing the shuttle vector pTCV-lac with promoterless lacZ (19), while TX5286 is TX5266 carrying pTEX5270 (gelE promoter::lacZ fusion in pTCV-lac) (20).…”

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

“…Seminal work has demonstrated the ability of GelE secreted from E. faecalis to degrade casein (40). E. faecalis V583 cells and CCM exhibited the ability to degrade casein ( Fig.…”