Quantitative determination of gelatinase activity among enterococci

Kanemitsu, Keiji; Nishino, Takeshi; Kunishima, Hiroyuki; Okamura, N; Takemura, Hiromu; Yamamoto, Hiroyuki; Kaku, Mitsuo

doi:10.1016/s0167-7012(01)00283-4

Cited by 49 publications

(36 citation statements)

References 18 publications

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“…The gelatinase assay was performed as described previously (13). Briefly, 5 l of an overnight culture and 5 l of supernatant were incubated on a gelatin plate (0.8% gelatin and 5% agar).…”

Section: Methodsmentioning

confidence: 99%

Influence of Culture Heterogeneity in Cell Surface Charge on Adhesion and Biofilm Formation by Enterococcus faecalis

et al. 2006

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Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms. Enterococcus faecalis strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of E. faecalis strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein (esp) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.

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“…The gelatinase assay was performed as described previously (13). Briefly, 5 l of an overnight culture and 5 l of supernatant were incubated on a gelatin plate (0.8% gelatin and 5% agar).…”

Section: Methodsmentioning

confidence: 99%

Influence of Culture Heterogeneity in Cell Surface Charge on Adhesion and Biofilm Formation by Enterococcus faecalis

et al. 2006

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“…Downstream from gelE, an open reading frame called sprE, coding for a putative serine protease (GenBank accession No Z12296), was identified (57). While gelatinase activity and the gelE gene have been utilized in a number of studies, including epidemiological ones (11,18,26,35,(61)(62)(63), and in animal models of infection (15,53), suggesting a possible role in microbial virulence and host response (33), until recently, little has been done to investigate sprE and the possible role of the predicted SprE protein or the presence of any other proteolytic activities in E. faecalis. Qin et al described the fsr locus, a regulatory system of E. faecalis (41,47,48) that is homologous to the S. aureus agr locus (49) that encodes a quorum sensing system regulating cotranscription of gelE and sprE from the common promoter (47,48).…”

mentioning

confidence: 99%

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

et al. 2005

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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...

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“…While some of these studies measured gelatinase activity by plate assay (1,5,7,10,28), others used DNA-based techniques to determine whether the gelE gene was present (25), and such techniques are now known to not reliably predict the gelatinase phenotype. In our study, we used the gelatinase plate assay, hybridization, and PCR to correlate the observed gelatinase phenotype with the underlying fsr and gelE genotypes.…”

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confidence: 99%

Molecular Epidemiology of the fsr Locus and of Gelatinase Production among Different Subsets of Enterococcus faecalis Isolates

Roberts¹,

Singh²,

Okhuysen³

et al. 2004

J Clin Microbiol

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We examined 215 Enterococcus faecalis isolates and found that neither the two-component regulatory locus fsr (E. faecalis regulator) nor gelatinase production was more common in disease-associated isolates than in isolates colonizing healthy individuals (ca. 60 to 65%). The majority of gelatinase-negative isolates, including 14 endocarditis isolates (of 80 isolates tested), contained the previously described 23.9-kb deletion and lacked fsrA and fsrB. While these findings indicate that neither fsr nor gelatinase is required for E. faecalis to cause infection, this study did not address whether fsr or gelatinase affects the severity of disease, as it does in animal models.Enterococci are normally commensal flora, but they can cause a wide range of diseases, including urinary tract infections, bloodstream infections, wound infections, and endocarditis (12, 13). Although there are over 20 species of enterococci (8), Enterococcus faecalis isolates cause the majority of infections (22). Despite the increasing number of cases of enterococcal infection and the remarkable ability of these organisms to resist antibiotics, relatively little about their pathogenesis is known compared to what is known about some other grampositive cocci.Despite its role as a possible virulence factor, gelatinase is not produced by all clinical isolates, and a number of studies have attempted to correlate gelatinase activity with disease caused by E. faecalis (3,5,7,17,20,24,28). Nakayama et al. recently reported that a 23.9-kb chromosomal deletion involving the fsr locus was commonly found in urine isolates and was also found in most gelatinase-negative strains (17). In contrast, another study reported that fsr was present in 100% of 12 endocarditis isolates (20). In the present study, we compared clinical isolates of E. faecalis from patients with endocarditis, urinary tract infections, or blood-borne infections to fecal isolates from healthy volunteers with respect to the presence of fsr and gelatinase production.A total of 215 E. faecalis isolates from clinical samples and from feces of healthy, community-based volunteers, collected between 1974 and 2003, were used in this study. The identification of all isolates as E. faecalis was initially done by biochemical tests and was confirmed by using an intragenic portion of the ace gene (18) as a probe for colony hybridization (data not shown). E. faecalis OG1RF (15), E. faecalis OG1SSp (6), E. faecalis V583 (23), and Enterococcus faecium TX0016 (2) are well-studied strains and were used in this study as controls. Brain heart infusion agar (Difco Laboratories, Detroit, Mich.) or brain heart infusion broth was used for growth.

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Quantitative determination of gelatinase activity among enterococci

Cited by 49 publications

References 18 publications

Influence of Culture Heterogeneity in Cell Surface Charge on Adhesion and Biofilm Formation by Enterococcus faecalis

Influence of Culture Heterogeneity in Cell Surface Charge on Adhesion and Biofilm Formation by Enterococcus faecalis

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

Molecular Epidemiology of the fsr Locus and of Gelatinase Production among Different Subsets of Enterococcus faecalis Isolates

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