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PubMed2004

Expression, purification, crystallization and preliminary crystallographic studies of the Enterococcus faecalis cytolysin repressor CylR2.

The expression of an exotoxin called cytolysin contributes to the virulence of Enterococcus faecalis, one of the organisms responsible for antibiotic resistant infections acquired in hospitals. The DNA-binding protein CylR2 is a transcriptional repressor of cytolysin. At a specific cell density, cytolysin triggers signaling events, which result in the dissociation of CylR2 from its DNA-binding site. CylR2 was overexpressed in Escherichia coli and purified and crystals diffracting to 1.9 A were obtained in two different crystal forms. One crystal form belongs to space group P4(1), with unit-cell parameters a = 63.7, b = 63.7, c = 41.2 A, alpha = beta = gamma = 90 degrees, and the other belongs to space group P1, with unit-cell parameters a = 36.9, b = 45.0, c = 47.7 A, alpha = 67, beta = 90, gamma = 66 degrees.

Bacterial Proteins↗

PubMed2004

Crystallization and preliminary crystallographic analysis of an Enterococcus faecalis repressor protein, CylR2, involved in regulating cytolysin production through quorum-sensing.

CylR2 is one of two regulatory proteins associated with the quorum-sensing-dependent synthesis of cytolysin in the common pathogen Enterococcus faecalis. The protein was expressed with a C-terminal six-histidine tag and purified to homogeneity with a cobalt-affinity column followed by size-exclusion chromatography. Both native and SeMet proteins were produced and crystallized. Complete X-ray diffraction data sets were collected from a native crystal, which diffracted to 2.3 angstroms resolution, and a SeMet crystal, which diffracted to 2.1 angstroms. The crystals were tetragonal, belonging to space group P4(1) or P4(3), with unit-cell parameters a = b = 66.2, c = 40.9 angstroms, alpha = beta = gamma = 90 degrees. Based on the calculated Matthews coefficient of 2.6 angstroms3 Da(-1) as well as analysis of anomalous difference Patterson maps, the asymmetric unit most likely contains two molecules of CylR2.

Cloning, Molecular↗

PubMed2004

The three-dimensional structure of catalase from Enterococcus faecalis.

Enterococcus faecalis haem catalase was crystallized using lithium sulfate at neutral pH. The crystals belong to space group R3, with unit-cell parameters a = b = 236.9, c = 198.1 A. The three-dimensional structure was determined by molecular replacement using a subunit of the Proteus mirabilis catalase structure. It was refined against 2.3 A synchrotron data to a free R factor of 21.8%. Like other catalases, the E. faecalis catalase is a homotetramer with a fold and structure similar to those of its structurally closest relative P. mirabilis. The solvent structure in the active site is identical in the four subunits but differs from that found in other catalases. The structural consequences of the Ramachandran outlier Ser196 are discussed.

Amino Acid Sequence↗

PubMed1999

Crystallization and preliminary X-ray characterization of VanA from Enterococcus faecium BM4147: towards the molecular basis of bacterial resistance to the glycopeptide antibiotic vancomycin.

A recombinant form of Enterococcus facieum BM4147 D-alanine-D-lactate ligase (VanA) has been prepared and crystallized. VanA was found to crystallize only in the presence of a phosphinate inhibitor analogue of D-alanine-D-alanine. The crystals grow in 40-45% ammonium sulfate, 0.1 M 3-(N-morpholino)-propanesulfonic acid pH 6.0 and reach dimensions of 0.4 x 0.2 x 0.1 mm. The crystals diffract to at least 2.5 A and are in the centred orthorhombic space group C222(1), with unit-cell dimensions a = 123.2, b = 225.4, c = 72.4 A.

Bacterial Proteins↗

PubMed2006

Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis.

The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 A resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 A, alpha = gamma = 90.0, beta = 119.1 degrees. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.

Adenosine Triphosphatases↗

PubMed2006

Overexpression, crystallization and preliminary X-ray crystallographic analysis of phosphopantetheine adenylyltransferase from Enterococcus faecalis.

Phosphopantetheine adenylyltransferase, an essential enzyme in the coenzyme A biosynthetic pathway, catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine, yielding 3'-dephospho-CoA and pyrophosphate. Enterococcus faecalis PPAT has been overexpressed in Escherichia coli as a fusion with a C-terminal purification tag and crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium HEPES pH 7.5, 0.8 M sodium dihydrogen phosphate and 0.8 M potassium dihydrogen phosphate. X-ray diffraction data were collected to 2.70 A at 100 K. The crystals belong to the primitive tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = b = 160.81, c = 225.68 A. Four copies of the hexameric molecule are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (V(M)) of 3.08 A(3) Da(-1) and a solvent content of 60.1%.

Bacterial Proteins↗

PubMed2004

Enterococcus faecalis mevalonate kinase.

Gram-positive pathogens synthesize isopentenyl diphosphate, the five-carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR-amplified and cloned into pET-28b the mvaK1 gene thought to encode mevalonate kinase, the fourth enzyme of the pathway. Following transformation of the construct EFK1-pET28b into Escherichia coli BL21(DE3) cells, the expressed C-terminally hexahistidine-tagged protein was purified on a nickel affinity support to apparent homogeneity. The purified protein catalyzed the divalent ion-dependent phosphorylation of mevalonate to mevalonate 5-phosphate. The specific activity of the purified kinase was 24 micromole/min/mg protein. Based on sedimentation velocity data, E. faecalis mevalonate kinase exists in solution primarily as a monomer with a mass of 32.2 kD. Optimal activity occurred at pH 10 and at 37 degrees C. Delta H(a) was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. K(m) values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg(2+)). Unlike mammalian mevalonate kinases, E. faecalis mevalonate kinase utilized all tested nucleoside triphosphates as phosphoryl donors. ADP, but not AMP, inhibited the reaction with a K(i) of 2.7 mM.

Adenosine Diphosphate↗

PubMed2005

Enterococcus faecalis phosphomevalonate kinase.

The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

Amino Acid Sequence↗

PubMed2001

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium.

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.

Amino Acid Sequence↗

PubMed2004

In vitro antimicrobial effect of chlorhexidine-impregnated gutta percha points on Enterococcus faecalis.

AIM: To evaluate the in vitro antimicrobial effect of chlorhexidine-impregnated gutta percha points, Roeko activ point (Roeko, Langenau, Germany) on Enterococcus faecalis. METHODOLOGY: Human maxillary premolar roots were prepared with.04 rotary ProFile instruments to a master apical file size 40, autoclave-sterilized and then infected with E. faecalis (ATCC 29212) for 3 weeks. Baseline controls were carried out verifying negligible effects of plain gutta percha cones on E. faecalis. Subsequent to intracanal placement of calcium hydroxide, 'activ points' or saline (positive control) and the 2-week incubation in 54 root specimens, dentine sampling at depths of 100 and 250 micro m was carried out using.04 rotary ProFile instruments at sizes 60 and 90 to assess the quantity of bacteria present. Inactivating agents were used prior to sampling and the colony-forming units (CFU) of E. faecalis were then plate-counted after culturing. Statistical analysis was completed using the paired t-test. RESULTS: In comparison to the positive control, treatment with calcium hydroxide (P = 0.000 and 0.000) or activ points (P = 0.000 and 0.002) produced significantly lower colony counts of E. faecalis at dentine depths of 100 and 250 micro m, respectively. Calcium hydroxide (2.10 x 102 CFU mL-1) was significantly more effective than activ points (1.58 x 103 CFU mL-1) at 100 micro m (P = 0.013), but not at 250 micro m (P = 0.353). Neither of these two medications was able to eliminate E. faecalis completely. CONCLUSIONS: Chlorhexidine-impregnated activ points did not possess an in vitro inhibitory activity strong enough to eliminate E. faecalis completely from infected dentinal tubules.

Anti-Infective Agents, Local↗

PubMed2004

Survival of Enterococcus faecalis in infected dentinal tubules after root canal filling with different root canal sealers in vitro.

AIM: To investigate the ability of different endodontic sealers and calcium hydroxide to kill bacteria in experimentally infected dentinal tubules. METHODOLOGY: Fifty-six human root segments were enlarged to size 2 (ISO size 090) Largo Peeso Reamer. After treatment with 17% EDTA and 5% NaOCl for 4 min each, the specimens were infected with Enterococcus faecalis for 3 weeks. The roots were divided into eight groups and filled with gutta-percha and AH Plus (AH); Grossman's sealer (GS); Ketac-Endo (KE); Apexit (AP); RoekoSeal Automix (RSA); or RoekoSeal Automix with an experimental primer (RP), or calcium hydroxide (CH) only. One group of specimens was left unfilled for control (CT). Following storage in humid conditions at 37 degrees C for 7 days, the root canals were re-established with new sterile Largo size 2. Dentine samples from each canal were then collected using a sterile size 5 (ISO size 150) Largo Peeso Reamer. The number of colony-forming units (CFU) was determined for each sample. RESULTS: The mean log10 CFU in all test groups was significantly lower (P < 0.05) than that in the CT group. Root filling with AH and GS killed bacteria (mean CFU = 0) in the dentinal tubules. The mean log10 CFU for the CH group (0.53) was lower than that of RSA, AP, RP and KE (1.36, 1.40, 1.46 and 1.94, respectively), but only the difference between the CH and the KE groups was statistically significant (P < 0.05). CONCLUSION: Root fillings in vitro with gutta-percha and AH or GS were effective in killing E. faecalis in dentinal tubules. Other endodontic sealers, as well as CH, were less effective.

Calcium Hydroxide↗

PubMed2004

Inter-hospital dissemination of glycopeptide-resistant Enterococcus faecalis in Brazil.

The antimicrobial susceptibility patterns of 73 glycopeptide-resistant Enterococcus faecalis isolates from nine hospitals in Brazil were analysed by the disk diffusion method and Etests. Isolates were typed by pulsed-field gel electrophoresis (PFGE), and vancomycin resistance genes were detected by PCR. The isolates shared a single major PFGE pattern, with six subtypes, and all were positive for vanA. These results indicate the occurrence of inter-hospital dissemination of glycopeptide-resistant E. faecalis in São Paulo, and raise concerns about the rapid dissemination of this pathogen throughout Brazil.

Anti-Bacterial Agents↗

PubMed1992

Proposal of a new scheme for the serological typing of Enterococcus faecalis strains.

No systematic study on serotyping of Enterococcus faecalis has been reported since 1964 when M.E. Sharpe conducted serotyping of group D streptococcus in U.K. So, we attempted to re-evaluate serotyping of E. faecalis. For this purpose, we received 42 Sharpe's strains and first examined for their biochemical characteristics as E. faecalis. Only 9 of the 42 strains were identified as E. faecalis. We raised rabbit antisera against a large number of E. faecalis strains, including the 9 Sharpe's strains, 2 strains obtained from CDC in U.S.A. and 36 strains isolated from patients hospitalized in different cities of Japan. From the results of cross-agglutination tests and absorption tests performed on these antisera using a large number of E. faecalis strains, we were able to classify 21 distinct serotype strains and to prepare 21 monospecific typing antisera by absorption of the antisera to the type strains with appropriate cross-agglutinating strains. When 832 E. faecalis strains were serotyped with the 21 typing antisera, 638 strains (76.7%) were typable. Thus, we propose a provisional scheme of 21 distinct serovars in E. faecalis.

Agglutination Tests↗

PubMed1993

Cytotoxic effect of hemolytic culture supernatant from Enterococcus faecalis on mouse polymorphonuclear neutrophils and macrophages.

We reconfirmed that the LD50s of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.

Animals↗

PubMed1996

Cloning and sequencing of the spore germination gene of Bacillus megaterium ATCC 12872: similarities to the NaH-antiporter gene of Enterococcus hirae.

The germination mutant TM-31 of Bacillus megaterium ATCC 12872, was isolated by transposon Tn917 insertional mutagenesis. Glucose, L-proline, L-leucine and KNO3 germinated TM-31 poorly. The DNA in the region of the Tn917 insertion was cloned, and its nucleotide sequence determined. One major open reading frame was present on the cloned DNA. The hydrophobic protein encoded is presumably membrane-associated. A homology search revealed that the gene encoded in the region of the Tn917 insertion is homologous to napA of Enterococcus hirae. napA codes for the NaH-antiporter. It is hypothesized that transport of cations must play an important role in spore germination in B. megaterium ATCC 12872.

Amino Acid Sequence↗

PubMed1996

Prophylactic effect of Enterococcus faecalis FK-23 preparation on experimental candidiasis in mice.

The prophylactic effects of heat-killed cells of Enterococcus faecalis FK-23 (FK-23 preparation) on experimental candidiasis were investigated in normal and leukopenic mice. In cyclophosphamide-induced leukopenic mice, oral or intraperitoneal administration of the FK-23 preparation at a daily dose of 1.25 or 5 mg/mouse for 3 consecutive days prior to Candida albicans infection significantly prolonged survival periods of the infected mice, and decreased viable counts of C. albicans recovered from their kidneys. In normal mice, the FK-23 preparation administered at dosages ranging from 0.63 to 10 mg/mouse/day for 3 consecutive days was ineffective, while in leukopenic mice, the FK-23 administered orally caused a facilitated recovery in the number of white blood cells including neutrophils. Furthermore, intraperitoneal administration of the FK-23 preparation into mice augmented the anti-Candida activity of immunocompromised peritoneal exudate cells obtained from the animals. These results suggested the potential usefulness of the FK-23 preparation as a prophylactic agent for the management of patients with opportunistic fungal infections.

Animals↗

PubMed2000

Oxalate-degrading Enterococcus faecalis.

An oxalate-degrading Enterococcus faecalis was isolated from human stools under anaerobic conditions. The bacteria required a poor nutritional environment and repeated subculturing to maintain their oxalate-degrading ability. The E. faecalis produced 3 proteins (65, 48, and 40 kDa) that were not produced by non-oxalate-degrading E. faecalis as examined by SDS-PAGE. Antibodies against oxalyl-coenzyme A decarboxylase (65 kDa) and formyl-coenzyme A transferase (48 kDa) obtained from Oxalobacter formigenes (an oxalate-degrading anaerobic bacterium in the human intestine) reacted with 2 of the proteins (65 and 48 kDa) from the E. faecalis as examined by Western blottings. This is the first report on the isolation of oxalate-degrading facultative anaerobic bacteria from humans.

Adult↗

PubMed2004

Effects of lysed Enterococcus faecalis FK-23 on allergen-induced serum antibody responses and active cutaneous anaphylaxis in mice.

BACKGROUND: Our previous studies have presented evidence that lysed Enterococcus faecalis FK-23 (LFK), a lysozyme and heat-treated probiotic product, can inhibit allergen-induced local accumulation of eosinophils in mice. OBJECTIVE: The purpose of this experimental study was to evaluate the influence of orally administrated LFK on the host immune responses. METHODS: BALB/c mice were sensitized subcutaneously, and challenged intraperitoneally by cedar pollen allergen. Blood and spleen samples were collected after oral administration of LFK 60 mg/day for 21 days. The serum levels of total and allergen-specific IgE and IgG2a antibodies and the production of IL-4, IL-5 and IFN-gamma generated by allergen-stimulated cultured splenocytes were determined. Additionally, the effect of LFK on active cutaneous anaphylaxis (ACA) induced by ovalbumin (OVA) challenge in mice was measured after 28 days LFK treatment. RESULTS: No significant differences in serum immunoglobulin levels, as well as in cytokine production of splenocytes were observed between LFK-treated and control mice (P>0.05). There was, however, an increasing tendency of allergen-specific IgG2a level in mice after LFK treatment for 21 days compared with controls (P=0.060). Furthermore, the serum ratio of specific IgE to IgG2a was found to be significantly decreased in the LFK group (P=0.005). In addition, a significant inhibition of OVA-induced ACA reaction was observed in mice that had been fed for 28 days with LFK compared with control mice (P=0.008). CONCLUSION: These results suggest that LFK shows an anti-inflammatory effect, which may be part of the mechanism for protection against IgE-mediated allergy.

Administration, Oral↗