Production of enterolysin A by a raw milk enterococcal isolate exhibiting multiple virulence factors

Rita M. Hickey1,3, Denis P. Twomey1,2, R. Paul Ross1 and Colin Hill2,3

1 Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
2 National Food Biotechnology Centre, University College Cork, Ireland
3 Microbiology Department, University College Cork, Ireland

Correspondence
R. Paul Ross
pross{at}moorepark.teagasc.ie


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Even though enterococci are a common cause of human infection they can readily be isolated from a range of food sources, including various meat and dairy products. An enterococcal strain, DPC5280, which exhibits a broad spectrum of inhibition against many Gram-positive bacteria was recently isolated from an Irish raw milk sample. Characterization of the inhibition revealed that the strain exhibits haemolytic activity characteristic of the two-component lantibiotic cytolysin and also produces a heat-labile antimicrobial protein of 34 kDa. The latter protein displayed cell wall hydrolytic activity, as evidenced by zymogram gels containing autoclaved lactococcal cells. N-terminal sequencing of the purified protein yielded the sequence ASNEWS which is 100 % identical to enterolysin A (accession no. AF249740), a protein which shares 28 and 29 % identity to the Gly-Gly endopeptidases, lysostaphin and zoocin A, respectively. Indeed, amplification of entL from DPC5280 and sequencing revealed that the protein is 100 % identical to enterolysin A. The DPC5280 strain also contained the determinants associated with multiple virulence factors, including gelatinase, aggregation substance and multiple antibiotic resistance. The linkage of this cell-wall-degrading enzyme to other virulence factors in enterococci may contribute to the competitiveness of pathogenic enterococci when found in complex microbial environments such as food and the gastrointestinal tract.


Abbreviations: AU, arbitrary units


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Despite the fact that foods containing enterococci have a long history of safe use, these organisms are not considered as GRAS (generally recognized as safe) organisms. They can readily be isolated from a range of food sources, including raw milk, and are often a constituent of some mixed starter strains used commercially. However, many strains can act as opportunistic pathogens, causing a variety of infections such as urinary tract infections, bacteremia and infective endocarditis (Murray, 1990; Moellering, 1992; Jett et al., 1994), and are of major importance in community-acquired and in hospital-acquired (nosocomial) infections (Jett et al., 1994; Low et al., 1994; Jones et al., 1997; Simjee & Gill, 1997). A contributing factor to their pathogenesis is their evolving resistance to antibiotics. For example, resistance to the antibiotic vancomycin is now widespread among members of the genus, which leaves few options for disease management (Aguirre & Collins, 1993; Knudtson & Hartman, 1993; Facklam & Sahm, 1995; Klein et al., 1998).

Considerable progress has recently been made in determining the traits responsible for pathogenesis of enterococci. For example, studies have shown that phenotypes such as production of cytolysin, which has both haemolytic activity (by lysing a broad spectrum of cells, including human, horse and rabbit erythrocytes) and bactericidal activity (against Gram-positive bacteria) play a role in the progression of enterococcal infection (Ike et al., 1984; Jett et al., 1992). Cytolysin enhances the virulence of Enterococcus faecalis in animal models, such as murine peritonitis and rabbit endophthalmitis (Ike et al., 1984; Jett et al., 1992; Chow et al., 1993; Jett et al., 1994). Other factors include aggregation substance, which is a pheromone-inducible surface protein of E. faecalis and promotes mating aggregate formation during bacterial conjugation (Clewell, 1993). This protein has been shown to enhance enterococcal adherence to renal cells (Joyanes et al., 2000) and to mediate internalization of E. faecalis by cultured human intestinal epithelial cells (Olmsted et al., 1994). In contrast, gelatinase is a protease that hydrolyses gelatin, collagen, casein, haemoglobin and other bioactive peptides (Coque et al., 1995), which suggests a role in inflammatory processes (Makinen et al., 1989). Other variable traits associated with enterococcal virulence are the enterococcal surface protein (Esp), which may contribute to the ability of E. faecalis to evade detection by the immune system (Shankar et al., 1999) and the EfaA proteins which are homologues of cell surface adhesions found on a number of streptococcal species (Lowe et al., 1995; Singh et al., 1998) and are expressed in serum. In addition, sex pheromones are peptides expressed prior to conjugation which induce genes on the plasmid of the donor strain to produce aggregation substance, thus increasing the frequency of plasmid transfer (Clewell 1990; Simjee & Gill, 1997).

Recently, Eaton & Gasson (2001) investigated the incidence of known virulence factors in medical, food and dairy starter Enterococcus strains. Not surprisingly, PCR and gene probe strategies revealed that medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than starter strains. All of the E. faecalis strains tested possessed multiple virulence determinants while Enterococcus faecium strains were generally free of virulence determinants. In addition, conjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. Thus, despite featuring in dairy fermentations for decades, the use of Enterococcus spp. in foods requires careful safety evaluation as the transfer of virulence determinants and antibiotic resistance to starter strains via natural conjugation mechanisms poses a potential risk in a mixed microbial environment.

In the present study, an E. faecalis strain which exhibited a broad spectrum of inhibition to Gram-positive bacteria, including Listeria, was isolated from Irish raw milk. This activity was found to be due to the production of both cytolysin and a heat-labile antimicrobial protein of 34 kDa. The latter protein was found to have cell-wall-degrading activity and N-terminal sequencing followed by genetic analysis revealed a protein which was 100 % identical to the cell-wall-degrading enzyme, enterolysin A (NCBI accession no. AF249740; Nilsen, 1999). Subsequently, it is shown that the DPC5280 strain also contains genes responsible for multiple virulence factors and multiple antibiotic resistance.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
Enterococcal strains were propagated in M17 (Difco) containing glucose (0·5 %, v/v) broth or agar (1·5 %, w/v). Standard cultures were prepared by inoculation of 5 ml GM17 broth with 5 µl of a frozen stock (-80 °C) and then incubation at 37 °C for 16–24 h. Lactococcus lactis subsp. lactis HP was employed as the DPC5280-sensitive indicator organism. Other micro-organisms used to examine the inhibitory spectrum and the antibiotic susceptibility of Enterococcus faecalis DPC5280 are listed in Table 1.


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Table 1. Bacterial strains used in this study

 
Characterization of the antimicrobial activity associated with E. faecalis DPC5280.
A preliminary characterization of the bacteriocin activity from E. faecalis DPC5280 was performed, using a cell-free, filter-sterilized (0·45 µm pore-size Millex-GV filter; Millipore) stationary phase GM17 culture supernatant which was tested for stability to heat, pH and the proteolytic enzyme proteinase K. For heat sensitivity, 1 ml samples were heated to 25, 50, 80 and 100 °C for 10 min each. To test for pH sensitivity, 1 ml aliquots of active supernatants were adjusted to different pH values (2, 5, 7 and 9) with 1 M NaOH or 1 M HCl. Active supernatant was tested for susceptibility to proteinase K (Sigma-Aldrich) by incubating with 5 µg proteinase K ml-1 in a 1 : 1 ratio at 4 °C for 6 h and using an agar well diffusion assay (Parente & Hill, 1992; Ryan et al., 1996). Plates were incubated at 30 °C for 24 h. The positive control used was the supernatant of E. faecalis FA2-2.pAD1 and the negative control was supernatant of E. faecalis OG1X.

Spectrum of inhibition.
To test the sensitivity of a strain to the antimicrobial activity produced by E. faecalis, 10 µl aliquots of a fresh overnight culture of E. faecalis DPC5280 were first spotted onto GM17 agar plates and incubated overnight at 37 °C. These plates were then overlaid with 3 ml soft agar seeded with 100 µl of the indicator strain (overnight culture) (Table 2). The sensitivity of a strain to the producer was scored according to the diameter of the zone of inhibition surrounding E. faecalis DPC5280. The experiment was performed in triplicate and the mean zone diameter was calculated. The positive control used was the supernatant of E. faecalis FA2-2.pAD1 and the negative control was the supernatant of E. faecalis OG1X.


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Table 2. Inhibitory spectrum of E. faecalis DPC5280 culture and partially purified enterolysin A

NZ, No zone; +, 1–5 mm; ++, 5–10 mm; +++, 10 mm and over.

 
Partial purification and analysis of enterolysin A.
All of the chromatographic media were purchased from Amersham-Pharmacia-Biotech. The non-haemolytic activity was purified from a 4 l GM17 broth culture of E. faecalis DPC5280 at 30 °C. The proteins from the supernatant were precipitated using a 55 % saturation of ammonium sulphate, resuspended in 50 ml 20 mM sodium phosphate buffer (pH 7) and desalted through a PD-10 column. This sample was then applied to a weak anion exchange column, DEAE Sephadex A-50, and allowed to elute by gravity flow. Many of the proteins associated with DPC5280, including cytolysin, were retained on the column while the eluent contained the antimicrobial activity capable of lysing lactococcal cells, while having no detectable cytolytic activity. The eluent was analysed by one-dimensional SDS-PAGE using a Mini Protean II cell unit (Bio-Rad) by the method of Laemmli (1970) with a 10 % acrylamide resolving gel. A prestained standard (wide range 6500–205 000 Da; Sigma) was used as a molecular mass marker. Following electrophoresis, the gel was stained for 1 h with 0·05 % Coomassie brilliant blue R250 and destained for 16 h. A corresponding gel was prepared using 100 ml of overnight culture of L. lactis HP which was then autoclaved and subsequently centrifuged and the resulting pellet was incorporated in the resolving gel (zymogram). This was used to view lytic activity upon renaturing as described by Leclerc & Asselin (1989) and Potvin et al. (1988). The gel was examined for the presence of a lytic zone after 3 h incubation at 37 °C.

Electroblotting and N-terminal amino acid analysis of enterolysin A proteins.
Proteins were electrotransferred from PAGE gels onto PVDF membranes (Bio-Rad) in CAPS buffer (Sigma), pH 11, using a Trans-Blot cell (Bio-Rad), according to manufacturer's instructions. Proteins were stained with Coomassie brillant blue R250, cut off from the membrane and sequenced on a Beckman LF 3000 microsequencer (Molecular Biology Unit, University of Newcastle upon Tyne). Database searches were performed with the program BLASTP.

Lytic activity spectrum and assay.
To test for sensitivity of a strain to partially purified enterolysin A, well diffusion assays were performed as described by Ryan et al. (1996). Molten agar was cooled to 48 °C and seeded with the indicator strains (overnight culture). The inoculated medium was dispensed into sterile Petri plates, allowed to solidify and dried. Wells (~4·6 mm diameter) were made in the seeded agar plates into which 50 µl aliquots of partially purified enterolysin A were dispensed and the plates were incubated overnight. The sensitivity of a strain to enterolysin A was scored according to the diameter of the zone of inhibition surrounding the wells. The experiment was performed in triplicate and the mean zone diameter was determined. DPC5280 supernatant was used as a positive control. The effect of the lytic activity was monitored by measuring the decrease in turbidity of a cell suspension of L. lactis HP. Cells were harvested in the exponential-growth phase at an OD600 of approximately 0·2. Aliquots were supplemented with 50 000 arbitrary units (AU) ml-1 (as described by Ryan et al., 1996) of enterolysin A in 20 mM sodium phosphate buffer (pH 7). As a control, the same volume of 20 mM sodium phosphate buffer was added to the other aliquots and the mixtures incubated at 30 °C. The OD600 of the cultures was measured spectrophotometrically at time intervals of 15 min over a 90 min period.

DNA manipulations.
Total DNA was extracted from overnight Enterococcus cultures using a modification of the method of Hoffman & Winston (1987). Briefly, enterococcal cells were subjected to vortex mixing in the presence of acid-washed glass beads, SDS, Triton X-100, phenol and chloroform. This mixture was then centrifuged to separate the DNA from cellular debris. The DNA was then precipitated using sodium acetate and ethanol, and subsequently washed using 70 % ethanol prior to resuspension in molecular standard distilled water. Plasmid DNA was isolated from E. faecalis DPC5280 using the method of Anderson & McKay (1983). Oligonucleotide primers for PCR were obtained from Genosys and are listed in Table 3. PCR amplifications were performed in 50 µl reaction mixtures using 2 µg DNA, 2 mM MgCl2, 50 pmol each primer and 1 U Taq polymerase (Bioline). Negative control PCRs with no template DNA were also performed. Samples were subjected to a cycle of denaturation (94 °C for 1 min), annealing (at an appropriate temperature for 1 min) and elongation (72 °C for 1 min) for 35 cycles using a Hybaid PCR express unit. Amplified 16S rDNA was purified from a 1 % agarose gel using a QIAquick Gel Purification Kit (Qiagen).


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Table 3. PCR primers used in this study

 
Design of primers.
PCR primers used in this study are listed in Table 3. Primers for gelE, cylMBA and entL were designed on the basis of published sequences and NCBI database sequences while primers for the other virulence genes were originally designed by Eaton & Gasson (2001). PCR primers for amplification of the 16S rRNA gene (CO1 and CO2), were designed originally by Beresford & Condon (1991). These primers were used to identify the species of Enterococcus which was isolated.

DNA sequencing and analysis.
Purified 16S rDNA was sequenced with an automated DNA sequencer (MWG-BIOTECH custom DNA sequencing service, Ebersberg, Germany) using the CO1 and CO2 primers. Sequence analysis was performed using DNAStar software and the program BLASTN.

Production of gelatinase and haemolysin.
Gelatinase activity was detected by the growth of E. faecalis DPC5280 on 3 % gelatin medium as described by Su et al. (1991). Gelatinase-positive colonies were identified by a turbid halo after 2 days incubation at 37 °C. For investigation of haemolysin production, E. faecalis DPC5280 was streaked onto fresh 5 % horse blood agar plates and grown for 24 h at 37 °C. Zones of clearing surrounding isolated colonies indicated haemolysin production.

Antibiotic susceptibility testing.
The antibiotic resistance phenotype of E. faecalis DPC5280 and other control enterococcal strains, listed in Table 4, was determined by the use of antibiotic susceptibility discs obtained from Oxoid. GM17 plates were seeded with 0·5 % inoculum of each enterococcal strain after overnight growth. Six antibiotic discs were placed on each plate and after overnight incubation at 37 °C, the diameter of the zone of inhibition around each disc was measured. The experiment was carried out in triplicate and the results are shown as the mean.


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Table 4. Summary of antibiotic resistance/sensitivity associated with E. faecalis DPC5280

Complete resistance (no zone of inhibition) was found with bacterial cell wall inhibitors cefotetan (30 µg) and cefoxitin (30), protein synthesis inhibitors clindamycin (10), gentamicin (30), kanamycin (30), spectinomycin (25), streptomycin (25) and sulphamethoxazole (25), DNA replication inhibitor nalidixic acid (30) and bacterial cell membrane inhibitor colistin sulphate (25). –, No zone, complete resistance.

 

   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Characterization of E. faecalis DPC5280
Initially, 500 Irish raw milk samples acquired immediately following milking were screened for bacteriocinogenic bacteria with a view to isolating novel antimicrobials. Of the 362 strains which exhibited activity, one, DPC5280, was chosen for further investigation due to its activity against Listeria and other Gram-positive bacteria (Table 2). Based on 16S rRNA sequencing of the strain it belongs to the E. faecalis species (99 % identity to E. faecalis, accession no. AJ420803 and 99 % identity E. faecalis, accession no. AB036835 over 1452 bp). The strain was found to have a relatively broad spectrum of inhibition which included all lactococci, pediococci, lactobacilli, listeriae and some of the enterococci tested. In contrast, neither Staphylococcus aureus nor any of the Gram-negative bacteria tested were inhibited by the strain. Initial characterization of the inhibition demonstrated that it was proteinase K sensitive and heat labile. Indeed, heating to 80 °C completely abolished any detectable antimicrobial activity in supernatants of the culture.

DPC5280 contains the genetic determinants for cytolysin production
Cultivation of DPC5280 on equine blood agar plates revealed that it had a haemolytic phenotype. Given that the haemolytic phenotype is often associated with production of cytolysin in enterococci, the strain was tested for cyl genetic determinants using primers designed for the cylLL, cylLS and cylM genes. In repeated PCRs, a product of 2659 bp was amplified (Fig. 1a) which was sequenced and shown to be 100 % identical to the entire cylLL, cylLS and cylM gene cluster found on the cytolysin encoding plasmid pAD1.



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Fig. 1. (a) Agarose gel showing PCR products generated with primers for known virulence factors. Lanes: 1+13, 1 kb ladder (New England Biolabs); 2+12, 100 bp ladder (New England Biolabs); 3, agg (aggregation protein) 1500 bp; 4, gelE (gelatinase) 700 bp; 5, cylMBA (cytolysin) 2600 bp; 6, esp (enterococcal surface protein) 933 bp; 7, efaAfs (E. faecalis cell wall adhesion) 705 bp; 8, efaAfm (E. faecium cell wall adhesion) 735 bp; 9, cpd (sex pheromone) 782 bp; 10, cob (sex pheromone) 1400 bp; 11, ccf (sex pheromone) 543 bp. (b) Plasmid profile of E. faecalis 5280 (lane 2) and molecular mass standard, the plasmid profile of L. lactis DRC3 (lane 1) as described by McKay & Baldwin (1984).

 
DPC5280 produces enterolysin A.
In an effort to purify the proteins responsible for the antimicrobial activity of the strain, ion exchange chromatography using DEAE Sephadex A-50 was performed on an ammonium sulphate precipitate of the culture supernatant. The eluent obtained after gravity flow had a surprisingly high antimicrobial activity of 10 000 AU ml-1 and had no associated haemolytic activity, which could be detected after washing the column with 1 M NaCl. The haemolytic activity had been retained on the column which therefore separated it from another antimicrobial activity. The proteins in the antimicrobial-containing eluent were subsequently separated on a one-dimensional gel which revealed three proteins of 35, 34 and 29 kDa (Fig. 2a). These were subsequently transferred to a PDVF membrane and sequenced. No sequence could be obtained for the 29 kDa protein possibly due to N-terminal blockage. The first 9 aa of the 35 kDa protein, GSEVTLKNS, were found to be identical to those of gelatinase (also called coccolysin), a zinc-dependent endopeptidase isolated from E. faecalis OG1-10 which is considered to be involved in pathogenicity of Enterococcus species (Coque et al., 1995; Kuhnen et al., 1988). The 34 kDa protein was found to have the sequence ASNEWS, which is identical to the N terminus of enterolysin A (AF249740; Nilsen, 1999). A corresponding zymogram using autoclaved L. lactis HP revealed a zone of lysis at approximately 34 kDa, corresponding with this second band in the stained gel (Fig. 2a'). Primers were designed based on the nucleotide sequences of gelatinase and enterolysin A in the database and amplification via PCR revealed products of 635 and 1721 bp, respectively (Figs 1a and 2b). These products were sequenced and revealed that the 635 bp PCR product displays 99 % identity to gelE from E. faecalis OG1-10 in a 635 bp sequencing run and that the enterolysin product was 100 % identical to entL from E. faecalis LMG 2333 over its entire length (AF249740; Nilsen, 1999).



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Fig. 2. (a) One-dimensional SDS-PAGE. The sample was partially purified enterolysin A. (a') Zymogram containing autoclaved L. lactis HP cells. The sample was partially purified enterolysin A which appears as a dark zone of clearing upon renaturing. (b) PCR of the entL gene. Lanes: 1, 1 kb molecular mass marker; 2, 100 bp molecular mass marker; 3, amplified entL gene.

 
Enterolysin A encodes a 343 aa pre-protein, with a sec-dependent signal peptide of 27 aa, while mature enterolysin A consists of 316 aa with a calculated molecular mass of 34 501 Da. The signal cleavage site is found at a double alanine at position 27. A map of the enterolysin structural gene is presented in Fig. 3. The N-terminal sequence of enterolysin A contains a domain which is found in members of the M37 family of metallopeptidases which have Zn2+ in their catalytic sites (Recsei et al., 1987; Sugai et al., 1997). Members of this family include lysostaphin (Schindler & Schuhardt, 1964; Schleifer & Kandler, 1972; Schleifer & Fischer, 1982; Baba & Schneewind, 1996) and zoocin A (Simmonds et al., 1997) whose conserved N-terminal domain provides the enzymic functions of the proteins and contains a His-Xxx-His motif which may serve as the Zn2+ ligand (Sugai et al., 1997).



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Fig. 3. Map of enterolysin A structural gene (entL) and novel downstream protein (entM). Amino acid homologies for different regions shown in boxes. Possible promoter -35 and -10 sites are underlined. Grey letters represent amino acids in the aligned sequences having homology to enterolysin sequences. ALE1 (D86328), LytM (L77194), A3 (CAA15796), ZooA (ACC46072), YomI (CAB14053) and Phig1e (CAA66745). Asterisks above the GW sequence in entL may be ChW-type repeats and occur at three points within the sequence (two of the repeats are almost identical). L.innA, Listeria innocua (AL596163); L.mono, Listeria monocytogenes (AL591973); L.innB, Listeria innocua (AL5919760).

 
In this study, database searches revealed that the N-terminal region of enterolysin A also shows homology (38 % identity in a 113 aa stretch) to the C-terminal region of an unfinished protein from the E. faecium genome (NC_002712; DOE Joint Genome Institute). This protein contains domains from the amidase 4 family, (mannosyl-glycoprotein endo-{beta}-N-acetylglucosamidase) and the lysozyme subfamily 2 in its N-terminal region (NCBI Conserved Domain Search; gnl|CDD|6589 and gnl|CDD|5777 respectively). While the N-terminal region of enterolysin A more than likely functions as an endopeptidase like that of lysostaphin and zoocin A, the C terminus more than likely represents the cell-wall-binding domain(s) and has no homology with these proteins. Such a result is not surprising since partially pure enterolysin A was found to lyse a broad range of Gram-positive bacteria (Table 2), unlike lysostaphin and zoocin A which are narrow spectrum enzymes. Database searches found that the C-terminal sequence of enterolysin A shows homology (Fig. 3) to the C-terminal sequences of N-acetylmuramoyl-L-alanine amidase from phage PL-1 (Kashige et al., 2000) (66 % identity in a 148 aa stretch) and the putative lysin from phage A2 (Garcia et al., 1997) (59 % identity in a 110 aa stretch), both phage of Lactobacillus casei. The activity spectrum of the lysins associated with these phage is narrow, with the PL-1 phage lysin being strongly effective against the host strain Lactobacillus casei ATCC 27092, but inactive or weakly active against several other Lactobacillus casei strains, Lactobacillus plantarum, Streptococcus pyogenes, Streptococcus mutans, Micrococcus luteus, Bacillus megaterium, Bacillus subtilis and Escherichia coli (Watanabe et al., 1984; Hayashida et al., 1987).

Interestingly, the C terminus of enterolysin A shares weak homology (21 % identity in a 138 aa stretch) with a protein from Clostridium acetobutylicum (NC_003030; Nolling et al., 2001) containing ChW-repeats (clostridial hydrophobic, with a conserved tryptophan; Nolling et al., 2001). ChW-repeat domains contain a distinct repetitive structure which may function in either substrate-binding or protein–protein interactions (Nolling et al., 2001), suggesting they may play a critical role in the cell-wall binding of enterolysin. Some of the ChW-repeat proteins contain additional domains such as glycosyl hydrolases or proteases, which implicates them in the degradation of polysaccharides and proteins. Several also contain domains that are involved in cell interactions, such as the cell adhesion domain (Kelly et al., 1999) and the leucine-rich repeat (internalin) domain (Marino et al., 1999). Modular structure with separate domains for catalytic activity and substrate binding is typical for lysins of Gram-positive bacteria (Riley, 1993; Baba & Schneewind, 1996) and similarity between bacteriophage and bacterial DNA allows shuffling of domains by recombination, restructuring both viral and bacterial genomes (Garcia et al., 1988; Lopez et al., 1992). A short threonine-proline (TP)-rich putative linker sequence is present between the domains of enterolysin A, whose role in the evolution of many protein families is well established by enabling a mix and match of many protein domains (Cooper & Salmond, 1993).

In a simultaneous effort to identify enterolysin from a two-dimensional electrophoretic gel, the same non-haemolytic fraction used in one-dimensional electrophoresis was analysed and a protein of similar molecular mass (but with a pI of approximately 6·5) to enterolysin A was subjected to N-terminal sequencing. Interestingly, the sequence obtained (SEDYNLLGVKNYDQYALGAPSGCEGASLLQGLQYKGKIPDWDL) was 100 % identical to a sequence encoded by a gene directly downstream of entL which displays homology to putative transcriptional regulators from Listeria innocua (38 % identity in a 70 aa stretch; AL596163), Listeria monocytogenes (38 % identity in a 70 aa stretch; AL591973) and Listeria innocua (42 % identity in a 49 aa stretch; AL596166). Given the proximity of these genes and the lack of evidence of an obvious strong intergenic promoter it is probable that the genes are co-transcribed.

To assess the effectiveness of enterolysin A activity, the decrease in OD600 of L. lactis HP culture upon exposure to the enzyme was measured over 90 min. Fig. 4 demonstrates that the addition of enterolysin A resulted in a rapid reduction in OD600 when compared with that of comparable control cultures. A similar result was observed by Nilsen (1999) on exposure of L. lactis IL1403 to enterolysin A. Indeed, following the 90 min period, all of the cells had been lysed by enterolysin A. Fig. 4 also shows a photo of the L. lactis HP cultures after the 90 min period, with a completely lysed culture on the left which has been treated with enterolysin A, while the untreated culture on the right remains turbid. From these results, it is evident that enterolysin A provides DPC5280 with an additional advantage against competing bacteria as lysis of other bacteria may release nutrients, which the strain can then utilize for growth.



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Fig. 4. Killing of L. lactis HP with purified enterolysin A. Bacteriolytic activities of enterolysin A determined by turbidimetry; 500 µl (10 000 AU ml-1) incubated with 5 ml L. lactis HP culture (dashed line). Control (solid line) incubated with 500 µl 20 mM sodium phosphate buffer (pH 7). Results are means of triplicate experiments. Photo of L. lactis HP culture in the presence (+) and absence (-) of enterolysin A.

 
DPC5280 contains multiple genes associated with enterococcal virulence.
Given that DPC5280 was found not only to produce enterolysin A but also exhibit haemolytic and gelatinase activity, the strain was characterized for possession of other virulence factors. Table 3 lists 9 genes associated with enterococcal virulence found on E. faecalis DPC5280 while Fig. 1 shows the PCR products obtained upon amplification of these genes and the plasmid profile of DPC5280. These genes include agg (aggregation protein), gelE (gelatinase), cylLLLSM (cytolysin), esp (enterococcal surface protein), efaAfs (E. faecalis cell wall adhesion), efaAfm (E. faecium cell wall adhesion), cpd (sex pheromone), cob (sex pheromone) and ccf (sex pheromone). Indeed, these results show that the strain possesses all the virulence-associated genes tested. In addition, gelatinase and haemolytic activity could be detected on plates, suggesting expression of these genes. These results correlate with those of Eaton & Gasson (2001), where E. faecalis strains isolated from food sources in certain cases contained many virulence determinants. These factors could influence enterococcal species-specific selection in colonization of the human intestinal tract and may confer enhanced abilities to cause disease beyond that intrinsic to the species background.

In addition, enterococci have emerged as major nosocomial pathogens in part because of their resistance to multiple antibiotics, which allows them to survive and subsequently infect patients. Considering the pathogenic potential of DPC5280, the spectrum of resistance to various antibiotics was determined. The relative sensitivities of E. faecalis DPC5280 and two control strains to 30 different antibiotics are presented in Table 4. The results demonstrate that E. faecalis DPC5280 and the control E. faecalis strains were resistant to some of the cell-wall-inhibiting antibiotics such as cefotetan and cefoxitin which belong to the cephalosporin class of antibiotics. This result may be expected as most enterococci have naturally occurring or inherent resistance to such cell-wall-active agents. All strains tested were found to be sensitive to ampicillin, penicillin G ({beta}-lactams) and vancomycin (glycopeptide). Resistance to these drugs would most likely be acquired after exposure to fixed concentrations of the antibiotics (Hodges et al., 1992). With regards to antibiotics which inhibit protein synthesis, E. faecalis DPC5280 was found to be resistant to chloramphenicol, erythromycin, minocycline, oxytetracycline and tetracycline when compared with the controls. Natural resistance to these agents is usually plasmid- or transposon-mediated and Teuber et al. (1996) reported a common multiple drug resistance type that included resistance to tetracycline, chloramphenicol, erythromycin and also gentamicin. All strains were found to be generally intrinsically resistant to the aminoglycosides such gentamicin and kanamycin.

The evolutionary development of resistance to many drugs has been attributed to the possession of broad host range and extremely mobile genetic elements like conjugative plasmids and transposons (e.g. pAM{beta}1 or Tn916; Clewell et al., 1995). Since E. faecalis DPC5280 possesses the determinants for sex pheromones, aggregation substance and cytolysin, it is likely the strain possesses one or more sex pheromone plasmids. Since sex pheromone plasmids may also carry one or more antibiotic genes (Clewell, 1990; Wirth, 1994), the plasmid profile (Fig. 1b) was determined which revealed the presence of two plasmids (78 and 27 kb). The smaller plasmid was partially sequenced and contained a gene for kanamycin resistance (R. M. Hickey and others, unpublished) which was 100 % identical to that found on the E. faecalis plasmid pJH1 (Trieu-Cuot & Courvalin, 1983).

Conclusions
A raw milk isolate, E. faecalis DPC5280 was found to contain multiple genes associated with virulence, including cytolysin and gelatinase, and was resistant to antibiotics which inhibit protein synthesis. In addition, this strain produces an endopeptidase, enterolysin A encoded by entL, which is homologous to other cell wall lytic enzymes such as lysostaphin and zoocin A. However, unlike these enzymes enterolysin A has a broad spectrum of activity and can lyse a wide range of Gram-positive bacteria. It is tempting to suggest that production of this potent antimicrobial may contribute to the pathogenic potential of some enterococcal strains since it could allow them to better compete in complex microbial environments such as the human gastrointestinal tract.


   ACKNOWLEDGEMENTS
 
R. H. is in receipt of a Teagasc Walsh Fellowship. This work was funded by the Irish Government under the National Development Plan 2000-2006.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 15 August 2002; revised 11 November 2002; accepted 28 November 2002.