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

Abstract

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 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.

Keyword(s): AU, arbitrary units
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.25949-0
2003-03-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/3/mic149655.html?itemId=/content/journal/micro/10.1099/mic.0.25949-0&mimeType=html&fmt=ahah

References

  1. Aguirre M., Collins M. D. 1993; Lactic acid bacteria and human clinical infection. J Appl Bacteriol 75:95–107
    [Google Scholar]
  2. Anderson D. G., McKay L. L. 1983; Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 46:549–552
    [Google Scholar]
  3. Baba T., Schneewind O. 1996; Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus . EMBO J 15:4789–4797
    [Google Scholar]
  4. Beresford T., Condon S. 1991; Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp. lactis . FEMS Microbiol Lett 62:319–323
    [Google Scholar]
  5. Chow J. W, Thal L. A, Perri M. B, Vazquez J. A, Donabedian S. M, Clewell D. B., Zervos M. J. 1993; Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocartitis. Antimicrob Agents Chemother 37:2474–2477
    [Google Scholar]
  6. Clewell D. B. 1990; Movable genetic elements and antibiotic resistance in enterococci. Eur J Clin Microbiol Infect Dis 9:90–102
    [Google Scholar]
  7. Clewell D. B. 1993; Bacterial sex pheromone-induced plasmid transfer. Cell 73:9–12
    [Google Scholar]
  8. Clewell D. B, Tomich P. K, Gawron-Burke M. C, Franke A. E, Yagi Y., An F. Y. 1982; Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J Bacteriol 152:1220–1230
    [Google Scholar]
  9. Clewell D. B, Flannagan S. E., Jaworski D. D. 1995; Unconstrained bacterial promiscuity: the Tn 916 -Tn 1545 family of conjugative transposons. Trends Microbiol 3:229–236
    [Google Scholar]
  10. Cooper V. J. C., Salmond G. P. C. 1993; Molecular analysis of the major cellulase (CelV) of Erwinia carotovora : evidence for an evolutionary ‘mix and match’ of enzyme domains. Mol Gen Genet 241:342–350
    [Google Scholar]
  11. Coque T. M, Patterson J. E, Steckelberg J. M., Murray B. E. 1995; Incidence of hemolysin, gelatinase, and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from the feces of hospitalized and community-based persons. J Infect Dis 171:1223–1229
    [Google Scholar]
  12. Eaton T. J., Gasson M. J. 2001; Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol 67:1628–1635
    [Google Scholar]
  13. Facklam R. R., Sahm D. F. 1995; Enterococcus . In Manual of Clinical Microbiology pp  308–314 Edited by Murray P. R., Baron E. J., Pfaller M. A., Tenover F. C., Yolken R. H. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  14. Garcia E, Garcia J. L, Garcia P, Arraras A, Sanchez-Puelles J. M., Lopez R. 1988; Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Proc Natl Acad Sci U S A 85:914–918
    [Google Scholar]
  15. Garcia P, Bascaran V, Rodriguez A., Suarez J. E. 1997; Isolation and characterization of promoters from the Lactobacillus casei temperate bacteriophage A2. Can J Microbiol 43:1063–1068
    [Google Scholar]
  16. Hayashida M, Watanabe K, Muramatsu T., Goto M. A. 1987; Further characterization of PL-1 phage-associated N -acetyl-muramidase of Lactobacillus casei . J Gen Microbiol 133:1343–1349
    [Google Scholar]
  17. Hodges T. L, Zighelboim-Daum S, Eliopoulos G. M, Wennersten C., Moellering R. C. Jr 1992; Antimicrobial susceptibility changes in Enterococcus faecalis following various penicillin exposure regimens. Antimicrob Agents Chemother 36:121–125
    [Google Scholar]
  18. Hoffman C. S., Winston F. 1987; A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli . Gene 57:267–272
    [Google Scholar]
  19. Ike Y, Craig R. A, White B. A, Yagi Y., Clewell D. B. 1983; Modification of Streptococcus faecalis sex pheromones after acquistion of plasmid DNA. Proc Natl Acad Sci U S A 92:12055–12059
    [Google Scholar]
  20. Ike Y, Hashimoto H., Clewell D. B. 1984; Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice. Infect Immun 45:528–530
    [Google Scholar]
  21. Jett B. D, Jensen H. G, Nordquist R. E., Gilmore M. S. 1992; Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis. Infect Immun 60:2445–2452
    [Google Scholar]
  22. Jett B. D, Huyke M. M., Gilmore M. S. 1994; Virulence of enterococci. Clin Microbiol Rev 7:462–478
    [Google Scholar]
  23. Jones R. N, Marshall S. A, Pfaller M. A, Wilke W. W, Hollis R. J, Erwin M. E, Edmond M. B., Wenzel R. P. 1997; Nosocomial enterococcal blood stream infections in the SCOPE program: antimicrobial resistance, species occurrence, molecular testing results, and laboratory testing accuracy. SCOPE Hospital Study Group. Diagn Microbiol Infect Dis 29:95–102
    [Google Scholar]
  24. Joyanes P, Pascual A, Martinez-Martinez L, Hevia A., Perea E. J. 2000; In vitro adherence of Enterococcus faecalis and Enterococcus faecium to urinary catheters. Eur J Clin Microbiol Infect Dis 19:124–127
    [Google Scholar]
  25. Kashige N, Nakashima Y, Miake F., Watanabe K. 2000; Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes. Arch Virol 145:1521–1534
    [Google Scholar]
  26. Kelly G, Prasannan S, Daniell S, Fleming K, Frankel G, Dougan G, Connerton L., Matthews S. 1999; Structure of the cell-adhesion fragment of intimin from enteropathogenic Escherichia coli . Nat Struct Biol 6:313–318
    [Google Scholar]
  27. Klein G, Pack A., Reuter G. 1998; Antibiotic resistance patterns of enterococci and occurrence of vancomycin-resistant enterococci in raw minced beef and pork in Germany. Appl Envir Microbiol 64:1825–1830
    [Google Scholar]
  28. Knudtson L. M., Hartman P. A. 1993; Antibiotic resistance among enterococcal isolates from environmental and clinical sources. J Food Prot 56:486–492
    [Google Scholar]
  29. Kuhnen E, Richter F, Richter K., Andries L. 1988; Establishment of a typing system for group D streptococci. Zentbl Bakteriol Hygiene A 267:322–330
    [Google Scholar]
  30. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  31. Leclerc D., Asselin A. 1989; Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can J Microbiol 35:749–753
    [Google Scholar]
  32. Lopez R, Garcia J. L, Garcia E, Ronda C., Garcia P. 1992; Structural analysis and biological significance of the cell wall lytic enzymes of Streptococcus pneumoniae and its bacteriophage. FEMS Microbiol Lett 79:439–447
    [Google Scholar]
  33. Low D. E, Willey B. M, Betschel S., Kreiswirth B. 1994; Enterococcus : pathogens of the 90s. Eur J Surg Suppl 573:19–24
    [Google Scholar]
  34. Lowe A. M, Lambert P. A., Smith A. W. 1995; Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci . Infect Immun 63:703–706
    [Google Scholar]
  35. Makinen P. L, Clewell D. B, An F., Makinen K. K. 1989; Purification and substrate specificity of a strongly hydrophobic extracellular metalloendopeptidase (‘gelatinase’) from Streptococcus faecalis (strain OG1-10. J Biol Chem 264:3325–3334
    [Google Scholar]
  36. Marino M, Braun L, Cossart P., Ghosh P. 1999; Structure of the lnlB leucine-rich repeats, a domain that triggers host cell invasion by the bacterial pathogen L. monocytogenes . Mol Cell 4:1063–1072
    [Google Scholar]
  37. McKay L. L., Baldwin K. A. 1984; Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage. Appl Envir Microbiol 47:68–74
    [Google Scholar]
  38. Moellering R. C. Jr 1992; Emergence of Enterococcus as a significant pathogen. Clin Infect Dis 14:1173–1176
    [Google Scholar]
  39. Murray B. E. 1990; The life and times of the Enterococcus . Clin Microbiol Rev 3:46–65
    [Google Scholar]
  40. Nilsen T. 1999 Novel enterococcal bacteriocins; optimization of production, purification, biochemical and genetic chacterization PhD thesis Agricultural University of Norway; Ås, Norway:
    [Google Scholar]
  41. Nolling J, Breton G, Omelchenko M. V. 16 other authors 2001; Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum . J Bacteriol 183:4823–4838
    [Google Scholar]
  42. Olmsted S. B, Dunny G. M, Erlandsen S. L., Wells C. L. 1994; A plasmid-encoded surface protein on Enterococcus faecalis augments its internalization by cultured intestinal epithelial cells. J Infect Dis 170:1549–1556
    [Google Scholar]
  43. Parente E., Hill C. 1992; Inhibition of Listeria in buffer, broth and milk by enterocin 1146, a bacteriocin produced by Enterococcus faecium . J Food Prot 55:503–508
    [Google Scholar]
  44. Piard J. C, Muriana P. M, Desmazeaud M. J., Klaenhammer T. R. 1992; Purification and partial characterisation of lacticin 481, a lanthionine-containing bacteriocin produced by Lactococcus lactis subsp. lactis CNRZ481. Appl Environ Microbiol 58:279–284
    [Google Scholar]
  45. Potvin C, Leclerc D, Tremblay G, Asselin A., Bellemare G. 1988; Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli . Mol Gen Genet 214:241–248
    [Google Scholar]
  46. Recsei P. A, Gruss A. D., Novick R. P. 1987; Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans . Proc Natl Acad Sci U S A 84:1127–1131
    [Google Scholar]
  47. Riley M. A. 1993; Molecular mechanisms of colicin evolution. Mol Biol Evol 10:1380–1395
    [Google Scholar]
  48. Ryan M. P, Rea M. C, Hill C., Ross R. P. 1996; An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Appl Environ Microbiol 62:612–619
    [Google Scholar]
  49. Schindler C. A., Schuhardt V. T. 1964; Lysostaphin: a new bacteriolytic agent for the Staphylococcus . Proc Natl Acad Sci U S A 51:414–421
    [Google Scholar]
  50. Schleifer K. H., Fischer U. 1982; Description of a new species of the genus Staphylococcus : Staphylococcus carnosus . Int J Syst Bacteriol 32:153–156
    [Google Scholar]
  51. Schleifer K. H., Kandler O. 1972; Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36:407–477
    [Google Scholar]
  52. Shankar V, Baghdayan A. S, Huycke M. M, Lindahl G., Gilmore M. S. 1999; Infection-derived Enterococcus faecalis strains are enriched in esp , a gene encoding a novel surface protein. Infect Immun 67:193–200
    [Google Scholar]
  53. Simjee S., Gill M. J. 1997; Gene transfer, gentamicin resistance and enterococci. J Hosp Infect 36:249–259
    [Google Scholar]
  54. Simmonds R. S, Simpson W. J., Tagg J. R. 1997; Cloning and sequence analysis of zooA , a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin. Gene 189:255–261
    [Google Scholar]
  55. Singh K. V, Coque T. M, Weinstock G. M., Murray B. E. 1998; In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification. FEMS Immunol Med Microbiol 21:323–331
    [Google Scholar]
  56. Su Y. A, Sulavik M. C, He P, Makinen K. K, Makinen P. L, Fiedler S, Wirth R., Clewell D. B. 1991; Nucleotide sequence of the gelatinase gene ( gelE ) from Enterococcus faecalis subsp. liquefaciens . Infect Immun 59:415–420
    [Google Scholar]
  57. Sugai M, Fujiwara T, Akiyama T, Ohara M, Komatsuzawa H, Inoue S., Suginaka H. 1997; Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. J Bacteriol 179:1193–1202
    [Google Scholar]
  58. Teuber M, Perreten V., Wirsching F. 1996; Antibiotikumresistente Bakterien: eine neue Dimension in der Lebensmittelmikrobiiologie. Lebensmittel-Technologie 29:182–199
    [Google Scholar]
  59. Trieu-Cuot P., Courvalin P. 1983; Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3′5′-aminoglycoside phosphotransferase type III. Gene 23:331–341
    [Google Scholar]
  60. Watanabe K, Hayashida M, Ishibashi K., Nakashima Y. 1984; An N -acetylmuramidase induced by PL-1 phage infection of Lactobacillus casei . J Gen Microbiol 130:275–277
    [Google Scholar]
  61. Wirth R. 1994; The sex pheromone system of Enterococcus faecalis . More than just a plasmid-collection mechanism?. Eur J Biochem 222:235–246
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.25949-0
Loading
/content/journal/micro/10.1099/mic.0.25949-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed