Surface Protein EF3314 Contributes to Virulence Properties of Enterococcus faecalis

Abstract

Purpose

Identification of putative new virulence factors as additional targets for therapeutic approaches alternative to antibiotic treatment of multi-resistant enterococcal infections.

Methods

The EF3314 gene, coding for a putative surface-exposed antigen, was identified by the analysis of the Enterococcus faecalis V583 genome for LPXTG-motif cell wall anchor surface protein genes. A non-polar EF3314 gene deletion mutant in the E. faecalis 12030 human clinical isolate was obtained. The wild type and the isogenic mutant strain were investigated for biofilm formation, adherence to Hela cells, survival in human macrophages and a Caenorhabditis elegans infection model. The aminoterminal portion of the EF3314 protein was overexpressed in E. coli to obtain mouse polyclonal antibodies for use in Western blotting and immunolocalization experiments.

Results

The EF3314 gene has an unusually high GC content (46.88% vs. an average of 37.5% in the E. faecalis chromosome) and encodes a protein of 1744 amino acids that presents a series of 14 imperfect repeats of 90 amino acids covering almost the entire length of the protein. Its global organization is similar to the alpha-like protein family of group B streptococci, enterococcal surface protein Esp and bio film associated protein Bap from S. aureus. The EF3314 gene was always present and specific for E. faecalis strains of human, food and animal origin. Differences in size depended on variable numbers of repeats in the repetitive region.

Conclusions

EF3314 is a newly described, surface exposed protein that contributes to the virulence properties of E. faecalis.

Keywords

E. faecalis Surface protein Adhesion Biofilm C. elegans infection model

Get full access to this article

View all access options for this article.

References

Murray

B.E.

, Weinstock

G.M.

. Enterococci: new aspects of an old organism Proc Assoc Am Physicians 1999; 111: 328–34.

Koch

, Hufnagel

, Huebner

. Treatment and prevention of enterococcal infections - alternative and experimental approaches. Expert Opin Biol Ther 2004; 7: 439–44.

Ruiz-Garbajosa

, del Campo

, Coque

T.M.

, Asensio

, Bonten

, Willems

, Baquero

, Canton

. Longer intestinal persistence of Enterococcus faecalis compared to Enterococcus faecium clones in intensive-care-unit patients. J Clin Microbiol 2009; 47: 345–51.

Baldassarri

, Creti

, Montanaro

, Orefici

, Arciola

. Pathogenesis of implant infections by enterococci Int J Artif Organs 2005; 28: 1101–9.

Shepard

B.D.

, Gilmore

M.S.

. Antibiotic-resistant enterococci: the mechanisms and dynamics of drug introduction and resistance. Microbes Infect 2002; 4: 215–24.

Werner

, Coque

T.M.

, Hammerum

A.M.

. Emergence and spread of vancomycin resistance among enterococci in Europe. Euro Surveill 2008; 13: 19046.

Arias

C.A.

, Murray

B.E.

. Emergence and management of drug-resistant enterococcal infections. Expert Rev Anti Infect Ther 2008; 6: 637–55.

Baldassarri

, Creti

, Recchia

. Virulence factors in enterococcal infections. Int J Artif Organs, 2006; 29: 402–6.

Baldassarri

, Bertuccini

, Creti

. Glycosaminoglycans mediate invasion and survival of Enterococcus faecalis into macrophages J Infect Dis 2005; 191: 1253–62.

10.

Park

S.Y.

, Shin

Y.P.

, Kim

C.H.

. Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b. J Immunol 2008; 181: 6328–36.

11.

Cox

C.R.

, Coburn

P.S.

, Gilmore

M.S.

. Enterococcal cytolysin: a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells. Curr Protein Pept Scei 2005; 6: 77–84.

12.

Creti

, Imperi

, Bertuccini

. Survey for virulence determinants among Enterococcus faecalis isolated from different sources J Med Microbiol 2004; 53: 13–20.

13.

Shankar

, Baghdayan

A.S.

, Gilmore

M.S.

. Modulation of virulence within pathogenicity island in vancomycin-resistant Enterococcus faecalis. Nature 2002; 417: 746–50.

14.

McBride

S.M.

, Coburn

P.S.

, Baghdayan

A.S.

. Genetic variation and evolution of the pathogenicity island of Enterococcus faecalis. J Bacteriol 2009; 191: 3392–402.

15.

Gilmore

M.S.

, Ferretti

J.J.

. The thin line between gut commensal and pathogen. Science 2003; 299: 1999–2002.

16.

Creti

, Koch

, Fabretti

, Baldassarri

, Huebner

. Enterococcal colonization of the gastro-intestinal tract: role of biofilm and environmental oligosaccharides. BMC Microbiol 2006; 6: 60–8.

17.

Fabretti

, Huebner

. Implant infections due to enterococci: role of capsular polysaccharides and biofilm. Int J Artif Organs 2005; 28: 1079–90.

18.

Theilacker

, Sanchez-Carballo

, Toma

. Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis. Mol Microbiol 2009; 71: 1055–69.

19.

Tendolkar

P.M.

, Baghdayan

A.S.

, Shankar

. Putative surface proteins encoded within a novel transferable locus confer a high-biofilm phenotype to Enterococcus faecalis. J Bacteriol 2006; 188: 2063–72.

20.

Di Rosa

, Creti

, Venditti

. Relationship between biofilm formation the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium. FEMS Microbiol Lett 2006; 256: 145–50.

21.

Arciola

C.R.

, Baldassarri

, Campoccia

. Strong biofilm production, antibiotic multi-resistance and high gelE expression in epidemic clones of Enterococcus faecalis from orthopaedic implant infections. Biomaterials 2008; 29: 580–6.

22.

Ballering

K.S.

, Kristich

C.J.

, Grindle

S.M.

, Oromendia

, Beattie

D.T.

, Dunny

G.M.

. Functional genomics of Enterococcus faecalis: multiple novel genetic determinants for biofilm formation in the core genome. J Bacteriol 2009; 191: 2806–14.

23.

Fiedler

, Wirth

. Transformation of Enterococcus faecalis and Enterococcus faecium by electroporation. In: Dunny

G.M.

, Geary

P.P.

, McKay

L.L.

(eds). Genetics and molecular biology of Streptococci lactococci and enterococci. Washington DC: American Society for Microbiology; 1991. p 301.

24.

Ausubel

F.M.

, Brent

, Kingstone

R.E.

. (eds). Current protocols in Molecular Biology. New York, NY: John Wiley & Sons, Inc.; 1998.

25.

Qin

, Teng

, Xu

, Singh

K.V.

, Weinstock

G.M.

, Murray

B.E.

. Targeted mutagenesis of enterococcal genes. Methods Cell Sci 1998; 20: 21–33.

26.

Baldassarri

, Cecchini

, Bertuccini

. Enterococcus spp produces slime and survives in rat peritoneal macrophages Med Microbiol Immunol (Berl) 2001; 190: 113–20.

27.

Hufnagel

, Koch

, Creti

, Baldassarri

, Huebner

. A putative sugar-binding transcriptional regulator in a novel gene locus in Enterococcus faecalis contributes to production of biofilm and prolonged bacteremia in mice. J Infect Dis 2004; 189: 420–30.

28.

Deighton

, Borland

. Regulation of slime production in Staphylococcus epidermidis by iron limitation. Infect Immun 1993; 61: 4473–9.

29.

Scorneaux

, Ouadrhiri

, Anzalone

, Tulkens

P.M.

. Effect of recombinant human gamma interferon on intracellular activities of antibiotics against Listeria monocytogenes in the human macrophage cell line THP-1. Antimicrob Agents Chemother 1996; 40: 1225–30.

30.

Garsin

D.A.

, Sifri

C.D.

, Mylonakis

. A simple model host for identifying Gram-positive virulence factors Proc Natl Acad Sci USA 2001; 98: 10892–7.

31.

Maadani

, Fox

K.A.

, Mylonakis

, Garsin

D.A.

. Enterococcus faecalis mutations affecting virulence in the Caenorhabtidis elegans model host. Infect Immun 2007; 75: 2634–7.

32.

Lachenauer

C.S.

, Creti

, Michel

J.L.

, Madoff

L.C.

. Mosaicism in the alpha-like protein genes of group B streptococci. Proc Natl Acad Sci USA 2000; 97: 9630–5.

33.

Creti

, Baldassarri

, Montanaro

, Arciola

C.R.

. The Alpha-like surface proteins: an example of an expanding family of adhesins. Int J Artif Organs 2008; 31: 834–40.

34.

Jackson

D.W.

, Simecka

J.W.

, Romeo

. Catabolite repression of Escherichia coli biofilm formation. J Bacteriol 2002; 184: 3406–10.

35.

Sifri

C.D.

, Mylonakis

, Singh

K.V.

. Virulence effect of Enterococcus faecalis protease genes and the quorum-sensing locus fsr in Caenorhabditis elegans and mice. Infect Immun 2002; 70: 5647–50.

36.

Paulsen

I.T.

, Banerjei

, Myers

G.S.

. Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 2003; 299: 2071–4.

37.

McBride

S.M.

, Fischetti

V.A.

, LeBlanc

D.J.

, Moellering

R.C.

Jr. , Gilmore

M.S.

. Genetic diversity among Enterococcus faecalis. Plos One 2007; 2: e582.

38.

Faguy

D.M.

. The controlled chaos of shifty pathogens. Curr Biol 2000; 10: R498–501.

39.

Creti

, Ceccarelli

, Bocchetta

. Evolution of translational elongation factor (EF) sequences: reliability of global phylogenies inferred from EF-1 alpha(Tu) and EF-2(G) proteins Proc Natl Acad Sci U S A 1994; 91: 3255–9.

40.

Wegmann

, O'Connell-Motherway

, Zomer

. Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363. J Bacteriol 2007; 189: 3256–70.

41.

Toledo-Arana

, Valle

, Solano

. The enterococcal surface protein Esp is involved in Enterococcus faecalis biofilm formation. Appl Environ Microb 2001; 67: 4538–45.

42.

Cucarella

, Solano

, Valle

, Amorena

, Lasa

, Penades

J.R.

. Bap a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol 2001; 183: 2888–96.

43.

, Kasper

D.L.

, Ausubel

F.M.

, Rosner

, Michel

J.L.

. Inactivation of the alpha C protein antigen gene bca by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus. Proc Natl Acad Sci USA 1997; 94: 13251–6.

44.

Baron

M.J.

, Filman

D.J.

, Prophete

G.A.

, Hogle

J.M.

, Madoff

L.C.

. Identification of a glycosaminoglycan binding region of the alpha C protein that mediates entry of group B Streptococci into host cells. J Biol Chem 2007; 282: 10526–36.

45.

Madoff

L.C.

, Michel

J.L.

, Gong

E.W.

, Kling

D.E.

, Kasper

D.L.

. Group B Streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein. Proc Natl Acad Sci USA 1996; 93: 4131–6.

46.

Mohamed

J.A.

, Huang

D.B.

. Biofilm formation by enterococci. J Med Microbiol 2007; 56: 1581–8.

47.

Deighton

, Borland

. Regulation of slime production in Staphylococcus epidermidis by iron limitation. Infect Immun 1993; 61: 4473–9.

48.

Anderson

J.M.

, Marchant

R.E.

. Biomaterials: factors favoring colonization and infection. In: Waldvogel

F.A.

, Bisno

A.L.

(eds.) Infections associated with indwelling medical devices, 3rd ed. Washington, DC: American Society for Microbiology; 2000. p 89–109.