Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis - PubMed (original) (raw)

Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis

Fang Teng et al. Infect Immun. 2009 Sep.

Abstract

We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

OG1RF and its derivatives. (A) Illustration of the epa gene cluster. In white are the genes belonging to the epa locus. (B) Disruptions in orfde2, epaA, epaB, epaE, epaM, and epaN (using pTEX4577) are denoted by an inverted triangle, while disruption of OG1RF_163 was made using Tn_917_. The name of the strain is in boldface, and the disrupted gene is indicated in parentheses. The arrows above the genes represent the transcripts determined by qRT-PCR. The asterisks indicate the locations of the qRT-PCR primers for each gene. (C) Growth curve of OG1RF and its derivatives in TSBG (biofilm medium). (D) Growth curve of OG1RF and its derivatives in BHIS. These experiments (C and D) were performed at least twice, and results from one representative experiment are shown.

FIG. 2.

FIG. 2.

Morphology of OG1RF and the epaB mutant cells by phase-contrast microscopy (A) and thin-section electron microcopy (B). Disruptions are denoted by an inverted triangle.

FIG. 3.

FIG. 3.

Polysaccharides of OG1RF and its derivatives. (A and B) Stains-All-stained polysaccharide extracts in 0.8% agarose gel; (C) Western blots with polysaccharide extracts and patient serum. Lane 1, OG1RF; lane 2, TX10113 (the orfde2 mutant); lane 3, TX10114 (the epaA mutant); lane 4, TX5179 (the epaB mutant); lane 5, TX5180 (the epaE mutant); lane 6, TX5391 (the epaM mutant); lane 7, TX5436 (the epaN mutant); lane 8, TX5179.1 (the _epaB_-complemented strain).

FIG. 4.

FIG. 4.

Biofilm formation by OG1RF and its derivatives. (A) TX10113, TX10114, TX5179, TX5180, TX5391, TX5436, and TX5179.1 are the orfde2, epaA, epaB, epaE, epaM, and epaN mutants and the _epaB_-complemented strain, respectively. (B) DAGF29 is the OG1RF_0163 transposon insertion mutant (9).

FIG. 5.

FIG. 5.

Phage sensitivity of OG1RF and its derivatives. NPV-1 phage was used to infect TX10113, TX10114, TX5179, TX5180, TX5391, TX5436, and TX5179.1, which are the orfde2, epaA, epaB, epaE, epaM, and epaN mutants and the _epaB_-complemented strain, respectively. DAGF29 is the OG1RF_0163 transposon insertion mutant (9). PFU are shown.

FIG. 6.

FIG. 6.

Electron microscopy of OG1RF and the epaB mutant infected by NPV-1 phage. (A) Cell morphology in the presence of phage; (B) attachment of phage (full head) to bacteria; (C) attachment of phage (empty head) to bacteria. In panel D, OG1RF cells are filled with phage particles and show early lysis, whereas no phage was seen inside TX5179. The arrows point to the phage.

FIG. 7.

FIG. 7.

Survival curves of OG1RF and its derivatives in the mouse peritonitis model. Six mice per group were used, and P = 0.0051 for OG1RF versus TX5391 (the epaM mutant) or TX5436 (the epaN mutant). The inocula for OG1RF, TX5391, and TX5436 were 3.4 × 108, 3.5 × 108, and 3.4 × 108 CFU, respectively. Disruptions are denoted by an inverted triangle.

References

    1. AlonsoDeVelasco, E., A. F. Verheul, J. Verhoef, and H. Snippe. 1995. Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines. Microbiol. Rev. 59591-603. -PMC -PubMed
    1. Arduino, R. C., K. Jacques-Palaz, B. E. Murray, and R. M. Rakita. 1994. Resistance of Enterococcus faecium to neutrophil-mediated phagocytosis. Infect. Immun. 625587-5594. -PMC -PubMed
    1. Bamford, D. H., and L. Mindich. 1980. Electron microscopy of cells infected with nonsense mutants of bacteriophage phi 6. Virology 107222-228. -PubMed
    1. Bourgogne, A., D. A. Garsin, X. Qin, K. V. Singh, J. Sillanpaa, S. Yerrapragada, Y. Ding, S. Dugan-Rocha, C. Buhay, H. Shen, G. Chen, G. Williams, D. Muzny, A. Maadani, K. A. Fox, J. Gioia, L. Chen, Y. Shang, C. A. Arias, S. R. Nallapareddy, M. Zhao, V. P. Prakash, S. Chowdhury, H. Jiang, R. A. Gibbs, B. E. Murray, S. K. Highlander, and G. M. Weinstock. 2008. Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF. Genome Biol. 9R110. -PMC -PubMed
    1. Bourgogne, A., S. G. Hilsenbeck, G. M. Dunny, and B. E. Murray. 2006. Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: the Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease. J. Bacteriol. 1882875-2884. -PMC -PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources