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Infection and Immunity, September 2009, p. 3759-3767, Vol. 77, No. 9
0019-9567/09/$08.00+0     doi:10.1128/IAI.00149-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Further Characterization of the epa Gene Cluster and Epa Polysaccharides of Enterococcus faecalis ^,

Fang Teng,^1,2, Kavindra V. Singh,^1,2 Agathe Bourgogne,^1,2 Jing Zeng,^1,2, and Barbara E. Murray^1,2,3*

Center for the Study of Emerging and Re-Emerging Pathogens, Division of Infectious Diseases,¹ Department of Internal Medicine,² Department of Microbiology and Molecular Genetics, The University of Texas Medical School, Houston, Texas³

Received 6 February 2009/ Returned for modification 10 March 2009/ Accepted 29 June 2009

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.

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* Corresponding author. Mailing address: Center for the Study of Emerging and Re-emerging Pathogens. University of Texas Medical School at Houston, 6431 Fannin, MSB 2.112, Houston, TX 77030. Phone: (713) 500-6745. Fax: (713) 500-5495. E-mail: bem.asst{at}uth.tmc.edu

Published ahead of print on 6 July 2009.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: A. Camilli

Present address: Centocor R&D, Inc., 145 King of Prussia Road, Radnor, PA 19087.

Present address: Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, No. 6, Tianshuiyuan Street, Chaoyang, Beijing 100026, China.

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Infection and Immunity, September 2009, p. 3759-3767, Vol. 77, No. 9
0019-9567/09/$08.00+0     doi:10.1128/IAI.00149-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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