Evidence that the Enterococcal Polysaccharide Antigen Gene ( epa ) Cluster Is Widespread in Enterococcus faecalis and Influences Resistance to Phagocytic Killing of E. faecalis

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Abstract
In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis ( epa ) from Enterococcus faecalis strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in Escherichia coli . Disruption of two open reading frames in the epa gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with E. faecalis endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF ( P ≤ 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF ( P ≤ 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the epa gene cluster of OG1RF (which is also present in the sequenced E. faecalis strain V583) was detected in 12 of 12 other clonally distinct E. faecalis strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an E. faecalis endocarditis patient serum, and sera from four other patients with E. faecalis endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the epa gene cluster is widespread among E. faecalis and confers some protection against human host defenses.