Analysis of a Gene Cluster ofEnterococcus faecalisInvolved in Polysaccharide Biosynthesis

Abstract

Previously, we described a gene cluster of Enterococcus faecalis OG1RF that produced an antigenic polysaccharide when cloned in Escherichia coli. The polysaccharide antigen was not detectable in E. faecalis strains, however. Here, we show by reverse transcriptase-PCR that the 16 genes in this region are transcribed in OG1RF. Gene disruption of orfde4, encoding a putative glycosyl transferase, and orfde6, a putative dTDPrhamnose biosynthesis gene, generated two OG1RF mutants. The mutants showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. In addition, two mucoid E. faecalis isolates from patients with chronic urinary tract infections were found to produce the polysaccharide antigen.Polysaccharides (PS) of bacterial pathogens play important roles during infection. The capsule of Streptococcus pneumoniae is considered its most important virulence factor, since it enables the organism to persist in the host by conferring resistance to phagocytosis (6,8). Capsular PS from Staphylococcus aureus types 5 and 8 were shown to induce cytokine release from human epithelial and endothelial cell lines (22). The serotype f PS (a rhamnose-glucose polymer) of Streptococcus mutans was found to stimulate the release of tumor necrosis factor ␣ (23). Furthermore, being the dominant immunogens on the bacterial surface, some PS (e.g., the O antigens of gram-negative bacteria and capsules of streptococci and staphylococci) have been used as the basis for both vaccine development and serological typing of clinical isolates.Enterococci are a leading cause of nosocomial infections and account for 5 to 15% of infective endocarditis in the United States (14), with most clinical isolates being Enterococcus faecalis. The development of multiple antibiotic resistance in enterococci in recent decades has posed a serious threat to effective therapy and raises awareness of the need for a better understanding of the pathogenicity of enterococci. Members of our group previously reported that a cloned gene cluster (epa, encoding proteins involved in the biosynthesis of an enterococcal PS antigen) of E. faecalis OG1RF produced a PS in Escherichia coli (27). The PS reacted with sera from four patients with enterococcal endocarditis. Sequence analysis showed that epa genes were similar to those involved in the biosynthesis and export of PS, including genes for rhamnose biosynthesis, glycosyl transferases, and ATP-binding cassette transporters. Insertion mutations in three of the genes, orfde4, orfde5, and orfde8, abolished immunoreactivity of the E. coli clone (27). However, we were not able to detect the PS antigen in several E. faecalis strains. Early studies of E. faecalis showed that some cell wall PS were antigenic and might be used for serological typing (1,9,19,21). Neither the chemical compositions nor the genetic basis of these PS have been elucidated. In addition, Bottone et al. (2) recently described the first isolation of mucoid E. faecalis strains from patients with chronic urinary tract infections.The biosynt...

Translocation of Enterococcus faecalis Strains across a Monolayer of Polarized Human Enterocyte-Like T84 Cells

“…While we have not characterized Epa biochemically, Hancock and Gilmore were able to detect three different cell wall carbohydrates in E. faecalis: a type-specific capsular polysaccharide, a rhamnopolysaccharide (possibly Epa), and a polymer likely representing an integral cell wall teichoic acid (7). Their model, in agreement with our earlier observations (27), suggests that the Epa polysaccharide is not on the cell surface, at least in vitro; however, Epa may be important for the overall integrity of membrane and/or cell wall structures, or conceivably, it could be exposed on the surface in vivo. Our present results showed that there was no detectable translocation of the epa mutant TX5179 across the T84 monolayers and that the translocation efficiency of the epa mutant TX5180 was lower (about 10-fold) than that of the wild-type strain OG1RF, indicating that some epa genes, especially orfde4, are important for successful translocation.…”

“…To further confirm the importance of epa genes for translocation, the epa mutant with a disruption in orfde4, TX5179, was complemented with extrachromosomal orfde4 and its downstream gene orfde5 (both genes are likely to be in the same operon [27]). The polysaccharide content of the complemented strain was compared with that of the mutant (TX5179) and wild-type OG1RF, and the polysaccharide smear missing in the mutant TX5179 appeared in the complemented strain, which showed a polysaccharide pattern similar to that of the wild-type strain OG1RF; the pAT18 shuttle vector did not affect the polysaccharide component (data not shown).…”

“…1B) and that is important for virulence in mice and for resistance to phagocytosis and/or killing by polymorphonuclear leukocytes (7,20,(25)(26)(27). The epa gene cluster has been shown to be widespread among E. faecalis strains (20), and Epa is thought to be a species-specific polysaccharide (7).…”

“…Translocation experiments were performed by methods described previously, with slight modifications (8,11,15). Briefly, 10 (27). orfde4 is disrupted in TX5179, and orfde6 is disrupted in TX5180.…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…In gram-positive bacteria, genes highly similar to those involved in the biogenesis of capsular polysaccharide and LPS are found (267,534). In B. subtilis, limited expression of the tagGH genes leads to anomalous morphology and a decrease in cell wall galactosamine and phosphate, two components of teichoic acids.…”