Capsular polysaccharide production in Enterococcus faecalis and contribution of CpsF to capsule serospecificity

Abstract

Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.

FIG. 1.

Dot blot analysis of the four putative serotypes of E. faecalis. Serotypes A and B (top) hybridize only to cpsA, cpsB, and the control gene hcp, which sits outside of the capsule locus. The serotype C strains (middle) hybridize to all the genes in the cps locus (cpsC to cpsK), as well as the cpsA, cpsB, and hcp genes. Serotype D strains (bottom) hybridize to all genes of the cps locus except cpsF.

FIG. 2.

(a) Strategy for the construction of plasmid pLT06, used in this study for construction of isogenic, in-frame deletion mutants of E. faecalis. See Materials and Methods for details. The erm marker from pCJK47 was replaced with the cat marker from pGB354, resulting in pKS05. oriT from pKS05 was replaced with an enterococcal origin of replication and the temperature-sensitive repA gene, resulting in pLT06. pLT06 was subsequently used to engineer all of the isogenic, in-frame deletion mutants used in this study. (b) Diagram of the generation of the in-frame, isogenic cpsF mutation using pLT08. Integration through homologous recombination of pLT08 into the E. faecalis genome took place at the nonpermissive temperature of 42°C. Strains harboring the integrated plasmid were serially passaged at the permissive temperature of 30°C in the absence of the selecting antibiotic Cm. Serial passaging induced the second site homologous recombination event and the excision of the plasmid. Bacteria were plated on medium containing ρ-chlorophenylalanine and X-Gal to screen for isolates that lost the plasmid. White colonies were screened by PCR for the deletion event, and isolated DNA was sequenced to confirm that an in-frame deletion had occurred.

FIG. 3.

Acrylamide gel stained with Stains-All, showing the presence/absence of capsule production in serotype A to D strains. The high-molecular-weight bands correspond to capsular polysaccharide as described previously (10). The serotype A strain 12030 (B) and the serotype B strain OG1RF (C) do not produce the capsule band. The serotype C strains V583 and FA2-2 (D and E) and the serotype D strains T-5 and T-18 (F and G) produce the high-molecular-weight capsule band.

FIG. 4.

CPS ELISA using MT2 antibodies to detect serotype C capsule. Serotype C strains V583 and FA2-2 show reactivity with the MT2 antibody. The cpsF deletion mutant LT01 is not detected by the antibody, but complementation of LT01 (FA2-2 ΔcpsF) with pLT10 (LT03) restores reactivity to the antibody. The serotype D strains T-5 and T-18 are not detected by the serotype C antibody. However, LT09 (T-5 plus pLT10) and LT10 (T-18 plus pLT10) are seroconverted to serotype C strains when complemented with cpsF. The serotype B strain OG1RF is not detected by the MT-2 antibody before or after (LT11) complementation with pLT10, indicating that serotype conversion cannot occur in a strain that does not produce capsular polysaccharide. O.D. 490, optical density at 490 nm.

FIG. 5.

Polyacrylamide gel stained with Stainz-all, showing the high-molecular-weight capsule bands of capsule mutants and complemented mutants. Lanes: A, V583; B, LT06 (V583 ΔcpsC); C, LT08 (V583 ΔcpsC plus pLT10); D, LT15 (V583 ΔcpsD); E, LT25 (V583 ΔcpsD plus pLT25); F, LT17 (V583 ΔcpsE); G, LT27 (V583 ΔcpsE plus pLT32); H, LT02 (V583 ΔcpsF); I, LT04 (V583 ΔcpsF plus pLT10); J, LT19 (V583 ΔcpsG); K, LT29 (V583 ΔcpsG plus pLT33); L, LT21 (V583 ΔcpsH); M, LT31 (V583 ΔcpsH plus pLT14); N, LT23 (V583 ΔcpsI); O, LT33 (V583 ΔcpsI plus pLT35). Only the genes cpsF and cpsH are not essential for capsule production. Deletion of the genes cpsC, cpsD, cpsE, cpsG, and cpsI completely abrogates capsule production. This observation supports the evidence that serotypes A and B do not produce capsule based on the absence of essential genes for capsule production in these strains. Complementation of these deletions restores capsule production.