Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid - PubMed (original) (raw)

Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid

Christian Theilacker et al. Infect Immun. 2006 Oct.

Abstract

A teichoic acid (TA)-like polysaccharide in Enterococcus faecalis has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from E. faecalis strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with d-Ala and kojibiose. We also detected a novel substituent at position C-2, [D-Ala-->6]-alpha-D-Glcp-(1-->2-[D-Ala-->6]-alpha-D-Glcp-1-->). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the E. faecalis strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to E. faecalis 12030 are directed predominantly to nonalanylated epitopes on the LTA molecule.

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Figures

FIG. 1.

1H NMR spectra of lipoteichoic acids isolated from Enterococcus faecalis strain 12030. (a) BuOH-LTA; (b) BuOH-LTA after incubation at pH 8.5 for 4 h. Spectra were recorded at 27°C in 2H2O relative to an internal standard of acetone (δH 2.225; δC 31.45). The letters refer to the glycerol residue and the glucose residues in kojibiose (Fig. 4; Table 1), and the numerals refer to the protons in the respective residues. The protons belonging to the glucose residues substituted by alanine are in lowercase.

FIG. 2.

Sections of the TOCSY spectra of lipoteichoic acids isolated from Enterococcus faecalis strain 12030. (a) BuOH-LTA; (b) BuOH-LTA after incubation at pH 8.5 for 4 h. Spectra were recorded at 27°C in 2H2O relative to an internal standard of acetone (δH 2.225; δC 31.45). The letters refer to the glycerol residue and the glucose residues in kojibiose (Fig. 4; Table 1), and the numerals refer to the protons in the respective residues. The protons belonging to the glucose residues substituted by alanine at position C-6 are in lowercase.

FIG. 3.

Section of the HMQC-TOCSY spectrum of LTA isolated from E. faecalis strain 12030. Spectra were recorded at 27°C in 2H2O relative to an internal standard of acetone (δH 2.225; δC 31.45). The letters refer to the glycerol residues and the glucose residues in kojibiose (Fig. 4), and the numerals refer to the protons and 13C atoms in the respective residues. The rectangles show different types of 1,3-poly(glycerol phosphate) residues due to various substitutions at position C-2.

FIG. 4.

Chemical structures of different repeating units of lipoteichoic acids isolated from Enterococcus faecalis strain 12030. (a) 1,3-Poly (glycerol phosphate); (b) 1,3-poly(glycerol phosphate) substituted at position C-2 of glycerol residues by

-Ala; (c) 1,3-poly(glycerol phosphate) substituted at position C-2 of glycerol residues by kojibiose; (d) 1,3-poly(glycerol phosphate) substituted at the position C-2 by [

-Ala→6]-α-

-Glc_p_-(1→2)-[

-Ala→6]-α-

-Glc_p_-(1→.

FIG. 5.

Binding of rabbit antiserum raised against antibiotic-killed whole bacterial cells (A) and of antiserum raised against enzyme-TA (B) against various coating antigens. Antigens used to coat ELISA plates are indicated. Data points represent averages of two determinations.

FIG. 6.

Inhibition ELISA of rabbit antiserum raised against antibiotic-killed whole bacterial cells (A) and of antiserum raised against enzyme-TA (B). The solid-phase antigen is the native BuOH-LTA, and the liquid phase antigen or inhibitor is as indicated. Data points represent averages of two determinations.

FIG. 7.

Inhibition of opsonophagocytic killing of rabbit antiserum raised against antibiotic-killed whole bacterial cells (A) or enzyme-TA (B). Both antisera were used at a 1:200 dilution. Inhibitors used are indicated. Points represent averages of four values and error bars the standard errors of the means. *, P < 0.01 for enzyme-TA versus BuOH-LTA; °, P < 0.05 for enzyme-TA versus BuOH-LTA, §, P < 0.01 for enzyme-TA versus BuOH-LTA with no alanine; +, P < 0.05 for enzyme-TA versus BuOH-LTA with no alanine.

FIG. 8.

Inhibition of opsonophagocytic killing of E. faecalis strain 12030 after absorption of the indicated antiserum with either the homologous wild-type strain or an isogenic Δ_dltA_ mutant lacking alanine incorporation into the LTA. Bars represent the means of four determinations and error bars the standard errors of the means.

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Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid - PubMed (original) (raw)