Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides

Abstract

Enterococcus faecalis is among the predominant causes of nosocomial infections. Surface molecules like d-alanine lipoteichoic acid (LTA) perform several functions in gram-positive bacteria, such as maintenance of cationic homeostasis and modulation of autolytic activities. The aim of the present study was to evaluate the effect of d-alanine esters of teichoic acids on biofilm production and adhesion, autolysis, antimicrobial peptide sensitivity, and opsonic killing. A deletion mutant of the dltA gene was created in a clinical E. faecalis isolate. The absence of d-alanine in the LTA of the dltA deletion mutant was confirmed by nuclear magnetic resonance spectroscopy. The wild-type strain and the deletion mutant did not show any significant differences in growth curve, morphology, or autolysis. However, the mutant produced significantly less biofilm when grown in the presence of 1% glucose (51.1% compared to that of the wild type); adhesion to eukaryotic cells was diminished. The mutant absorbed 71.1% of the opsonic antibodies, while absorption with the wild type resulted in a 93.2% reduction in killing. Sensitivity to several cationic antimicrobial peptides (polymyxin B, colistin, and nisin) was considerably increased in the mutant strain, confirming similar results from other studies of gram-positive bacteria. Our data suggest that the absence of d-alanine in LTA plays a role in environmental interactions, probably by modulating the net negative charge of the bacterial cell surface, and therefore it may be involved in the pathogenesis of this organism.

FIG. 1.

Genetic organization of the dlt operon in E. faecalis.

FIG. 2.

¹H NMR spectra of LTA isolated from E. faecalis strains 12030 (a) and 12030ΔdltA (b). Spectra were recorded at 27°C in ²H₂O. Residue A, →2)-α-d-glucopyranose-(1→; residue B, α-d-glucopyranose-(1→; residue C, →1)-Gro-(3→ P→, with Ala at C-2 (the Arabic numerals refer to the protons in the respective residues). The shifts at δ 1.629 and 5.388 are missing in the 12030 ΔdltA mutant, indicating the absence of bound d-alanine. The shift at δ ∼1.9 represents a contamination from the chromatography buffer (NH₄OAc). (FA), fatty acid; HOD, deuterated water.

FIG. 3.

The wild type (WT) and the dltA mutant (Mut) strain were tested for biofilm production in media without (TSB) and with 1% glucose (TSB-G). The amount of biofilm is expressed as the biofilm index (9). Error bars represent standard errors of the means (n = 3).

FIG. 4.

Opsonic killing of the wild-type (WT) strain E. faecalis 12030 with serum raised against whole bacterial cells. The serum was absorbed (abs) with the homologous wild-type strain (black bar) or with the dltmutant (white bar). Error bars represent standard errors of the means.

FIG. 5.

E. faecalis 12030 (black bars) and the dlt mutant (white bars) were tested for their ability to adhere to and/or invade HEp-2 cells. While neither strain was able to survive within epithelial cells, there was a significant difference between the ability of the wild-type (WT) and that of the mutant to adhere to cells when the adherence step was prolonged from 1 to 2 h. Error bars represent standard errors of the means. ns, not significant.

FIG. 6.

Transmission electron microscopy of wild-type E. faecalis 12030 (a) and the dlt mutant (b).

FIG. 6.

Transmission electron microscopy of wild-type E. faecalis 12030 (a) and the dlt mutant (b).