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.

Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages

“…The n-butanol method and HIC on octyl-sepharose were used to extract and purify LTA. The butanol method can be used to extract structurally intact LTA and has been widely used to extract LTA from numerous types of Gram-positive bacteria, such as Staphylococcus aureus, Streptococcus pneumoniae, E. faecalis and Streptococcus gordonii (6,19,23,24). Furthermore, SDS-PAGE and Coomassie Blue G-250 staining were used to detect protein contamination.…”

“…Among them, LTA is the key virulence factor due to its major role in pathogenicity. It has been demonstrated that E. faecalis LTA can stimulate leukocytes to release certain mediators associated with inflammatory response (5) and plays a critical role in biofilm formation (6). E. faecalis LTA is also known to inhibit the repair mechanism of periapical bone by decreasing the proliferation of human osteoblast-like cells and inducing apoptosis (7).…”

“…Its basic structure is comprised of a glycolipid moiety and 1,3-polyglycerolphosphate substituted at position C-2 with D-alanine and kojibiose residues (8). The alanyl esters with D-configured glycerophosphate influence the antibiotic action, pathogenesis, adhesion, biofilm formation and virulence of Gram-positive bacteria (6,9). The dlt operon consists of 4 genes: dltA, dltB, dltC and dltD, which are responsible for D-alanine synthesis (10).…”

“…The dlt operon consists of 4 genes: dltA, dltB, dltC and dltD, which are responsible for D-alanine synthesis (10). The inactivation of the dlt genes resulted in the absence of D-alanyl residues of LTA and further decreased the virulence features of E. faecalis (6).…”

Regulation of d-alanylation of lipoteichoic acid in Streptococcus gordonii

“…The dltD gene encodes a membraneassociated protein, which facilitates the binding of Dcp and Dcl for ligation of Dcp with D-alanine. Disruption of any one of the dlt genes will eliminate D-alanylation of LTA.D-Alanylation confers a positive charge upon LTA and has been demonstrated to have a role in the regulation of autolytic activity (Fischer et al, 1981), the binding of Mg 2+ (Archibald et al, 1973), intragenic coaggregation (Clemans et al, 1999), biofilm formation (Fabretti et al, 2006;Gross et al, 2001), adherence to mammalian cells (Abachin et al, 2002;Weidenmaier et al, 2004) There is evidence that bacteria can modulate the level of D-alanyl esters on LTA. An increase in the level of D-alanyl ester on LTA has been reported under low external pH conditions (Ellwood & Tempest, 1972;MacArthur & Archibald, 1984), and a decrease in D-alanyl ester contents has been reported when metal cations, such as Mg 2+ , Ca 2+ , Na + and K + , are present in the culture medium (Fischer & Rösel, 1980;Heptinstall et al, 1970;Kiriukhin & Neuhaus, 2001;Koch et al, 1985; Koprivnjak et al, 2006;Reusch & Neuhaus, 1971).…”