Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum

Abstract

The biochemical basis for the acquired or natural resistance of various gram-positive organisms to glycopeptides was studied by high-pressure liquid chromatographic analysis of their peptidoglycan UDPMurNAc-peptide precursors. In all cases, resistance was correlated with partial or complete replacement of the C-terminal D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide by a new precursor with a modified C terminus. Nuclear magnetic resonance analysis by sequential assignment showed that the new precursor encountered in Enterococcus faecium D366, a strain belonging to the VANB class, which expresses low-level resistance to vancomycin, was UDP-MurNAc-L-Ala--y-D-Glu-L-Lys-D-Ala-D-lactate, identical to that previously found in the VANA class, which expresses high-level resistance to vancomycin. High-pressure liquid chromatographic analyses, composition determinations, and digestion by R39 D,D-carboxypeptidase demonstrated the exclusive presence of the new precursor in LactobaciUlus casei and Pediococcus pentosaceus, which are naturally highly resistant to glycopeptides. The low-level natural resistance of Enterococcus gaUlinarum to vancomycin was found to be associated with the synthesis of a new precursor identified as a UDP-MurNAcpentapeptide containing a C-terminal D-serine. The distinction between low and high levels of resistance to glycopeptides appeared also to depend on the presence or absence of a substantial residual pool of a D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide.Glycopeptides are very important antibiotics widely used against gram-positive organisms in the clinical setting. Their mechanism of action is based essentially on their capacity to bind specifically to the D-alanyl-D-alanine C terminus of the disaccharide pentapeptide (24), which is the structural monomer unit of cell wall peptidoglycan (27). The formation of such a complex inhibits peptidoglycan synthesis by interfering with the transglycosylation and transpeptidation reactions leading to nascent peptidoglycan (27). It was previously assumed that except under very abnormal growth conditions (15), the Dalanyl-D-alanine terminal dipeptide was a feature present in the precursors of the peptidoglycans of all bacteria (28). Recently, clinical isolates of enterococci which have acquired resistance to glycopeptides (19,29,34) have been separated into two major classes, VANA and VANB. In the VANA class, the genes responsible for the resistance are plasmid borne and lead to inducible cross-resistance between vancomycin and teicoplanin (5, 29). In the VANB class, the resistance genes are chromosome borne and associated with inducible resistance to vancomycin only (34). There is a third class, designated VANC, which corresponds to natural resistance to vancomycin and to which Enterococcus gallinarum belongs (30,33 N-acetyldepsipeptide (10). Taking into account all of these results, we were prompted to investigate (i) whether the biochemical basis of the glycopeptide resistance in the VANB class resembles that of VANA and (ii) whether the presence o...

D-Alanyl-D-Lactate and D-Alanyl-D-Alanine Synthesis by D-Alanyl-D-Alanine Ligase from Vancomycin-resistant Leuconostoc mesenteroides EFFECTS OF A PHENYLALANINE 261 TO TYROSINE MUTATION

“…This enzyme has the activity shown in Fig. 4B (20,21). These activities pinpoint this chromosomal D-Ala-D-Ala ligase as a key molecular determinant in the antibiotic resistance phenotype.…”

“…In these cases, PG intermediates terminating in DAla-D-Lac were also detected (20,21), suggesting a common evolutionary mechanism and a possible origin for at least the VanH and -A genes. Polymerase chain reaction (PCR) analysis has been used to identify and sequence fragments of the ddl genes in such organisms (22) Cloning, Subcloning, and Site-directed Mutagenesis-Cloning was carried out by amplification of a DNA fragment by using two rounds of PCR of genomic DNA purified from L. mesenteroides ATCC 8293.…”

“…A yield of 30 mg of enzyme from ϳ5 g (wet weight) of E. coli was obtained. (20,21), it may be that LmDdl2 functions in depsipeptide ligase mode in vivo.…”

“…Activity g/ml) (20,21) was subcloned into plasmid pET22b behind the T7 promoter as noted under "Experimental Procedures," expressed in E. coli BL21(DE3), and purified as summarized in Fig. 2.…”

Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583

“…Strains of E. fuecium and E. faecalis which display variable levels of inducible resistance to vancomycin (MIC from 4 to 1,000 &ml) and retain susceptibility to teicoplanin (MIC < 2 @ml) are of the VanB type [14,18] [8,9]. Analysis of the peptidoglycan composition of VanB and VanC type strains allowed identification of D-Lac and D-serine, respectively, as C-terminal residues of peptidoglycan precursors [4,9,17]. These observations imply that VanB and VanC are also o-Ala:D-Ala ligases of altered substrate specificity.…”