Peter E. Reynolds

Glycopeptide resistance mediated by enterococcal transposon Tn1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine

Cloning and nucleotide sequencing indicated that transposon Tn 1546 from Enterococcus faecium BM4147 encodes a 23365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2‐2 derivative producing VanH, VanA and VanX accumulated mainly UDP‐MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys‐D‐Ala‐D‐Lac (pentadepsipeptide) and small amounts of UDP‐MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys‐D‐Ala‐D‐Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D‐dipeptidase that hydrolysed D‐Ala‐D‐Ala. Pentadepsipeptide, pentapeptide and D‐Ala‐D‐Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D‐dipeptidase that hydrolyses D‐Ala‐D‐Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D‐Ala‐D‐Ala‐containing peptidoglycan precursors at the cell surface.

QUANTITATIVE ANALYSIS OF THE METABOLISM OF SOLUBLE CYTOPLASMIC PEPTIDOGLYCAN PRECURSORS OF GLYCOPEPTIDE-RESISTANT ENTEROCOCCI

Transposon Tn1546 from Enterococcus faecium BM4147 mediates high‐level resistance to the glycopeptide antibiotics vancomycin and teicoplanin. Tn1546 encodes a dehydrogenase (VanH) and a ligase (VanA) that synthesize D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac), a DD‐dipeptidase (VanX) that hydrolyses D‐Ala‐D‐Ala and a two‐component regulatory system (VanR–VanS) that controls transcription of the vanHAX operon. Strains of Enterococcus faecalis harbouring various copy numbers of the vanRSHAX cluster were tested to determine if there was a correlation between the levels of resistance to glycopeptides, the levels of expression of the corresponding resistance genes and the relative proportions of the different cytoplasmic peptidoglycan precursors. Increased transcription of the vanHAX operon was associated with increased incorporation of D‐Ala‐D‐Lac into peptidoglycan precursors to the detriment of D‐Ala‐D‐Ala, and with a gradual increase in the vancomycin‐resistance levels. More complete elimination of D‐Ala‐D‐Ala‐containing precursors was required for teicoplanin resistance. The VanY and VanZ proteins also encoded by Tn1546 were not effectors of the regulation of the vanHAX operon but contributed to vancomycin and teicoplanin resistance, respectively. Differences at the regulatory level accounted for phenotypic diversity in acquired glycopeptide resistance by production of D‐lac‐ending precursors.

Penicillin-binding proteins of β-lactam-resistant strains of Staphylococcus aureus: Effect of growth conditions

Methidllin‐resistant clinical isolates of Staphylococcus aureus are intrinsically resistant to β‐lactam antibiotics in that the resistance mechanism is unrelated to the possession of β‐lactamases. We have demonstrated that a new, high‐molecular‐mass penicillin‐binding protein (PBP) is present in these strains with a low affinity for β‐lactams and that its amount is regulated by the growth conditions. The new PBP from all strains that have been examined has an identical mobility on SDS gel electrophoresis and is the only PBP still present in an uncomplexed state with β‐lactams (and therefore the only functional PBP) when these strains are grown in media containing concentrations of β‐lactam antibiotics sufficient to kill sensitive strains.

The vanZ gene of Tn1546 from enterococcus faecium BM4147 confers resistance to teicoplanin

A five-gene cluster from Tn1546 confers resistance to the glycopeptide antibiotics vancomycin (Vm) and teicoplanin (Te) by synthesis of pentadepsipeptide peptidoglycan precursors terminating in D-lactate, which replaces D-alanine in the same position of precursors utilized by susceptible enterococci. Cloning and nucleotide sequencing indicated that Tn1546 contains an additional gene, designated vanZ, which confers low-level Te resistance, in the absence of the genes required for pentadepsipeptide synthesis. Analysis of cytoplasmic peptidoglycan precursors, accumulated in the presence of ramoplanin, showed that VanZ-mediated Te resistance does not involve incorporation of a substituent of D-alanine into the precursors.

Mechanisms of glycopeptide resistance in enterococci

Inducible resistance to high levels of glycopeptide antibiotics in clinical isolates of enterococci is mediated by Tn1546 or related transposons. Tn1546 encodes the VanH dehydrogenase which reduces pyruvate to D-lactate (D-Lac) and the VanA ligase which catalyses synthesis of the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). The depsipeptide replaces the dipeptide D-Ala-D-Ala leading to production of peptidoglycan precursors which bind glycopeptides with reduced affinity. In addition, Tn1546 encodes the VanX dipeptidase and the VanY D,D-carboxypeptidase that hydrolyse the dipeptide D-Ala-D-Ala and the C-terminal D-Ala residue of the cytoplasmic precursor UDP-MurNAC-L-Ala-gamma-D- Glu-L-Lys-D-Ala-D-Ala, respectively. These two proteins act in series to eliminate D-Ala-D-Ala-containing precursors. VanX is required for resistance whereas VanY only slightly increases the level of resistance mediated by VanH, VanA and VanX.

Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors.

The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.

The vanG glycopeptide resistance operon from Enterococcus faecalis revisited.

Acquired VanG‐type resistance to vancomycin (MIC = 16 µg ml−1) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d‐alanine‐d‐serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3′ end encoded VanG, a d‐Ala:d‐Ser ligase, VanXYG, a putative bifunctional d,d‐peptidase and VanTG, a serine racemase: VanG and VanTG were implicated in the synthesis of d‐Ala:d‐Ser as in VanC‐ and VanE‐type strains. Upstream from the structural genes for these proteins were vanWG with unknown function and vanYG containing a frameshift mutation which resulted in premature termination of the encoded protein and accounted for the lack of UDP‐MurNAc‐tetrapeptide in the cytoplasm. Without the frameshift mutation, VanYG had homology with Zn2+ dependent d,d‐carboxypeptidases. The 5′ end of the gene cluster contained three genes vanUG, vanRG and vanSG encoding a putative regulatory system, which were co‐transcribed constitutively from the PYG promoter, whereas transcription of vanYG,WG,G,XYG,TG was inducible and initiated from the PYG promoter. Transfer of VanG‐type glycopeptide resistance to E. faecalis JH2‐2 was associated with the movement, from chromosome to chromosome, of genetic elements of c. 240 kb carrying also ermB‐encoded erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donor and transconjugants revealed the same 4 bp direct repeats and 22 bp imperfect inverted repeats that delineated the large element.

Requirement of the VanY and VanX D,D-peptidases for glycopeptide resistance in enterococci

Transposon Tn1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D‐peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N‐terminal membrane anchor of the protein. The enzyme was a Zn2+‐dependent D,D‐carboxypeptidase that cleaved the C‐terminal residue of peptidoglycan precursors ending in R‐D‐Ala‐D‐Ala or R‐D‐Ala‐D‐Lac but not the dipeptide D‐Ala‐D‐Ala. The specificity constants kcat/Km were 17‐ to 67‐fold higher for substrates ending in the R‐D‐Ala‐D‐Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP‐MurNAc‐tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high‐level glycopeptide resistance in a medium supplemented with D‐Ala. The enzyme could not replace the VanX D,D‐dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D‐Ala:D‐Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D‐carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D‐Ala‐containing medium. Thus, VanX and VanY had non‐overlapping functions involving the hydrolysis of D‐Ala‐D‐Ala and the removal of D‐Ala from membrane‐bound lipid intermediates respectively.

Sequence of the vanB and ddl genes encoding d-alanine:d-lactate and d-alanine:d-alanine ligases in vancomycin-resistant Enterococcus faecalis V583

A pair of degenerate oligodeoxyribonucleotides was used to amplify, by the polymerase chain reaction (PCR), DNA fragments internal to genes encoding D-Ala:D-Ala ligase-related proteins of vancomycin-resistant (VmR) Enterococcus faecalis V583. Cloning and nucleotide sequencing of the PCR products indicated that fragments of two genes, designated vanB and ddl, were co-amplified. The vanB gene was previously shown to be present in Enterococcus strains expressing VanB-type VmR [Quintiliani Jr. et al., J. Infect. Dis. 8 (1993) 943-950]. The ddl gene was detected by Southern hybridization in all VmR and VmS strains of En. faecalis, but not in representatives of 17 other species of Enterococcus. The vanB and ddl genes were cloned in bacteriophage lambda and sequenced. There was extensive similarity (76% amino-acid identity) between the product of vanB and the VmR protein, VanA. The product of ddl, the D-Ala:D-Ala ligases, DdlA and DdlB, of Escherichia coli and the resistance proteins, VanA and VanB, were more distantly related (32-40% aa identity). After induction of VmR, En. faecalis V583 synthesized the cell wall precursor, UDP-N-acetylmuramyl-tetrapeptide-D-lactate, indicating that the mechanism of glycopeptide resistance in strains with the VanA and VanB phenotype is similar.

Vancomycin Resistance in Enterococci Due to Synthesis of Precursors Terminating in d-Alanyl-d-Serine

the intrinsically glycopeptide-resistant Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens (VanC type) and is present in the VanE and VanG types in which E. faecalis has acquired the genes encoding vancomycin resistance. Some of the enzymes implicated in this second resistance mechanism are different from those involved in high-level resistance, indicating a second route by which resistance has evolved. The basis of resistance results from the sixfold-lower affinity of vancomycin for acyl-D-Ala-D-Ser than for acyl-D-Ala-D-Ala (13) because of the increased bulk of the hydroxymethyl group of serine relative to the methyl group of alanine.