Sylvie Dutka-Malen

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.

Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a d-alanine:d-alanine ligase-related protein necessary for vancomycin resistance

The amplification product obtained with DNA from vancomycin-resistant (VmR) Enterococcus gallinarum BM4174 and a pair of degenerate oligodeoxyribonucleotides that correspond to conserved amino acid (aa) motifs in Escherichia coli D-alanine (D-Ala):D-Ala ligases and in En. faecium VmR protein (VanA) was used as a probe to clone the vanC gene of that strain. The vanC product, with a calculated Mr of 37,504, exhibits 29 to 38% aa identity with VanA and E. coli ligases. Insertional inactivation of vanC led to Vm sensitivity of BM4174 suggesting that the gene may encode a D-Ala:D-Ala ligase of altered specificity.

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.

Evolution of structure and substrate specificity ind-alanine:d-Alanine ligases and related enzymes

Thed-alanine:d-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesized-alanyl-d-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesized-alanyl-d-lactate or Dalanyl-d-serine. The sequence of internal fragments of eight structurald-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (d-alanine:d-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and Dd1A and DdlB from enteric bacteria andHaemophilus influenzae constituted separate clusters.

Structural relationship between the vancomycin resistance protein VanH and 2-hydroxycarboxylic acid dehydrogenases

Sequencing upstream from the vanA gene of enterococcal plasmid pIP816 that confers Vm resistance revealed the presence of an ORF which could code for a protein of 322 aa designated VanH. Extensive aa similarity was detected between VanH and 2-hydroxycarboxylic acid dehydrogenase. We discuss possible roles for VanH in the synthesis of a novel type of peptidoglycan precursors with lower affinity for Vm.

Resistance to Quinupristin-Dalfopristin Due to Mutation of L22 Ribosomal Protein in Staphylococcus aureus

ABSTRACT The mechanism of resistance to the streptogramin antibiotics quinupristin and dalfopristin was studied in a Staphylococcus aureus clinical isolate selected under quinupristin-dalfopristin therapy, in four derivatives of S. aureus RN4220 selected in vitro, and in a mutant selected in a model of rabbit aortic endocarditis. For all strains the MICs of erythromycin, quinupristin, and quinupristin-dalfopristin were higher than those for the parental strains but the MICs of dalfopristin and lincomycin were similar. Portions of genes for domains II and V of 23S rRNA and the genes for ribosomal proteins L4 and L22 were amplified and sequenced. All mutants contained insertions or deletions in a protruding β hairpin that is part of the conserved C terminus of the L22 protein and that interacts with 23S rRNA. Susceptible S. aureus RN4220 was transformed with plasmid DNA encoding the L22 alteration, resulting in transformants that were erythromycin and quinupristin resistant. Synergistic ribosomal binding of streptogramins A and B, studied by analyzing the fluorescence kinetics of pristinamycin IA-ribosome complexes, was abolished in the mutant strain, providing an explanation for quinupristin-dalfopristin resistance.

Cloning and heterospecific expression of the resistance determinant vanA encoding high-level resistance to glycopeptides in Enterococcus faecium BM4147.

Fragments of plasmid pIP816, which confers high-level glycopeptide resistance in Enterococcus faecium BM4147, were cloned into a conjugative gram-negative-gram-positive shuttle vector. The resulting hybrids were transferred by conjugation from Escherichia coli to Enterococcus faecalis and Bacillus thuringiensis. A 4-kilobase EcoRI fragment from pIP816 was found to confer vancomycin resistance in these hosts but not in E. coli or Bacillus subtilis. Images