A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium.
Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.
Depardieu
Florence
F
Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France.
Courvalin
Patrice
P
Msadek
Tarek
T
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004151:Dipeptides / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D016984:Enterococcus faecium / Q000187:drug effects / Q000502:physiology*; D006020:Glycopeptides / Q000494:pharmacology; D000071677:Histidine Kinase; D008025:Ligases / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009876:Operon; D010447:Peptide Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D017334:Teicoplanin / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014640:Vancomycin / Q000494:pharmacology
D001426:Bacterial Proteins; D004151:Dipeptides; D006020:Glycopeptides; D011994:Recombinant Proteins; D014157:Transcription Factors; C073902:VanR protein, bacteria; C073901:VanS protein, Enterococcus; C002956:alanylalanine; D017334:Teicoplanin; D014640:Vancomycin; D011494:Protein Kinases; D000071677:Histidine Kinase; D010744:Phosphoric Monoester Hydrolases; D010447:Peptide Hydrolases; D008025:Ligases