Resistance to Glycopeptide Antibiotics in the Teicoplanin Producer Is Mediated by van Gene Homologue Expression Directing the Synthesis of a Modified Cell Wall Peptidoglycan

Abstract

Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal D-alanyl-D-alanine of the nascent peptidoglycan with D-alanyl-D-lactate or D-alanyl-D-serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 g/ml and a MIC of vancomycin of 90 g/ml during vegetative growth. The vanH, vanA, and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in D-alanyl-D-lactate. No trace of precursors ending in D-alanyl-D-alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 g/ml), the MIC of teicoplanin remained in the range of 25 to 35 g/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 g/ml.Glycopeptide antibiotics are produced by actinomycetes and inhibit the synthesis of bacterial cell wall by blocking peptidoglycan assembly. They bind to the D-alanyl-D-alanine (DAla-D-Ala) C terminus of the nascent peptidoglycan and prevent it from being utilized in the following cross-linking reactions catalyzed by transglycosylases and transpeptidases (18,38,44). The structurally related glycopeptides vancomycin and teicoplanin have been used in clinical settings since 1958 and 1988, respectively. These drugs are still extensively used against multiresistant enterococci and methicillin-resistant staphylococci. Concern about vancomycin-resistant enterococci has been increasing during the last decade, and highly vancomycin resistant Staphylococcus aureus isolates have recently appeared in clinical specimens (10, 11).Two resistance phenotypes, VanA and VanB, have been extensively studied in enterococci and are considered of main importance, since the genes responsible for resistance (van genes) are inducible and transferable and confer high-level resistance to vancomycin (at concentrations up to 1,000 g/ml or more) (42). The vanA gene cluster also confers high-level resistance to teicoplanin (4,9,15,16) (16,45,51). The genes vanH, vanA/vanB, and vanX are organized in a cluster and are oft...

Novel Mechanism of Glycopeptide Resistance in the A40926 Producer Nonomuraea sp. ATCC 39727

“…Glycopeptide production was estimated by HPLC performed on a 5-m-particle-size Ultrasphere ODS (Beckman) column Peptidoglycan precursor extraction and analysis. Extraction of UDP-linked peptidoglycan precursors was performed as already described (4,25). In brief, mycelium was harvested by centrifugation, suspended in distilled water at a final concentration of 0.1 g of fresh weight per ml of water, and boiled for 20 min.…”

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes

“…We reasoned that the differential response of actinomycetes of the Streptomycetaceae, Streptosporangiaceae and Micromonosporaceae to the treatments of either the digestion or the regeneration of their cell walls might be explained by investigating their cell wall structure. In a previous study, 24 we reported in A. teichomyceticus the presence of cell wall precursors with structure UDP-N-glycolylmuramyl-Gly-D-Glu-mDap-D-Ala-D-Lac and UDP-N-glycolylmuramylGly-D-Glu-mDap-D-Ala-D-Lac hydroxylated on the meso-Diaminopimelic acid. Previous findings also indicated the coexistence of these two peptidoglycan precursors in other Actinoplanes species and in Micromomospora species.…”

“…ATCC 39727 and Streptomyces coelicolor A3(2) by a method previously described. 24 In brief, mycelium, grown in vegetative medium to the exponential phase, was incubated for 1 h with ramoplanin (approximately eightfold the ramoplanin MIC value for each strain) to block cell wall final assembly and thus to amplify the intracellular pool of uridinediphospho-linked precursors. 24 Then, cells were harvested, Media were prepared in de-ionized water and pH adjusted to 7.2 before sterilization.…”

“…The samples were analyzed by reversed-phase HPLC and ESI-MS on an LCQ-Deca spectrometer equipped with an ion trap analyzer (Thermo Finnigan, San Josè, CA, USA) as described in Beltrametti. 24 …”

“…These basic observations strengthened the hypothesis that the difference observed in protoplast formation among our strains could be attributed to differences in the cell wall structure and precursors. To further get an insight into the possible structural relationship among strains displaying similar behavior in protoplast formation, we took advantage of an LC-MS method previously used in our laboratories 24 for the analysis of cell wall cytoplasmatic precursors. Among the strains reported in this study, only Nonomuraea sp.…”