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

摘要

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d-alanyl-d-alanine of lipid II with d-alanyl-d-lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide , which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward during vegetative growth was 4 μg/ml, but this increased to ca. 20 μg/ml during production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for production contains a gene, vanY (ORF7), potentially encoding an enzyme capable of removing the terminal d-Ala residue of pentapeptide peptidoglycan precursors. Analysis of UDP-linked peptidoglycan precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala and only traces of the pentapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala-d-Ala. This suggested a novel mechanism of glycopeptide resistance in Nonomuraea sp. ATCC 39727 that was based on the d,d-carboxypeptidase activity of vanY. Consistent with this, a vanY-null mutant of Nonomuraea sp. ATCC 39727 demonstrated a reduced level of glycopeptide resistance, without affecting productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae, resulted in higher levels of glycopeptide resistance.