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. 2010 Jun;54(6):2465-72.
doi: 10.1128/AAC.00106-10. Epub 2010 Mar 22.

Novel mechanism of glycopeptide resistance in the A40926 producer Nonomuraea sp. ATCC 39727

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Novel mechanism of glycopeptide resistance in the A40926 producer Nonomuraea sp. ATCC 39727

Giorgia Letizia Marcone et al. Antimicrob Agents Chemother. 2010 Jun.

Abstract

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 A40926, 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 A40926 during vegetative growth was 4 microg/ml, but this increased to ca. 20 microg/ml during A40926 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 A40926 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 A40926 productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae, resulted in higher levels of glycopeptide resistance.

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Figures

FIG. 1.
FIG. 1.
Population analysis profile of Nonomuraea sp. ATCC 39727. Colony forming units in the presence of A40926 (♦), teicoplanin (▪), and vancomycin (▴) are shown. Results are the average of three independent experiments in which the standard deviation (SD) was less than 5%.
FIG. 2.
FIG. 2.
A40926 resistance during fermentation of Nonomuraea sp. ATCC 39727. Symbols: ▪, dry weight; •, A40926 production; ○, MIC values. Results are the average of three independent experiments in which the SD was less than 5%.
FIG. 3.
FIG. 3.
Minimal bactericidal concentrations (MBCs) of A40926 (♦), teicoplanin (▪), and vancomycin (▴). The glycopeptides do not show bactericidal activity against Nonomuraea sp. ATCC 39727, in contrast to the nonglycopeptide control antibiotic kanamycin (•). Results are the average of three independent experiments in which the SD was less than 5%.
FIG. 4.
FIG. 4.
Population analysis profile of Nonomuraea sp. ATCC 39727 ΔvanY mutant. Colony-forming units in the presence of A40926 (♦), teicoplanin (▪), and vancomycin (▴) are shown. Results are the average of three independent experiments in which the SD was less than 5%.
FIG. 5.
FIG. 5.
Comparison of A40926 production in 2-liter-batch fermentations of Nonomuraea sp. ATCC 39727 (A and B) and the ΔvanY mutant (C and D). Panels A and C: time course of pH, ▴, solid line; pO2, ▴, dashed line; glucose, ▪, dotted line; growth curve measured as dry weight, •, dashed line. Panels B and D, production of A40926 measured by HPLC analysis expressed as μg/ml, filled bars.
FIG. 6.
FIG. 6.
Map of pIJ86ΩvanY plasmid.
FIG. 7.
FIG. 7.
Population analysis profile of Streptomyces venezuelae containing pIJ86 (light lines) or pIJ86ΩvanY (dark lines). Colony-forming units in the presence of A40926 (A), teicoplanin (B), and vancomycin (C) are shown. Results are the average of three independent experiments in which the SD was less than 5%.

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References

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