Alternative Pathway to a Glycopeptide-Resistant Cell Wall in the Balhimycin Producer Amycolatopsis balhimycina.
Balhimycin, a vancomycin-type glycopeptide, is a lipid II targeting antibiotic produced by Amycolatopsis balhimycina. A. balhimycina has developed a self-resistance mechanism based on the synergistic action of different enzymes resulting in modified peptidoglycan. The canonical resistance mechanism against glycopeptides is the synthesis of peptidoglycan precursors ending with acyl-d-alanyl-d-lactate (d-Ala-d-Lac) rather than acyl-d-alanyl-d-alanine (d-Ala-d-Ala). This reprogramming is the result of the enzymes VanH, VanA, and VanX. VanH and VanA are required to produce d-Ala-d-Lac; VanX cleaves cytosolic pools of d-Ala-d-Ala, thereby ensuring that peptidoglycan is enriched in d-Ala-d-Lac. In A. balhimycina, the ΔvanHAXAb mutant showed a reduced glycopeptide resistance in comparison to the wild type. Nevertheless, ΔvanHAXAb was paradoxically still able to produce d-Ala-d-Lac containing resistant cell wall precursors suggesting the presence of a novel alternative glycopeptide resistance mechanism. In silico analysis, inactivation studies, and biochemical assays led to the characterization of an enzyme, Ddl1Ab, as a paraloguous chromosomal d-Ala-d-Lac ligase able to complement the function of VanAAb in the ΔvanHAXAb mutant. Furthermore, A. balhimycina harbors a vanYAb gene encoding a d,d-carboxypeptidase. Transcriptional analysis revealed an upregulated expression of vanYAb in the ΔvanHAXAb mutant. VanYAb cleaves the endstanding d-Ala from the pentapeptide precursors, reducing the quantity of sensitive cell wall precursors in the absence of VanXAb. These findings represent an unprecedented coordinated layer of resistance mechanisms in a glycopeptide antibiotic producing bacterium.
Interfaculty Institute of Microbiology and Infection Medicine Tuebingen (IMIT), Microbiology/Biotechnology, University of Tuebingen , 72076 Tuebingen, Germany.
Hans-Joerg
HJ
Frasch
Michael G. Degroote Institute for Infectious Disease Research, Biochemistry and Biomedical Sciences, McMaster University , MDCL-2301, 1280 Main Street West, Hamilton, Ontario L8S4L8, Canada.
Lindsay
L
Kalan
Interfaculty Institute of Microbiology and Infection Medicine Tuebingen (IMIT), Microbiology/Biotechnology, University of Tuebingen , 72076 Tuebingen, Germany.
Regina
R
Kilian
Interfaculty Institute of Microbiology and Infection Medicine Tuebingen (IMIT), Microbiology/Biotechnology, University of Tuebingen , 72076 Tuebingen, Germany.
Tobias
T
Martin
Michael G. Degroote Institute for Infectious Disease Research, Biochemistry and Biomedical Sciences, McMaster University , MDCL-2301, 1280 Main Street West, Hamilton, Ontario L8S4L8, Canada.
Gerard D
GD
Wright
Interfaculty Institute of Microbiology and Infection Medicine Tuebingen (IMIT), Microbiology/Biotechnology, University of Tuebingen , 72076 Tuebingen, Germany.
Evi
E
Stegmann
cell wall analysis; d-Ala-d-Lac-ligase; glycopeptide producer; lipid II; vanHAX; vanY