ATP hydrolysis and pristinamycin IIA inhibition of the Staphylococcus aureus Vga(A), a dual ABC protein involved in streptogramin A resistance.
In Gram-positive bacteria, a large subfamily of dual ATP-binding cassette proteins confers acquired or intrinsic resistance to macrolide, lincosamide, and streptogramin antibiotics by a far from well understood mechanism. Here, we report the first biochemical characterization of one such protein, Vga(A), which is involved in streptogramin A (SgA) resistance among staphylococci. Vga(A) is composed of two nucleotide-binding domains (NBDs), separated by a charged linker, with a C-terminal extension and without identified transmembrane domains. Highly purified Vga(A) displays a strong ATPase activity (K(m) = 78 mum, V(m) = 6.8 min(-1)) that was hardly inhibited by orthovanadate. Using mutants of the conserved catalytic glutamate residues, the two NBDs of Vga(A) were shown to contribute unequally to the total ATPase activity, the mutation at NBD2 being more detrimental than the other. ATPase activity of both catalytic sites was essential for Vga(A) biological function because each single Glu mutant was unable to confer SgA resistance in the staphylococcal host. Of great interest, Vga(A) ATPase was specifically inhibited in a non-competitive manner by the SgA substrate, pristinamycin IIA (PIIA). A deletion of the last 18 amino acids of Vga(A) slightly affected the ATPase activity without modifying the PIIA inhibition values. In contrast, this deletion reduced 4-fold the levels of SgA resistance. Altogether, our results suggest a role for the C terminus in regulation of the SgA antibiotic resistance mechanism conferred by Vga(A) and demonstrate that this dual ATP-binding cassette protein interacts directly and specifically with PIIA, its cognate substrate.
Institut de Chimie des Substances Naturelles, CNRS UPR 2301, avenue de la Terrasse, Gif-sur-Yvette 91190 and the. Electronic address: Eric.Jacquet@icsn.cnrs-gif.fr.
Eric
E
Jacquet
Institut de Chimie des Substances Naturelles, CNRS UPR 2301, avenue de la Terrasse, Gif-sur-Yvette 91190 and the.
Jean-Marie
JM
Girard
Institut de Chimie des Substances Naturelles, CNRS UPR 2301, avenue de la Terrasse, Gif-sur-Yvette 91190 and the.
Odile
O
Ramaen
Institut de Chimie des Substances Naturelles, CNRS UPR 2301, avenue de la Terrasse, Gif-sur-Yvette 91190 and the.
Olivier
O
Pamlard
Institut de Chimie des Substances Naturelles, CNRS UPR 2301, avenue de la Terrasse, Gif-sur-Yvette 91190 and the.
Hélène
H
Lévaique
Unité de Biochimie Structurale, Paris 75724 Cedex 15, France.
Jean-Michel
JM
Betton
Unité des Membranes Bactériennes, Institut Pasteur, CNRS URA 2172, 25 rue du Docteur Roux, Paris 75724 Cedex 15, France.
Elie
E
Dassa
Unité des Membranes Bactériennes, Institut Pasteur, CNRS URA 2172, 25 rue du Docteur Roux, Paris 75724 Cedex 15, France.
Olivier
O
Chesneau
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins / Q000737:chemistry*; D004352:Drug Resistance, Microbial; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism*; D025364:Streptogramin A / Q000737:chemistry* / Q000494:pharmacology*
D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C075964:VgA protein, Staphylococcus aureus; D000255:Adenosine Triphosphate; D025364:Streptogramin A; D000251:Adenosine Triphosphatases