Aminoglycoside resistance resulting from tight drug binding to an altered aminoglycoside acetyltransferase

Abstract

The aacA29b gene, which confers an atypical aminoglycoside resistance pattern to Escherichia coli, was identified on a class 1 integron from a multidrug-resistant isolate of Pseudomonas aeruginosa. On the basis of amino acid sequence homology, it was proposed that the gene encoded a 6'-N-acetyltransferase. The resistance gene was cloned into the pET23a(+) vector, and overexpression conferred high-level resistance to the usual substrates of the aminoglycoside N-acetyltransferase AAC(6')-I, except netilmicin. The level of resistance conferred by aacA29b correlated perfectly with the level of expression of the gene. The corresponding C-terminal six-His-tagged AAC(6')-29b protein was purified and found to exist as a dimer in solution. With a spectrophotometric assay, an extremely feeble AAC activity was detected with acetyl coenzyme A (acetyl-CoA) as an acetyl donor. Fluorescence titrations of the protein with aminoglycosides demonstrated the very tight binding of tobramycin, dibekacin, kanamycin A, sisomicin (K(d), </=1 micro M) and a weaker affinity for amikacin (K(d), approximately 60 micro M). The binding of netilmicin and acetyl-CoA could not be detected by either fluorescence spectroscopy or isothermal titration calorimetry. The inability of AAC(6')-29b to efficiently bind acetyl-CoA is supported by an alignment analysis of its amino acid sequence compared with those of other AAC(6')-I family members. AAC(6')-29b lacks a number of residues involved in acetyl-CoA binding. These results lead to the conclusion that AAC(6')-29b is able to confer aminoglycoside resistance by sequestering the drug as a result of tight binding.

FIG. 1.

Sequence alignment of AAC(6′)-29b and some members of the major AAC(6′)-I subfamily. The regions predicted to interact with acetyl-CoA based on the structure of AAC(6′)-Iy in complex with CoA are indicated by asterisks. Amino acids cited in the text are in boldface.

FIG. 2.

Structures of aminoglycosides used in this study.

FIG. 3.

Titration of AAC(6′)-29b-His₆ with tobramycin. Curves from the bottom to the top are the fluorescence spectra of buffer baseline AAC(6′)-29b-His₆ in the presence of 0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 0.7, 3, 9, 40, or 300 μM tobramycin. (Inset) Representation of the plot used to determine the association constant.