Probing the role of R2-loop deletions and substituions on the functional properties of the acquired AmpC-type β-lactamase CMY-2.

Background: CMY-2 is a plasmid-encoded AmpC-type β-lactamase (pAmpC) belonging to the CIT cluster which shows the broadest dissemination and represents an important mechanism of β-lactam resistance in Enterobacteriaceae. The crystal structure of CMY-10, a pAmpC belonging to the MOX cluster, revealed subtle differences in the R2-loop of the enzyme (due to a 3-AA deletion) that were associated with a higher hydrolytic activity towards imipenem. In this work, we wanted to probe the impact of modifications in the R2-loop of CMY-2 on the functional features of the enzyme. Methods: Wild-type cmy-2 gene was cloned in vector pLB-II and propagated in E. coli DH5α. Using mutagenic PCR, several CMY-2 laboratory variants were obtained in which (a) a 3-AA deletion was introduced in the R2-loop (CMY-2Δ319-321, CMY-2Δ320-322, etc.) or (b) the whole R2 loop of CMY-2 was substituted by that found in the structurally-equivalent position of CMY-10 (CMY-2loop10). The resulting recombinant vectors were transformed in E. coli. The β-lactam susceptibility of the recombinant E. coli strains carrying the cloned cmy-2 mutants were determined according to CLSI or using E-test. The hydrolytic activity of the various CMY-2 variants were investigated by standard kinetic measurements. Results: The various E. coli strains producing the CMY-2 laboratory variants were resistant to ampicillin and cephalothin, indicating that these modified proteins were functional. However, the 3-AA deletion in CMY-2 (such as in mutant CMY-2Δ319-321) apparently increased the susceptibility to FOX and CAZ, while imipenem MIC was unchanged. Similar results were observed with E. coli strains producing the CMY-2loop10 variant. Moreover, enzymatic assays did not show significant differences in imipenem hydrolysis between the wild-type and the CMY-2 variants. Conclusions: Several CMY-2 variants carrying deletions or substitutions in the R2-loop were obtained and characterized. Despite these variants were functional, they did not decrease imipenem susceptibility when produced in E. coli, indicating that other mutations might be required in CMY-2 to enhance its activity towards imipenem.