The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases

Abstract

An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-,I-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing I-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the I-lactamase II of Bacillus cereus and the CfiA I-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-,I-lactamases.In recent classification schemes for ,B-lactamases, a distinct grouping has been reserved for those enzymes requiring a metal cofactor for activity (metallo-1-lactamases) (1, 4, 5). The prototype for this group of enzymes is the 1-lactamase II of Bacillus cereus, which has long been studied as a model of metallo-13-lactamases and for which molecular sequence data, along with enzymologic and crystallographic data, are available (3,12,17,27). In the molecular classification of 1-lactamases, a class B was devised for this enzyme (1).Interest in this group of enzymes has grown recently since an increasing number of metallo-1-lactamases have been described in several gram-negative species which share the important characteristic of being able to hydrolyze carbapenem compounds (2,7,9,22,25,26,31). Carbapenems are new P-lactam antibiotics of great therapeutic potential, since they are not hydrolyzed by most bacterial 3-lactamases and show a broad spectrum of activity.Information currently available on carbapenem-hydrolyzing (CH) metallo-1-lactamases is limited, in most cases, to a few biochemical features and suggests the existence of different molecular species within this group of enzymes (5). Molecular sequence data are presently available only for the CfiA P-lactamase of Bacteroides fragilis TAL2480 (28) and for the ,B-lactamase II of B. cereus 569/H (12) and 5/B/6 (17). A strong sequence similarity exists between these two enzymes (28), suggesting that the CfiA enzyme also belongs to molecular class B...

Distribution and phenotypic and genotypic detection of a metallo-β-lactamase, CphA, among bacteraemic Aeromonas isolates

“…Determination of in vitro susceptibility to carbapenems by either the disc diffusion or dilution techniques among carbapenemase-producing Aeromonas isolates has often failed to yield a carbapenem-resistant phenotype (Massidda et al, 1991;Rossolini et al, 1996). Likewise, our cphA + aeromonads rarely exhibited the imipenem-non-susceptible phenotype by the disc diffusion, Etest or agar dilution techniques with the recommended inoculum.…”

“…X57102), i.e. ANY-SSD/F (F: 59-GCTTAGAGCTCCTAAGGAGCAAGATGAAAGGTTGG-39; R: 59-GCATAGGTACCTTATGACTGGGGTGCGGCCTTG-39) (Massidda et al, 1991;Talavera et al, 2006), with similar conditions for the rpoB gene amplification except for the annealing temperature, which was changed to 53 uC. The procedures for colony hybridization followed the manufacturer's instructions (DIG DNA Labelling kit; Roche), in which the hybridization probes were designed from the amplified DNA products of isolates of A. aquariorum A2-5, A. veronii A2-7 and A. hydrophila A2-11, around 720 bp.…”

“…The most commonly found MBL in Aeromonas species is CphA, which was first named in 1991 (Massidda et al, 1991). In addition to CphA, other MBLs have been identified in aeromonads, including ImiS (Walsh et al, 1998), IMP-19 (Neuwirth et al, 2007) and VIM (Libisch et al, 2008).…”

“…In addition to CphA, other MBLs have been identified in aeromonads, including ImiS (Walsh et al, 1998), IMP-19 (Neuwirth et al, 2007) and VIM (Libisch et al, 2008). CphA, a metalloenzyme, is inhibited by metal chelators, such as EDTA (Massidda et al, 1991;Segatore et al, 1993). Compared with other class B enzymes, CphA MBL has a very specific substrate profile, being active on penem and carbapenem only, and not on penicillin and cephalosporins (Segatore et al, 1993).…”

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

“…For Bacillus cereus metallo-␤-lactamase (BcII), 1 one metal-binding site contains three His (H-site); the other one contains 1 Asp, 1 Cys, and 1 His as the metal ligating residues (DCH site) as derived from x-ray crystallography (1). For CphA, 1 His from the H-site (His-116) is supposed to be replaced by an Asn (2). Various thiol-carboxylate compounds were identified as potent inhibitors (3).…”