Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop
Sandrine Dahyot, Hedi Mammeri
Sandrine Dahyot
Service de Bactériologie, Centre Hospitalo-Universitaire d'Amiens, Hôpital Nord, Faculté de Médecine d'Amiens, Université de Picardie Jules Verne, Amiens, France
Hedi Mammeri
The Citrobacter freundii isolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borne blaAmpC-CHA gene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded the Escherichia coli TOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resulting E. coli TOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase of C. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficient E. coli strain. Moreover, it exhibited increased catalytic efficiency (kcat/Km) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (kcat), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.
Many enterobacterial species, such as Citrobacter freundii, produce regulated Ambler class C β-lactamases (AmpCs) that confer resistance to narrow-spectrum cephalosporins, such as cephalothin and cefoxitin. Spontaneous mutations affecting the regulatory genes, most frequently the ampD gene, which codes for an amidase (16, 21), cause constitutive overproduction of the enzyme and resistance to extended-spectrum cephalosporins (ESCs), such as cefotaxime and ceftazidime, and aztreonam. Zwitterionic cephalosporins, such as cefepime, and carbapenems, such as imipenem and doripenem, which penetrate very efficiently through the outer membrane of the Enterobacteriaceae and are usually poor substrates for AmpC β-lactamases, remain active in vitro against organisms producing high levels of cephalosporinases (13).
Since the 1980s, plasmid-mediated AmpC β-lactamases, such as the CMY-2 enzyme, which derives from the chromosome-borne AmpC β-lactamase of C. freundii, have emerged among enterobacterial clinical isolates and have spread worldwide (4, 38). They are often expressed in large amounts (41, 38), thus conferring a phenotype of resistance similar to those displayed by chromosomal AmpC-overproducing strains.
During the last decade of the 20th century, the production of cephalosporinases with broadened substrate activity was reported in several enterobacterial species (32), such as Enterobacter cloacae (3, 33, 44), Enterobacter aerogenes (2), Serratia marcescens (14, 28, 40), Escherichia coli (10, 12, 23, 26, 29, 30), and C. freundii (1), and in Acinetobacter baumannii and Pseudomonas aeruginosa (42, 43). Those extended-spectrum AmpC (ESAC) β-lactamases differed from wild-type cephalosporinases by amino acid insertions, deletions, or substitutions in the Ω loop, which constitutes one boundary of the R1 binding site, or in the R2 loop, the H-9 and the H-11 helices, which surround the R2 binding site (9, 18, 20, 32, 39).
Structural modifications in the Ω loop are responsible for increased rates of hydrolysis of ESCs (increased kcat values), whereas structural alterations in the R2 binding site account for enhanced affinity to ESCs and carbapenems (lower Km values) (27, 32). All ESAC β-lactamases confer high level of resistance to ceftazidime and significantly reduce susceptibility to cefotaxime, ceftriaxone, and cefepime (32). The weak carbapenemase activity exhibited by several ESAC enzymes increased the MICs of ertapenem and imipenem in combination with outer membrane impermeability (12, 27). Moreover, the tripeptide deletion in the R2 binding site of the AmpCD variant increased the affinity (lower Ki value) for clavulanate and tazobactam (10).
We describe in this report a novel ESAC β-lactamase that had a structural alteration located in the vicinity of the Y-X-N motif. To the best our knowledge, this was the first time that a structural modification responsible for hydrolysis spectrum extension occurred outside the Ω loop, the R2 loop, or the H-9 or H-11 helix. Site-directed mutagenesis, enzyme purification, and biochemical determination were performed to characterize the effects induced by the R148H substitution.
Bacterial strains and plasmids.The clinical isolate C. freundii CHA, which was identified using the API 20E system (bioMérieux, Marcy l'Etoile, France), was recovered from a 44-year-old man admitted at the Teaching Hospital of Amiens (Amiens, France) and diagnosed with an intragastric migration of an adjustable gastric band system (Lap-Band) inserted 3 years ago. The patient underwent a laparotomy immediately to remove the Lap-Band. On the fourth postoperative day, the patient developed peritonitis resulting from a surgical wound infection. A specimen of peritoneal fluid was sent for microbiologic testing, and a probabilistic antibiotic treatment with piperacillin-tazobactam and gentamicin was started. An AmpC-overexpressing E. cloacae isolate, which was resistant to ticarcillin, ceftazidime, and cefotaxime and presented reduced susceptibility to piperacillin combined with tazobactam, was recovered together with a wild-type C. freundii isolate. The antibiotic treatment was modified once the microbiological documentation became available, and cefepime was substituted for the previous antibiotics for 10 days. On the 4th postoperative day, novel peritonitis was diagnosed and a subtotal enterectomy was performed. The C. freundii clinical isolate CHA was recovered from the peritoneal discharge. It was resistant or intermediate to all β-lactams except carbapenems and was susceptible to aminoglycosides, cotrimoxazole, and fluoroquinolones. This isolate was selected for further study on the basis of its unusual phenotype of resistance consistent with ESAC production. The infection was treated successfully by imipenem for 10 days in combination with amikacin within the first 3 days.
The wild-type E. coli TOP10 strain (Invitrogen, Cergy Pontoise, France) and the porin-deficient E. coli HB4 strain (27) were used as recipients in transformation experiments. The lack of membrane permeability of the E. coli HB4 strain was previously shown to magnify the weak β-lactamase activity of Ambler class C enzymes against poor substrates, such as carbapenems (25, 27).
The pCMY-2 recombinant plasmid, which was used for the site-directed mutagenesis experiment, was extracted from the E. coli HB4(pCMY-2) recombinant clone, which was previously described (25).
Antimicrobial agents and MIC determination.The antibiotic agents and their sources have been described elsewhere (5). MICs were determined by an agar dilution technique on Mueller-Hinton agar (Sanofi-Diagnostics Pasteur, Paris, France) or cloxacillin (250 mg/liter)-containing Mueller-Hinton agar plates with an inoculum of 104 CFU per spot and were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (6).
Cloning experiments and sequence analysis.The coding region of the chromosome-borne ampC gene of C. freundii was amplified using the primers CMY-2-ATG (5′-ATGATGAAAAAATCGTTATGC-3′) and CMY-2-TAA (5′-TTATTGCAGCTTTTCAAGAATGC-3′) and subsequently sequenced on both strands as previously described (26). Cloning of the PCR product, which was polished using Pfu DNA polymerase (Fermentas, St Rémy Les Chevreuse, France), was attempted using a PCR-BluntII-Topo kit according to the manufacturer's instructions (Invitrogen). A shotgun cloning experiment of the chromosome-borne ampC gene was also performed as previously described (5). Briefly, whole-cell DNA of the clinical isolate was partially digested by Sau3AI. The restriction fragments were ligated into the BamHI restriction site of the pBK-CMV plasmid vector (Stratagene, Amsterdam, The Netherlands), and the recombinant plasmids were introduced by electroporation into the E. coli TOP10 strain. Recombinants were selected on Mueller-Hinton agar plates supplemented with 30 μg/ml kanamycin and 100 μg/ml amoxicillin. All the cloned fragments were subjected to confirmatory sequencing. The nucleotide and deduced protein sequences were analyzed with software available over the Internet from the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).
Site directed-mutagenesis and transformation experiments.The recombinant plasmids used for mutagenesis experiments were purified using the Qiagen Midi kit (Qiagen). Site-directed mutagenesis was performed using the Quick Change kit (Stratagene) according to the manufacturer's instruction and the primers CMY-2-R148H-f (5′-GACTCCGGGCGCTAAGCACCTTTACGCTAACTCC-3′) and CMY-2-R148H-r (5′-GGAGTTAGCGTAAAGGTGCTTAGCGCCCGGAGTC-3′) to mutate the blaCMY-2 gene, along with the primer pair CHAR-r (5′-CAATGGACTCCGGGCGCTAAGAGGCTTTACGCTAACTCCAGC-3′) and CHAR-f (5′-GCTGGAGTTAGCGTAAAGCCTCTTAGCGCCCGGAGTCCATTG-3′) to mutate the blaAmpC-CHA gene. The mutated recombinant plasmids were subsequently introduced into the E. coli TOP10 and HB4 strains. A sequence analysis of the inserts confirmed the presence of the expected mutations, which led to the R148H and H148R replacements in the mature CMY-2-R148H and AmpC-CHA-H148R β-lactamases, respectively. The porin-deficient E. coli HB4 strain was used as a recipient to magnify the eventual carbapenemase activity of β-lactamases (27).
β-Lactamase purification.E. coli TOP10 recombinant clones were grown overnight at 37°C in 4 liters of Trypticase soy (TS) broth containing amoxicillin (100 μg/ml) and kanamycin (30 μg/ml), resuspended in 40 ml of 100 mM phosphate buffer (pH 7), disrupted by sonication, and centrifuged at 20,000 × g for 1 h at 4°C, as described previously (5). β-Lactamase extracts were filtered through a 0.45-mm-pore-size filter (Millipore, Saint-Quentin-en-Yvelines, France), dialyzed overnight at 4°C against 20 mM Tris (pH 7.5), and loaded on a preequilibrated Q-Sepharose column (Amersham Pharmacia Biotech). The flowthrough fractions containing the β-lactamase were recovered and dialyzed against 50 mM phosphate buffer (pH 6) before loading onto a preequilibrated S-Sepharose column (Amersham Pharmacia Biotech). The enzymes were eluted by a linear NaCl gradient (0 to 1 M) in the same buffer. Eluted fractions with the highest β-lactamase activity (nitrocefin test) were pooled and dialyzed against 100 mM phosphate buffer (pH 7). To assess the purity of the extracts, purified enzymes were subjected to SDS-PAGE analysis (19).
Kinetic measurements.Purified β-lactamases were used to determine the kinetic parameters (Km and kcat) of cephaloridine, cephalothin, imipenem, ceftazidime, cefotaxime, and cefepime at 30°C in 100 mM sodium phosphate (pH 7.0). The rates of hydrolysis were determined with an Ultrospec 2100 spectrophotometer and were analyzed using the Swift II software program (GE Healthcare, Aulnay-sous-Bois, France). Km and kcat values were determined by analyzing the β-lactam hydrolysis under initial rate conditions by using the Eadie-Hofstee linearization of the Michaelis-Menten equation as previously described (7). When the Km values were very low, Ki was determined instead of Km using cephaloridine as the substrate, and the kcat values were determined from initial rates at saturating substrate concentrations ([S] ≫ Km).
Protein structure analysis.A representative figure of the structure of the wild-type AmpC enzyme of the C. freundii GN346 strain, which was crystallized in previous work (Protein Data Bank [PDB] identifier 1RGY) (34), was generated using the UCSF Chimera software package, version 1.5.3, in order to determine the location of the amino acid replacement responsible for hydrolysis spectrum extension (37).
Nucleotide sequence accession number.The nucleotide sequence of the complete blaAmpC-CHA gene of C. freundii CHA has been deposited in the GenBank nucleotide database under accession no. JN400117.
The C. freundii clinical isolate CHA was resistant or intermediate to all β-lactams, including cefepime, whereas it remained susceptible to carbapenems (Table 1). The mechanism-based inhibitors, clavulanate and tazobactam, failed to restore susceptibility to amoxicillin and piperacillin, respectively. In contrast, the susceptibility to β-lactams of C. freundii CHA was restored by cloxacillin, in agreement with Ambler class C β-lactamase production.
MICs of β-lactams for the clinical strain Citrobacter freundii CHA, the E. coli TOP10(pAmpC-CHA), E. coli TOP10(pAmpC-CHA-H148R), E. coli TOP10(pCMY-2), E. coli TOP10(pCMY-2-R148H), E. coli HB4(pCMY-2), and E. coli HB4(pCMY-2-R148H) recombinant clones, and the E. coli TOP10 and HB4 recipient strains
PCR amplification of the chromosome-borne blaAmpC-CHA gene of C. freundii isolate CHA yielded a 1,146-bp amplification product that contained the entire coding region. Sequence analysis revealed five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, compared to the wild-type AmpC β-lactamase of C. freundii strain GN346 (GenBank accession number BAA02494). All these substitutions, except the R148H replacement, were distant from the reactive Ser-64 residue and were not expected to influence binding or catalysis.
Despite several attempts, cloning of the PCR product failed. A shotgun cloning experiment was therefore carried out and yielded the E. coli TOP10(pAmpC-CHA) recombinant strain. DNA sequence analysis of the 1.5-kb insert of pAmpC-CHA confirmed an open reading frame encoding the AmpC-CHA β-lactamase. MICs for the E. coli TOP10(pAmpC-CHA) clone mirrored those of the parental isolate, thus indicating that the AmpC-CHA β-lactamase constituted the main mechanism of resistance (Table 1).
Site-directed mutagenesis experiments followed by transformation assays were carried out to revert the R148H replacement in the AmpC-CHA variant. This yielded the E. coli TOP10(pAmpC-CHA-H148R) recombinant strain, which was fully susceptible to cefepime, thus demonstrating that the replacement of the Arg-148 residue, which is well conserved among AmpC β-lactamases, accounted for the hydrolysis spectrum extension of AmpC-CHA, while the other amino acid substitutions found in this enzyme were probably related to a polymorphism of the blaAmpC gene.
The amino acid substitution at position 148 occurred in the loop containing the Y150-X151-N152 motif (Fig. 1), which is the counterpart of the SDN motif in class A β-lactamases (15, 17, 22). So far, structural determinants of ESAC β-lactamases were identified in the Ω-loop, the R2 loop, and the H-9 and H-11 helices. Our report describes for the first time a structural modification in the loop containing the Y-X-N motif as a source of hydrolysis spectrum extension. This loop, which is constituted by 12 amino acids in class C enzymes instead of three residues in class A β-lactamases, defines one edge of the active site between the R2 loop and the flexible part of the Ω loop. It contains Asn-152, which forms a hydrogen bond with the carbonyl of the acylamide's linkage of the R1 side chain of β-lactam (8, 18, 22), the Tyr-150 residue, which contributes to the catalytic process (36, 39), and the Arg-148 residue, which defines one edge of the R2 binding site (Fig. 1) (8, 18, 22).
Representation of the secondary structures surrounding the amino acid at position 148. Left panel, Native AmpC β-lactamase of C. freundii GN346 (PDB 1RGY); Right panel, AmpC variant of the AmpC β-lactamase of C. freundii GN346 presenting the R148H substitution. The AmpC variant was modeled from the crystallographic structure of the AmpC β-lactamase of C. freundii strain GN346 (PDB 1RGY) by the introduction of the amino acid substitution R148H using the UCSF Chimera software package, version 1.5.3 (37). Its position was optimized by energy minimization.
To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase of C. freundii. This yielded the E. coli HB4(pCMY-2-R148H) and E. coli TOP10(pCMY-2-R148H) strains, which produced the CMY-2-R148H variant. Compared to the parental enzyme, the CMY-2-R148H variant conferred increased MICs of ESCs, including cefepime (16 μg/ml instead of 0.5 μg/ml), once expressed in the wild-type E. coli TOP10 strain, whereas it did not change the MICs of carbapenems once expressed in the porin-deficient E. coli HB4 strain (Table 1).
The CMY-2 and CMY-2-R148H β-lactamases were purified to near-homogeneity (>95%), as deduced from SDS-PAGE analysis. The purification yielded two extracts, containing 0.44 mg/ml and 0.48 mg/ml of proteins, respectively. The kinetic parameters are presented in Table 2. The Arg148His substitution in CMY-2 reduced the catalytic efficiency (kcat/Km) against good substrates such as cephalothin, while increasing the kcat/Km against "poor" substrates, such as ceftazidime, which was due to an increased kcat value for that compound. In contrast, the biochemical properties toward imipenem were unchanged. The kinetic parameters of the CMY-2 and CMY-2-R148H β-lactamases for cefepime were not determinable, since hydrolysis was not linear under the initial rate conditions throughout the course of the reaction (10 min) regardless of the substrate concentration (100 μM, 200 μM, 300 μM, 400 μM, and 500 μM).
Kinetic parameters of the wild-type CMY-2 β-lactamase and its extended-spectrum CMY-2-R148H variant
The biochemical alterations induced by the R148H substitution were not in agreement with a modification of the Tyr-150 residue that was previously shown to reduce the catalytic efficiency against ESCs (11). They also did not agree with structural alterations in the R2 binding site that increased affinity for ESCs and imipenem (lower Km value) (12, 24, 32). In contrast, the biochemical parameters of AmpC CHA were similar to those of ESACs that presented structural modifications in the DAEX third motif (residues 217 to 220), which is located in the R1 binding site (14, 31, 35). The Arg148His replacement might force the Y-X-N loop to adopt a novel conformation, modifying the interaction of the Asn-152 residue with the R1 lateral side chain of ESCs, whereas the binding of carbapenems, which lack the R1 side chain at position 6 in the β-lactam nucleus, remained unaffected.
Concluding remarks.This study describes a novel clinically occurring ESAC β-lactamase in C. freundii. Ahmed et al. previously reported a CMY-37-producing C. freundii clinical isolate. This chromosome-borne derivative differed from its progenitor by at least seven amino acid replacements (1). Unfortunately, the structural alteration accounting for the hydrolysis spectrum extension of CMY-37 was not precisely identified. Our study provides for the first time the biochemical and molecular characterization of an ESAC enzyme responsible for ceftazidime resistance in a C. freundii clinical isolate. It confirms the emergence of this mechanism of resistance among inducible AmpC-producing enterobacterial species (2, 3, 14) and subsequently raises the issue of their identification. Indeed, a previous study suggested that the phenotypic approach could fail to reliably differentiate ESAC β-lactamases from overexpressed class C enzymes (23). Only molecular methods, such as PCR, sequencing, and cloning assays, could accurately identify those mechanisms.
This study sheds new light on the structure/activity relationship of AmpC β-lactamases. In addition to the R1 and R2 binding sites, the loop containing the Y-X-N motif has been identified as a third hot spot for mutations accounting for hydrolysis spectrum extension. This highlights the evolutionary potential of Ambler class C enzymes and underscores the need for accurate molecular survey of AmpC-producing enterobacterial isolates.
We thank Stephanie Trudel for technical assistance in the sequencing experiment.
Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH grant P41 RR001081).
Received 30 August 2011.
Returned for modification 24 September 2011.
Accepted 26 December 2011.
Accepted manuscript posted online 9 January 2012.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
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Antimicrobial Agents and Chemotherapy Feb 2012, 56 (3) 1151-1156; DOI: 10.1128/AAC.05630-11
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