Effect of D240G substitution in a novel ESBL CTX-M-27

R. Bonnet1,*, C. Recule2, R. Baraduc1, C. Chanal1, D. Sirot1, C. De Champs1 and J. Sirot1

1 Laboratoire de Bactériologie, Faculté de Médecine, Service de Bactériologie-Virologie, 28 Place Henri-Dunant, 63001 Clermont-Ferrand Cedex; 2 Laboratoire de Bactériologie, CHU de Grenoble, Chemin Maquis du Grésivaudan, 38 700 La Tronche, France

Received 27 January 2003; returned 14 February 2003; revised 13 March 2003; accepted 13 March 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Escherichia coli clinical strain Gre-1 collected in 2000 from a French hospital harboured a novel CTX-M-encoding gene, designated blaCTX-M-27. CTX-M-27 differed from CTX-M-14 only by the substitution D240G and was the third CTX-M enzyme harbouring this mutation after CTX-M-15 and CTX-M-16. The Gly-240-harbouring enzyme CTX-M-27 conferred to E. coli higher MICs of ceftazidime (MIC, 8 versus 1 mg/L) than did the Asp-240-harbouring CTX-M-14 enzyme. Comparison of CTX-M-14 and CTX-M-27 showed that residue Gly-240 decreased Km for ceftazidime (205 versus 940 µM), but decreased hydrolytic activity against good substrates, such as cefotaxime (kcat, 113 versus 415 s–1), probably owing to the alteration of ß3 strand positioning during the catalytic process.

Keywords: CTX-M, ß-lactamase, D240G mutation, ESBL


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The first extended-spectrum ß-lactamase (ESBL) of the CTX-M type (MEN-1/CTX-M-1) was reported at the beginning of the 1990s.1,2 Initially characterized in Europe, CTX-M-producing strains have been observed over a wide geographic area including the Near and Far East,38 South America,3,912 Africa13 and Europe.1,2,1424 CTX-M enzymes have been observed in different species of the Enterobacteriaceae family such as: Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter freundii, Citrobacter amalonaticus, Enterobacter aerogenes and Enterobacter cloacae; and in the species Vibrio cholerae El Tor.12

The CTX-M enzymes form a rapidly growing family that comprises currently at least 34 enzymes of which 19 have been described in the last 3 years. They are generally much more active against cefotaxime than against ceftazidime and aztreonam.1 The flexibility of the ß3 strand and {Omega} loop, and residues Asn-104, Ser-237, Asp-240 and Arg-276 are involved in the cefotaxime-hydrolyzing activity of CTX-M enzymes.1,5,1719,25,26

There have been recent reports of CTX-M mutants exhibiting an increased enzymic activity against ceftazidime: the P167S mutant of CTX-M-18 (also designated CTX-M-14), designated CTX-M-19,21 and D240G mutants of CTX-M-3 and CTX-M-9, designated CTX-M-15 and CTX-M-16, respectively.8,10,14

In France, we isolated a CTX-M-producing strain that produced a novel D240G variant designated CTX-M-27 and derived from CTX-M-14 as well as CTX-M-19. The biochemical characterization of the two ß-lactamases CTX-M-14 and CTX-M-27 and molecular modelling give insights into the role of the D240G substitution in CTX-M enzymes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Clinical strain

Table 1 shows the strains and plasmids used in this study. Clinical strain Gre-1 was isolated in 2000 from a patient hospitalized in Grenoble, France. E. coli transformants producing CTX-M-1416 were used for comparison.


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Table 1.  Strains and plasmids used in the study
 
Susceptibility to ß-lactams

MICs were determined by a dilution method on Mueller–Hinton agar (Sanofi Diagnostics Pasteur, Marnes la Coquette, France) with an inoculum of 104 cfu per spot. Antibiotics were provided as powders by SmithKline Beecham Pharmaceuticals, Nanterre, France (amoxicillin, ticarcillin and clavulanate), Lederle Laboratories, Paris-La Défence, France (piperacillin and tazobactam), Eli Lilly, Paris, France (cefalothin), Roussel-Uclaf, Paris-La Défence, France (cefotaxime, cefpirome), Glaxo Wellcome, Marly-le-Roi, France (ceftazidime), and Sanofi Winthrop, Gentilly, France (aztreonam).

Detection of ESBLs was carried out with the standard double disc synergy tests as described previously.27 Antibiotic discs for agar tests were obtained from Sanofi Diagnostics Pasteur.

Isoelectric focusing

Isoelectric focusing was carried out with polyacrylamide gels containing ampholines with a pH range of 3.5–10 as previously described.10 ß-Lactamases of known pIs were used as standards: TEM-1 (pI 5.4), TEM-24 (pI 6.5), SHV-1 (pI 7.6) and SHV-5 (pI 8.2).

Amplification of CTX-M-encoding genes

The detection of CTX-M-encoding genes was carried out with the primers CTX-MA (5'-CGCTTTG CGATGTGCAG-3') and CTX-MB (5'-ACCGCGATATCGTTGGT-3') (temperature of annealing of 54°C). They amplify a 550 bp internal fragment from positions 264–814 (blaCTX-M-1 numbering), which correspond to conserved regions of blaCTX-M-type genes. The complete ORF of the blaCTX-M-27 gene was amplified with the primers CTX-M-9A (5'-CTGATGTAACACGGATTGAC-3') and CTX-M-9C (5'-AGCGCCCCATTATTGAGAG-3') (temperature of annealing of 54°C).

ß-Lactamase gene cloning

The recombinant DNA manipulations were carried out as described by Sambrook et al.28 T4 DNA ligase was purchased from Boehringer Mannheim, Germany. The CTX-M-encoding sequence was cloned as follows: the complete ORF, which was amplified with proof-reading Taq polymerase Tfu (Appligene Oncor, Illkirch, France) and primers reported above, was ligated in the SmaI site of the phagemid pBK-CMV (Stratagene, La Jolla, CA, USA). E. coli DH5{alpha}28 was transformed by electroporation. The transformants harbouring the recombinant CTX-M-encoding plasmid were selected on Mueller–Hinton agar supplemented with 2 mg of cefotaxime per L.

DNA sequencing

The sequence was determined by direct sequencing of PCR products, carried out by the dideoxy chain termination procedure of Sanger et al.29 on an ABI 1377 automatic sequencer using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq DNA polymerase FS (Perkin-Elmer/Applied Biosystems Division, Foster City, CA, USA).

ß-Lactamase preparation

The CTX-M-producing E. coli DH5{alpha} was grown in 6 L of brain heart infusion broth containing cefotaxime at 2 mg/L for 18 h at 37°C. The bacteria collected by centrifugation were suspended with MES–NaOH 20 mM (pH 5.5) and disrupted by ultrasonic treatment (4 x 30 s, each time at 20 W). After centrifugation (10 000g for 10 min at 4°C), nucleic acids were precipitated by addition of 0.2 M [7% (v/v)] spermine and centrifuged at 48 000g for 60 min at 4°C. The clarified supernatant was dialysed overnight against MES–NaOH 20 mM (pH 5.5). The CTX-M purification was carried out as previously described10 by ion-exchange chromatography with an SP Sepharose column (Amersham Biosciences Europe, Orsay, France) and gel-filtration chromatography with a Superose 12 column (Amersham Pharmacia Biotech). The total protein concentration was estimated by the Bio-Rad protein assay (Bio-Rad, Richmond, CA, USA), with bovine serum albumin (Sigma Chemical Co., St Louis, MO, USA) used as a standard.

The purity of CTX-M extracts was estimated as previously described10 by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and staining with Coomassie Blue R-250 (Sigma Chemical Co.). The renaturation of proteins and the detection of the ß-lactamase activity were carried out as previously described10 with renaturation buffer Tris–HCl (100 mM)–Triton X-100 [2% (v/v); pH 7.0] and 0.5 mM nitrocefin (Oxoid, Paris, France) in 100 mM phosphate buffer (pH 7.0), respectively.

Determination of ß-lactamase kinetic constants

The steady-state kinetic parameters (Km and kcat) of the ß-lactamases were obtained by an improved computerized micro-acidimetric method derived from the technique of Labia et al.30 using 702 SM Titrino pHstat (Metrohm, Herisau, Swiss). They were determined by the analysis of the complete hydrolysis time-courses and the kinetic progress curves were fitted by non-linear least-squares regression. The concentrations of the inhibitors (clavulanate and tazobactam) required to inhibit enzyme activity by 50% (IC50s) were determined as previously described with penicillin G.10 The specific activity and IC50s were monitored with penicillin G (200 mM) as the reporter substrate. The kinetic constants were determined three times.

Sequence analysis

The nucleotide sequence and the deduced protein sequence were analysed with the software available over the Internet at the National Center of Biotechnology (http://www.ncbi.nlm.nih.gov/). A hydrophobic blot was obtained with the method of Nielsen et al.31 Multiple sequence alignment and pairwise comparisons of sequences were carried out with the help of ClustalW 1.74 software.32

Molecular modelling

Molecular modelling was carried out using Hyperchem v6.3 software (Hypercube Inc., Gainesville, FL, USA) on the basis of the crystallographic structure of the Glu-166->Ala Toho-1 mutant25 by introducing the mutations as part as an automated procedure. The residues, the catalytic water molecule and the water molecules of the Toho-1 crystal were minimized using the Amber96 parameters33 and a distance-dependent dielectric constant by conjugate gradient energy minimization until the r.m.s. gradient was <0.1 kcal/mol.

Nucleotide sequence accession number

The blaCTX-M-27 nucleotide sequence data appear in the GenBank nucleotide sequence database under accession number AY156923.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Characterization of the CTX-M-27-producing clinical isolate

E. coli clinical isolate Gre-1 exhibited resistance to broad-spectrum cephalosporins (MIC of cefotaxime, 256 mg/L; MIC of ceftazidime, 16 mg/L; MIC of aztreonam, 32 mg/L) (Table 2) and a positive double-disc synergy test. Isoelectric point determination with benzylpenicillin as substrate revealed the presence of two different ß-lactamases (Table 1), but with cefotaxime as substrate, only one enzyme, of pI value 8.2, showed strong cefotaxime-hydrolysing activity.


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Table 2.  MICs for clinical strain E. coli Gre-1 and the corresponding transformant E. coli DH5{alpha} (pClGre-1) in comparison with CTX-M-14-producing transformant E. coli DH5{alpha} (pClCF-1)16
 
Cloning and DNA sequencing of ß-lactamase genes

The strain Gre-1 exhibited a positive amplification with primers CTX-MA and CTX-MB, which were designed to conserve sequences of blaCTX-M genes. DNA sequencing of PCR products showed that strain Gre-1 harboured a blaCTX-M-14-type gene. The complete ORF of the blaCTX-M gene was amplified with the primers CTX-M-9A and CTX-M-9C and cloned downstream of the LacZ promoter of plasmid pBK-CMV.

The sequencing of the blaCTX-M ORF revealed that strain Gre-1 harboured a new gene, designated blaCTX-M-27, which differed from blaCTX-M-1416 by the substitution A -> G at ORF position 725. On the basis of amino acid sequence alignment (data not shown) with CTX-M signal peptide sequences previously determined by direct amino acid sequencing1,6 and from hydropathy plots, the deduced amino acid sequence comprised a signal peptide of 28 amino acids. Thus, the putative mature enzyme CTX-M-27 consisted of 263 amino acid residues with a calculated molecular weight of 27 915 Da. This sequence differed by one or two substitutions at positions 231 and 240 (according to the numbering of Ambler et al. 34) from those of CTX-M-9 (A231V, D240G), CTX-M-14 (D240G) and CTX-M-16 (A231V). Thus, CTX-M-27 was the third CTX-M enzyme harbouring the substitution D240G after CTX-M-15 and CTX-M-16,8,10, which derive from CTX-M-3 and CTX-M-9, respectively.

ß-Lactam susceptibility of the CTX-M-27-producing E. coli transformant

MICs of ß-lactams for the E. coli DH5{alpha} transformants producing CTX-M-27 (pClGre-1) and CTX-M-14 (pClCF-1)16 are listed in Table 2. The CTX-M-producing strains exhibited a high level of resistance to amino- and carboxy-penicillins (MICs > 2048 mg/L), piperacillin (MIC, 256 mg/L), cefalothin and cefuroxime (MICs, 512–1024 mg/L). Similar levels of resistance to cefotaxime (MICs, 16 mg/L) and aztreonam (MICs, 4–8 mg/L) were observed. However, the Gly-240-harbouring enzyme CTX-M-27, like CTX-M-15 and CTX-M-16,8,10 exhibited higher MICs of ceftazidime than its parental enzyme CTX-M-14 (8 versus 1 mg/L), which contains the residue Asp-240.

Clavulanate and tazobactam restored partially or totally the activities of the ß-lactams (MICs, 0.06–16 mg/L).

Kinetic constants

The purified protein appeared on SDS–polyacrylamide gels as a band of ~28 kDa for CTX-M-27 (Figure 1). The specific activity of purified (≥97% pure) CTX-M-27 was 22 µmol min–1 mg–1 of protein with 200 mM benzylpenicillin as the substrate.



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Figure 1. Electrophoresis analysis of CTX-M-27 purified extracts. (a) SDS–PAGE stained with Coomassie Brilliant Blue R-250. (b) Zymogram detection of ß-lactamase activity with nitrocefin after renaturation treatment of SDS–PAGE. Lanes: 1, protein molecular mass reference; 2, purified extract of ß-lactamase CTX-M-27; 3, clarified extract of ß-lactamase CTX-M-27.

 
The kinetic constants of CTX-M-14 and CTX-M-27 are shown in Table 3. Common enzymic features were observed. There were better affinities for penicillins (Km, 6–48 µM) than for cephalosporins, cefalothin was the best substrate (kcat for cefalothin 10- to 20-fold higher than that for penicillin), and kcat for cefotaxime (113–415 s–1) was higher than for ceftazidime (3 s–1) and aztreonam (0.4–10 s–1). The hydrolytic properties of enzyme CTX-M-27 were therefore similar to those of previously reported CTX-M enzymes with regard to the higher catalytic activity against cefotaxime than against ceftazidime and aztreonam.


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Table 3.  Substrate profile of ß-lactamases CTX-M-14,16 and CTX-M-27
 
However, CTX-M-27 had lower kcat values than CTX-M-14 with all substrates except ceftazidime for which the hydrolytic activity was closely related to that observed with previously reported CTX-M.1,6,7,10,15,16,18 Shimamura et al.26 reported an interaction between the Asp-240 side chain and the amino-thiazole ring of cefotaxime in the crystallographic acyl-intermediate of Toho-1, which accounts for its activity against cefotaxime. Residue Gly-240, which is devoid of side chains, is unable to establish this interaction and may therefore decrease the hydrolytic activity of CTX-M-27 against cefotaxime and against substrates devoid of the amino-thiazole ring such as penicillins, cefuroxime and cefalothin.

The Gly-240-harbouring enzyme CTX-M-27, as previously observed with CTX-M-16,10 had a lower Km value than the Asp-240-harbouring enzyme CTX-M-14, with regard to ceftazidime. This decrease in Km could explain the higher MIC value of ceftazidime observed for the CTX-M-27-producing E. coli than for the CTX-M-14 producer.

In TEM and SHV ESBLs, residues Lys and Arg at position 240 are known to increase the enzymic activity against ceftazidime. Lys and Arg are positively-charged residues that can form an electrostatic bond with the carboxylic acid group on oxyimino substituents of ceftazidime.35,36 In CTX-M-27, neutral residue Gly-240 is not able to form electrostatic interactions with ß-lactams but could favour the accommodation of ceftazidime. In Asp-240-harbouring enzyme CTX-M-14, the acyl-amide group of ceftazidime is probably sterically encumbered by the side chain of Asp-240, which, as a result, could alter the interactions between the ceftazidime C-7ß-amid group and residues Ser-237 and Asn-132. The residue Gly-240, which is devoid of side chains, could decrease this steric hindrance.

Substitution D240G did not modify the susceptibility to ß-lactamase inhibitors. The two enzymes CTX-M-14 and CTX-M-27 were susceptible to tazobactam (IC50, 0.008 and 0.007 µM, respectively), clavulanate (IC50, 0.030 and 0.020 µM, respectively) and, to a lesser extent, sulbactam (IC50, 3.5 and 3.4 µM, respectively).

Molecular modelling

Ibuka et al. reported that the ß3 strand of CTX-M enzymes has numerous Gly residues and is therefore probably more flexible than that of TEM penicillinases.25 Residue Gly-240 in CTX-M-27 may further increase the flexibility of the ß3 strand and alter its positioning during the catalytic process. To investigate the diminution of the hydrolytic activity associated with residue Gly-240 in CTX-M-27, the enzymes CTX-M-14, CTX-M-27 and CTX-M-1 were modelled by geometric optimization after the introduction of amino acid substitutions and of the catalytic water molecule in the crystallographic structure of Toho-1. The major differences observed between the models obtained and the TEM-1 crystallographic structure (Figure 2c)37 are shown in Figure 2 (a and b). In the CTX-M enzymes, the loop connecting the ß5 strand and the H11 helix exhibited two additional residues and was oriented towards the C-terminal extremity of the ß3 strand unlike that in the TEM-type enzyme. In the CTX-M-14 model, the residue Asn-270 of this loop established a hydrogen bond with the Asp-240 side chain (Figure 2a). This interaction could favour the correct positioning of the ß3 strand residues during the catalytic process. In the crystal structure of Toho-1 acyl-enzyme intermediates,26 the interaction between residues 270 and 240 is mediated by a water molecule. However, the calculated energy from the structure exhibiting indirect binding was higher than that obtained from the structure exhibiting direct interaction of residues 270 and 240. No interaction between residues 240 and 270 was seen in CTX-M enzymes belonging to the CTX-M-1 group, because they are devoid of residue Asn-270. However, these enzymes harbour residue Lys-271, which could interact with residue Asp-240, and could replace the Asn-270 (data not shown).



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Figure 2. Structure of enzymes CTX-M-14 (a), and CTX-M-27 (b) obtained by molecular modelling and the crystallographic structure of the TEM-1 enzyme (c).35 Two black dots indicate the positions of the water molecules: W1, the catalytic water molecule maintained by the lateral chains of residues 166, 170 and 70; and W2, the water molecule of the oxyanionic hole formed by the main chains of residues 70 and 237. The hydrogen bonds between residues 170, 240 and 270 are indicated by dashed lines.

 
Following the substitution D240G, interaction between the residues 240 and 270 or 271 was absent in CTX-M-27 (Figure 2b). The absence of this interaction in CTX-M-27 could work towards modifying the flexibility of the C-terminal of the ß3 strand. During the catalytic process, this flexibility could favour the accommodation of substrates, but could alter the geometry of the oxyanionic hole and/or the positioning of the catalytic water molecule as the result of modifications of the interactions between residues 240 and Asn-170.

In conclusion, residue Gly-240 in CTX-M-27 decreases the Km for ceftazidime but could decrease hydrolytic activity against good substrates, probably by modifying ß3 strand-residue positioning during the hydrolytic process.


    Acknowledgements
 
We thank Rolande Perroux, Marlène Jan and Dominique Rubio for technical assistance. This work was supported in part by a grant from the Ministère de l’Education Nationale, de la Recherche et de la Technologie.


    Footnotes
 
* Corresponding author. Fax: +33-4-73-27-74-94; E-mail: Richard.Bonnet{at}u-clermont1.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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