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JAC Advance Access published online on February 14, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn031
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Structure–function studies of arginine at position 276 in CTX-M β-lactamases

Francisco José Pérez-Llarena1, Mónica Cartelle1, Susana Mallo1, Alejandro Beceiro1, Astrid Pérez1, Rosa Villanueva1, Antonio Romero2, Richard Bonnet3 and Germán Bou1,*

1 Servicio de Microbiología, Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain 2 Departamento de Ciencia de Proteínas, Centro Investigaciones Biológicas (CSIC), Madrid, Spain 3 Laboratoire de Bactériologie, CHU Clermont-Ferrand, Faculté de Medecine, Clermont-Ferrand, France

* Corresponding author. Tel: +34-981-176087; Fax: +34-981-176097; E-mail: germanbou{at}canalejo.org

Received 12 April 2007; returned 30 May 2007; revised 3 November 2007; accepted 3 January 2008


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Objectives: In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to β-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis.

Methods: By site-directed mutagenesis and by use of the blaCTX-M-1 gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of β-lactams alone and in combination with β-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (kcat, Km, kcat/Km) towards cefalotin and cefotaxime were determined. Clavulanic acid IC50 values were also assessed with all enzymes.

Results: No increase in MICs of β-lactam/β-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the clavulanic acid IC50 values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His.

Conclusions: Arg-276 in CTX-M-type β-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.

Key Words: clavulanate , β-lactamase inhibition , cefotaxime hydrolysis


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Since 1995, there has been a dramatic increase worldwide in the presence of a new family of plasmid-mediated, extended-spectrum β-lactamases (ESBLs), named CTX-M, characterized by their preferential hydrolysis of cefotaxime.1 These enzymes have been isolated from a wide range of clinical bacteria, particularly from members of the family Enterobacteriaceae.1 So far 67 different CTX-M enzymes have been described (www.lahey.org) and grouped into five different groups on the basis of amino acid sequence similarities: CTX-M-1, -2, -8, -25 and -9 groups.1

Kinetic studies have shown that CTX-M-type β-lactamases hydrolyse cefalotin and cefaloridine in preference to penicillin, and also cefotaxime in preference to ceftazidime. However, it is important to point out that during recent years, allelic variants of CTX-M enzymes with an improved capacity for ceftazidime hydrolysis have been reported in clinical strains2–5 and that this feature is associated with mutations at specific amino acid positions such as Pro-167 to Ser and Asp-240 to Gly. In addition, and in combination with other amino acid residues, positions Arg-164 to His, Asp-179 to Gly and Arg-276 to Ser have been shown to be relevant in ceftazidime hydrolysis in laboratory-obtained mutants.6

Three amino acid substitutions at positions Met-69, Arg-244 and Asn-276 are important in conferring a phenotype of inhibitor resistance in TEM-type β-lactamases.7 Regarding SHV-type enzymes, and unlike with TEM-type enzymes, replacements at these positions are not associated with a clear inhibitor-resistant phenotype, although they lower SHV β-lactamase efficiency.8

Position Arg-276 in CTX-M enzymes may be equivalent to position Arg-244, which is not present in CTX-M enzymes but is found in other class A β-lactamases such as TEM- and SHV-type enzymes.9,10 Arg-244 appears to be critical for β-lactam hydrolysis and inhibition by mechanism-based β-lactamase inhibitors, such as clavulanic acid and sulbactam.10 Residue 276 is located in the terminal {alpha}-11 helix of CTX-M enzymes.11 In TEM-type β-lactamases, residue 244 is conserved on the B4 β-strand (which is anchored to Asn-276 by two hydrogen bonds) and it may interact with the carboxylic acid group of β-lactams.10 Although these arginine residues are spaced more than 20 residues apart from each other in the primary sequence, the spatial disposition of the guanidinium side-chain groups, Arg-276 in the Toho-1 structure12 and Arg-244 in non-ESBL TEM-type,13 is ~1.5 Å (Figure 1).


Figure 1
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  		Figure 1. Superposition of CTX-M-14 (dark orange) and E. coli TEM-1 (greenish yellow) β-lactamases, highlighting the spatial disposition of the arginine residue, which is at position 276 in CTX-M-14 and other ESBLs but at position 244 in TEM, SHV and other class A β-lactamases. The figure was generated with MOLSCRIPT21 and RASTER 3D.22

 
As regards TEM-IRT-derivative enzymes, it has been reported that replacement of Arg-244 with Gly, His, Cys and Ser abrogates inhibition of TEM-type enzymes by the classical inhibitors clavulanic acid and sulbactam.14 When Arg-244 is replaced by an amino acid with a short side chain, such as cysteine, serine, glycine or histidine, the enzyme–substrate interaction is modified and affinity for the substrate decreases. The shorter side chains of these residues are unable to activate the water molecule involved in the process of inactivation of clavulanic acid.15

As regards the structural basis for inhibitor resistance in CTX-M-type enzymes, Aumeran et al.16 reported that substitution of Ser-130 to Gly in CTX-M-9 induced a 40–650-fold increase in 50% inhibitory concentrations of clavulanic, sulbactam and tazobactam, but decreased hydrolytic activity against cefotaxime. Gazouli et al.17 reported that substitution of serine for alanine at position 237 (Ambler numbering) in CTX-M-4 rendered CTX-M-4 slightly less susceptible to inhibition by clavulanate and tazobactam, and that the altered CTX-M-4 also showed a 3-fold reduction in the relative rate of hydrolysis of cefotaxime with respect to that of CTX-M-4 wild-type (wt).

To clarify the putative role of Arg-276 of CTX-M-type enzymes in susceptibility to clavulanic acid as well as its involvement in oxyimino-cephalosporin hydrolysis, we aimed to replace the Arg-276 of CTX-M-1 with different residues: (i) the previously reported Asn in CTX-M-4;9 (ii) the residues reported in IRT enzymes as important replacements (Gly, His, Cys and Ser); and (iii) an amino acid with a bulky side chain (Trp). β-Lactam MICs and kinetic parameters with selected antibiotics were determined for CTX-M-1 and its mutant-derived enzymes.


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Antibiotics and other chemicals

Nitrocefin was obtained from Oxoid (Basingstoke, Hants, UK). Cefotaxime and cefalotin were purchased from Sigma (St Louis, MO, USA). Clavulanic acid was a gift from GlaxoSmithKline (Brentford, London, UK). The antibiotic extinction molar coefficients were –4200, –7120 and 15 900 M–1cm–1 for cefotaxime, cefalotin and nitrocefin, respectively. The wavelength used was 260 nm for cefotaxime and cefalotin and 495 nm for nitrocefin. Isopropyl-β-D-thiogalactopyranoside was obtained from Roche (Basel, Switzerland).

Plasmid and mutagenesis experiments

The blaCTX-M-1-encoding plasmid pMC-35 was used to introduce the mutations into blaCTX-M-1 by site-directed mutagenesis, as previously described.18 The Arg-276 mutants were created with the oligonucleotides indicated in Table 1 (the replaced codons are underlined). All mutated as well as blaCTX-M-1 wt genes were cloned at BamHI and EcoRI restriction sites, identically orientated with respect to the lacZ gene into plasmid pBGS18 (harbouring a kanamycin-resistant gene) and transformed into Escherichia coli TG1 as previously reported.5 All mutations were confirmed by sequencing on both strands by standard procedures.


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  		Table 1. Nucleotide sequences of the primers used for PCR

 
MIC analysis

The MICs of β-lactams with and without β-lactamase inhibitors were determined by Etest (AB Biodisk, Solna, Sweden) and interpreted in accordance with the manufacturer’s recommendations.

β-Lactamase purification

To purify the CTX-M-1 wt enzyme as well as its derived mutants, the blaCTX-M genes were cloned into pGEX-6P-1 vector (BamHI and EcoRI restriction sites) with the primers CTX-M-1spF and CTX-M-1spR (Table 1), which allowed a fusion protein between glutathione S-transferase (GST) and the CTX-M enzymes. The β-lactamase was purified to homogeneity with the GST gene fusion system (Amersham Pharmacia Biotech, Europe GmbH) in accordance with the manufacturer’s instructions. Purity of protein was assessed at this stage by SDS–PAGE and Coomassie Blue staining, and the gel showed a band of 28 kDa (>95% purity). Purity was taken into account for the determination of kcat values.

Kinetic experiments

The CTX-M-1 wt enzyme and the six CTX-M-1 derivative mutants were further used in biochemical studies with cefotaxime and cefalotin as antibiotic substrates. The experiments were carried out at 25°C in a Nicolete Evolution 300 spectrophotometer (Thermo Electron Corporation, Waltham, MA, USA). The tests were repeated three times in PBS with 20 mg/L BSA. The Km values were calculated as Ki values in competitive assays, with nitrocefin as the substrate.19 The kcat values were determined under zeroth-order conditions ([S]>>Km). The IC50 values of purified enzymes were obtained according to a previously described method.20


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Antibiotic susceptibility testing

The MICs of the antibiotics tested with E. coli TG1 strain harbouring CTX-M-1 as well as with Arg-276 to Trp, Asn, Cys, Gly, His and Ser mutants are shown in Table 2. High MICs of amoxicillin, piperacillin, cefalotin, cefuroxime and cefotaxime as well as cefepime and aztreonam were obtained with E. coli expressing CTX-M-1. The addition of clavulanic acid to amoxicillin, cefotaxime and ceftazidime as well as tazobactam to piperacillin almost totally restored the activity of β-lactams against all mutants and with all combinations of β-lactams tested. Replacement of Arg-276 with Trp, Cys, Gly or Ser reduced the MICs of oxyimino-cephalosporins (this was more evident with cefotaxime) and piperacillin, although no inhibitor-resistant phenotype was observed. Slight decreases in cefotaxime MICs were observed with Arg-276 to Asn and Arg-276 to His mutants. These results reveal interesting structural features at this position as different replaced amino acids confer great differences in the MICs of oxyimino-cephalosporins.


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  		Table 2. MIC values for E. coli TG1 harbouring pBGS18 with the indicated genes

 
Overall, no increased resistance in the combination of β-lactams with either inhibitor (clavulanic acid or tazobactam) was observed with any of the mutants studied.

Biochemical studies

The CTX-M-1 enzyme as well as its mutants Arg-276 to Trp, Asn, Cys, Gly, His and Ser were purified for kinetic studies. Inhibition profiles of all enzymes (parental and mutants) showed very similar IC50 values with clavulanic acid (data not shown). Therefore, and in agreement with the MIC values, the overall results suggest that the six replacements at position Arg-276 are not involved in resistance to inhibitors in CTX-M-type enzymes.

The kinetic constants of the six mutants and of the parental enzyme CTX-M-1 are shown in Table 3. The results showed that the catalytic efficiency (kcat/Km) of the CTX-M-1 mutants (Arg-276 to Trp, Cys, Gly and Ser) was clearly reduced with respect to cefotaxime, which is consistent with the microbiological susceptibility data. Arg-276 to Gly kcat/Km value for cefotaxime was five times lower than those obtained with CTX-M-1 (the lowest value among all mutants), whereas Arg-276 to Trp, Cys and Ser kcat/Km values for cefotaxime were clearly reduced when compared with that of CTX-M-1. This reduction in kcat/Km values with the specific mutants was mainly due to the decreased kcat, although lower affinity to the antibiotic (higher Km values) was also detected with the Arg-276 to Cys mutant (Table 3). Overall results of kinetic data (Table 3) showed a good agreement with microbiological results (MIC values; Table 2).


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  		Table 3. Kinetic parameters for CTX-M-1 wt and some of its mutants

 

   Discussion
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The main aim of this study was to assess whether or not the Arg-276 position in CTX-M enzymes can be considered equivalent to Arg-244 reported in other inhibitor-resistant class A β-lactamases.

To clarify this, we replaced the Arg-276 in CTX-M-1 β-lactamase with six different amino acids, the previously described Asn in CTX-M-4,9 as well as Ser, Cys, His and Gly, previously reported as important in TEM IRT-derivative enzymes,14 and another amino acid with a long side chain (Trp), to investigate the importance of the side chain in the functionality of the position. Interestingly, different amino acid replacements caused different phenotypic effects, which revealed the importance of the substituent in the enzyme function. Although the replacements of Arg-276 with Trp, Cys, Gly and Ser were clearly associated with lower MICs of oxyimino-cephalosporins, the Arg-276 to Asn and His replacements only slightly affected cefotaxime MICs. In any case, MICs of β-lactam plus inhibitors (clavulanic acid or tazobactam) were almost identical to those of CTX-M-1 wt with all amino acid replacements.

Biochemical data were consistent with microbiological MIC values. The results obtained with the purified enzymes CTX-M-1 and their mutants Arg-276 to Trp, Cys, Gly and Ser showed impaired catalytic efficiency towards cefotaxime.

To investigate the role of residue 276, the replacements Arg-276 to Trp and Gly, representative of two phenotypes, for which cefotaxime MICs of, respectively, 6 and 2 mg/L were obtained, were chosen for modelling with the previously reported structure of CTX-M-14 enzyme.11 The mutations were studied by using molecular dynamics with CNS software.23

The Arg-276 position appears to be too far from the active centre to have any effect on the enzyme activity. In addition, the activities of the Asn and His mutants (with near-wt activities) clearly differ from those of the Trp, Cys, Gly and Ser mutants, which are impaired in their catalysis of cefotaxime. This suggests that the side chain of Arg-276 does not play a direct role in cefotaxime hydrolysis although is important for the structural integrity of the protein, probably by interacting with surrounding residues instead of the antibiotic. In this regard, when either Arg-276 to Gly or Trp replacement occurs, this causes a shift in the β3 strand (Figure 2). This displacement is more apparent with Arg-276 to Gly and causes steric hindrance, which hinders entrance of the cefotaxime antibiotic to the active centre of the enzyme. However, when Arg-276 to Trp replacement occurs, there is also a slight β3 strand shift, although lower than that with Gly (Gly and Trp MICs are 2 and 6 mg/L, respectively).


Figure 2
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  		Figure 2. Close-up view of the active-site cleft in native CTX-M-14 and two selected mutants (Arg-276 to Trp and Arg-276 to Gly) with a docked cefotaxime molecule. The mutations were manually fitted onto the structure and then subjected to energy minimization using the CNS program.23 For clarity, different colours have been used only at the region (β3 strand) where the largest displacement occurs upon simulation: native (orange), Arg-276 to Trp (lime) and Arg-276 to Gly (green). Residues Ser-237 and Trp-276 as well as the cefotaxime molecule are shown in ball-and-stick mode.

 
Very few studies have addressed the issue of CTX-M-type enzymes and inhibitor resistance. Gazouli et al.9 reported that substitution of Asn by Arg-276 in the cefotaxime-hydrolysing CTX-M-4 β-lactamase conferred lower levels of resistance to cefotaxime, ceftriaxone and aztreonam, although the levels of resistance to penicillins remained almost intact. The E. coli transformant harbouring this CTX-M-4 mutant enzyme yielded CTX-M-4 that was slightly less susceptible to inhibition by clavulanate and tazobactam. Replacement of Ser-237 to Ala in CTX-M-4 had a similar effect to that obtained with the Arg-276 to Asn.17 By homology with inhibitor-resistant TEM/SHV-type enzymes, Aumeran et al.16 replaced the Ser-130 to Gly in CTX-M-9, which yielded a CTX-M-type enzyme that exerted a 40–650-fold increase in IC50 values but decreased hydrolytic activity against cefotaxime. That study is probably the only report that clearly shows that increased resistance to β-lactamase inhibitors in CTX-M-type enzymes is associated with a mutation. No changes in the resistance to inhibitors were observed with the Arg-276 replacements, at least with the six amino acids studied here, although important modifications in the hydrolytic profile of oxyimino-cephalosporins were detected with some, which reveals the importance of the Arg-276 position in the extension of enzyme activity in CTX-M-type enzymes, rather than it being involved in susceptibility to β-lactamase inhibitors.


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This work was supported by the Consellería de Innovación, Industria y Comercio, Xunta de Galicia (PGIDIT04BTF916028PR), Fondo de Investigaciones Sanitarias. (PI040514 and PI061368), Ministerio de Educación y Ciencia (BFU2005-05055) and Spanish Network for the Research in Infectious Diseases (REIPI RD06/008). M. C. and A. B. are in receipt of a scholarship from SEIMC. F. J. P.-L. is in receipt of a research support contract from ISCIII.


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None to declare.


   Acknowledgements
 
We thank M. Galleni for critical advice regarding kinetic experiments.


   References
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1 . Bonnet R. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother (2004) 48:1–14.[Free Full Text]

2 . Poirel L, Naas T, Le Thomas I, et al. CTX-M-type extended-spectrum β-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrob Agents Chemother (2001) 45:3355–61.[Abstract/Free Full Text]

3 . Bonnet R, Dutour C, Sampaio JL, et al. Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240->Gly. Antimicrob Agents Chemother (2001) 8:2269–75.

4 . Bonnet R, Recule C, Baraduc R, et al. Effect of D240G substitution in a novel ESBL CTX-M-27. J Antimicrob Chemother (2003) 52:29–35.[Abstract/Free Full Text]

5 . Cartelle M, Tomas M, Molina F, et al. High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution. Antimicrob Agents Chemother (2004) 48:2308–13.[Abstract/Free Full Text]

6 . Delmas J, Robin F, Carvalho F, et al. Prediction of the evolution of ceftazidime resistance in extended-spectrum β-lactamase CTX-M-9. Antimicrob Agents Chemother (2006) 50:731–8.[Abstract/Free Full Text]

7 . Stapleton PD, Shannon KP, French GL. Construction and characterization of mutants of the TEM-1 β-lactamase containing amino acid substitutions associated with both extended-spectrum resistance and resistance to β-lactamase inhibitors. Antimicrob Agents Chemother (1999) 43:1881–7.[Abstract/Free Full Text]

8 . Randegger CC, Hachler H. Amino acid substitutions causing inhibitor resistance in TEM β-lactamases compromise the extended-spectrum phenotype in SHV extended-spectrum β-lactamases. J Antimicrob Chemother (2001) 47:547–54.[Abstract/Free Full Text]

9 . Gazouli M, Legakis NJ, Tzouvelekis LS. Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A β-lactamase CTX-M-4. FEMS Microbiol Lett (1998) 169:289–93.[Web of Science][Medline]

10 . Knox JR. Extended-spectrum and inhibitor-resistant TEM-type β-lactamases: mutations, specificity, and three-dimensional structure. Antimicrob Agents Chemother (1995) 39:2593–601.[Web of Science][Medline]

11 . Chen Y, Delmas J, Sirot T, et al. Atomic resolution structures of CTX-M β-lactamases: extended spectrum activities from increased mobility and decreased stability. J Mol Biol (2005) 348:349–62.[CrossRef][Web of Science][Medline]

12 . Ibuka A, Taguchi A, Ishiguro M, et al. Crystal structure of the E166A mutant of extended-spectrum β-lactamase Toho-1 at 1.8 Å resolution. J Mol Biol (1999) 285:2079–87.[CrossRef][Web of Science][Medline]

13 . Jelsch C, Lenfant F, Masson JM, et al. β-Lactamase TEM1 of E. coli crystal structure determination at 2.5 Å resolution. FEBS Lett (1992) 299:135–42.[CrossRef][Web of Science][Medline]

14 . Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev (2001) 14:933–51.[Abstract/Free Full Text]

15 . Chaibi EB, Sirot D, Paul G, et al. Inhibitor-resistant TEM β-lactamases: phenotypic, genetic and biochemical characteristics. J Antimicrob Chemother (1999) 43:447–58.[Abstract/Free Full Text]

16 . Aumeran C, Chanal C, Labia R, et al. Effects of Ser130Gly and Asp240Lys substitutions in extended-spectrum β-lactamase CTX-M-9. Antimicrob Agents Chemother (2003) 47:2958–61.[Abstract/Free Full Text]

17 . Gazouli M, Tzelepi E, Sidorenko SV, et al. Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A β-lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis. Antimicrob Agents Chemother (1998) 42:1259–62.[Abstract/Free Full Text]

18 . Ho SN, Hunt HD, Horton RM, et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene (1989) 77:51–9.[CrossRef][Web of Science][Medline]

19 . Galleni M, Francecchini N, Quinting B, et al. Use of the chromosomal class A β-lactamase of Mycobacterium fortuitum D316 to study potentially poor substrates and inhibitory β-lactam compounds. Antimicrob Agents Chemother (1994) 38:1608–14.[Abstract/Free Full Text]

20 . Sirot D, Recule C, Chaibi EB, et al. A complex mutant of TEM-1 β-lactamase with mutations encountered in both IRT-4 and extended-spectrum TEM-15, produced by an Escherichia coli clinical isolate. Antimicrob Agents Chemother (1997) 41:1322–5.[Abstract]

21 . Kraulis PJ. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr (1991) 24:946–50.[CrossRef][Web of Science]

22 . Merritt EA, Bacon DJ. Raster 3D: photorealistic molecular graphics. Methods Enzymol (1997) 277:505–24.[Web of Science][Medline]

23 . Brunger AT, Adams PD, Clore GM, et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr (1998) 54:905–21.[CrossRef][Medline]


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