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Keywords:

  • imipenem;
  • resistance;
  • AmpC;
  • ertapenem;
  • cephalosporinase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Only a few plasmid-borne AmpC (pAmpC) β-lactamases, such as CMY-2, can account for carbapenem resistance in Enterobacteriaceae in combination with outer membrane impermeability. The aim of this study was to elucidate the contribution of Asn-346, which is well conserved among carbapenem-hydrolyzing pAmpCs, to the hydrolysis spectrum of CMY-2. Site-directed mutagenesis experiments were carried out to replace Asn-346 with glycine, alanine, valine, glutamate, aspartate, serine, threonine, glutamine, tyrosine, isoleucine, lysine, and histidine. The recombinant plasmids were transferred into wild-type and porin-deficient Escherichia coli strains. Asn-346 replacement reduced significantly the MICs of all β-lactams, except the Asn-346-Ile substitution that increased the MICs of cephalosporins, whereas it decreased those of carbapenems. The biochemical characterization, along with a molecular modeling study, showed that the size and the polarity of the side chain at position 346 assisted substrate binding and turnover. This study shows for the first time that the amino acid at position 346 contributes to the β-lactamase activity of cephalosporinases. Asparagine and isoleucine residues, which are well conserved at position 346 among AmpC-type enzymes, modulate their hydrolysis spectrum in an opposing sense. Ile-346 confers higher level of cephalosporins resistance, whereas Asn-346 confers carbapenem resistance in combination with outer membrane impermeability.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Ambler class C (AmpC) β-lactamases are serine active enzymes widely encountered among clinically relevant Gram-negative families, such as Enterobacteriaceae. The hydrolysis reaction consists of two steps, acylation and deacylation (Oefner et al., 1990; Galleni et al., 1995; Beadle et al., 2002). In the acylation step, Ser-64 attacks the β-lactam ring carbon and forms a covalent acyl-enzyme complex. In the deacylation step, the catalytic water reacts with the covalent ester bound, leading to the release of the hydrolyzed product. Deacylation is the rate-determining step, and the kcat and kcat/Km describe the deacylation and acylation steps, respectively (Mazzella & Pratt, 1989).

AmpC β-lactamases inactivate preferentially narrow-spectrum cephalosporins, such as cephaloridin, and to a lesser extend, extended-spectrum cephalosporins (ESCs), such as ceftazidime and cefepime and carbapenems, such as ertapenem and imipenem (Philippon et al., 2002). The conformational change adopted by ESCs and carbapenems after acylation accounts for their partial resistance to AmpC β-lactamases. After the nucleophilic attack, the rotation of the dihydrothiazine or oxacephem ring, respectively, result in a displacement of the C-4/C-3 carboxylate (Fig. 1) (Beadle & Shoichet, 2002; Nukaga et al., 2004), which picks up an interaction with the Asn-346 residue (Fig. 1). This conformational change, in turn, forces the electrophilic acyl center to rotate away from the point of hydrolytic attack thus precluding deacylation (Powers et al., 2001; Beadle & Shoichet, 2002).

image

Figure 1. Representation of the substrate conformation during the catalytic process. (a) Native cephalothin bound in the oxyanion hole of the AmpC β-lactamase of E. coli K-12 (PDB 1KVL) (Beadle et al., 2002); (b) acyl-enzyme adduct formed with ceftazidime from the AmpC β-lactamase of E. coli K-12 (PDB 1IEL) (Powers et al., 2001); (c) acyl-enzyme adduct formed with imipenem from the AmpC β-lactamase of E. coli K-12 (PDB 1LL5) (Beadle & Shoichet, 2002).

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Interestingly, only a few plasmid-borne AmpC (pAmpC) β-lactamases can account for carbapenem resistance in Enterobacteriaceae in combination with outer membrane impermeability. CMY-2, ACT-1, and DHA-1 enzymes display a significant carbapenemase activity, whereas FOX-1 and ACC-1 β-lactamases fail to confer carbapenems resistance (Mammeri et al., 2010). Multiple sequence alignment revealed that Asn-346, which is located at the top of the helix H-11 (Fig. 1), is well conserved among carbapenem-hydrolyzing pAmpCs, whereas FOX-1 and ACC-1 have an Ile residue at position 346.

To elucidate the role of the amino acid at position 346 in the catalytic activities of class C β-lactamases, we undertook an investigation of the effects that mutagenesis had on the kinetics of hydrolysis, and we describe our results in relation to the known crystallographic structure of a wild-type enzyme. The CMY-2 β-lactamase, which displays the highest carbapenemase activity among pAmpCs (Mammeri et al., 2010), and for which the three-dimensional structure had been established by X-ray crystallography, was used as a model.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Bacterial strains

Strain E. coli TOP10 (pCMY-2), which harbored the recombinant plasmid pCMY-2 containing the coding region of the blaCMY-2 gene, was used as a source of ampC gene (Mammeri et al., 2010).

The wild-type strain E. coli TOP10 (Invitrogen, Cergy Pontoise, France) and the mutant strain E. coli HB4, which lacks both porins OmpC and OmpF (Mammeri et al., 2008), were used as recipient strains in transformation experiments. The lack of permeability of E. coli HB4 has been shown to highlight the weak hydrolytic activity against poor substrates, such as carbapenems (Mammeri et al., 2008). By reducing the antibiotic concentration inside the periplasm, porin change may amplify the β-lactamase effects toward weakly hydrolyzed substrates.

Site-directed mutagenesis experiments

The pCMY-2 recombinant plasmid was extracted using the Qiagen plasmid midi kit (Qiagen, Courtaboeuf, France). The mutagenesis experiments were performed using the site-directed mutagenesis kit (Agilent Technologies, Massy, France), the recombinant plasmid pCMY-2 as template, and the primer pairs indicated in Table 1. It gave rise to the recombinant plasmids pCMY-2-N346I, pCMY-2-N346G, pCMY-2-N346A, pCMY-2-N346V, pCMY-2-N346E, pCMY-2-N346D, pCMY-2-N346S, pCMY-2-N346T, pCMY-2-N346Q, pCMY-2-N346Y, pCMY-2-N346K, and pCMY-2-N346H, respectively. A sequence analysis of the inserts confirmed the presence of the expected mutation. These 12 recombinant plasmids encoded 12 variant β-lactamases that differed from the native enzyme by the Asn-346-Ile, Asn-346-Gly, Asn-346-Ala, Asn-346-Val, Asn-346-Glu, Asn-346-Asp, Asn-346Ser, Asn-346-Thr, Asn-346-Gln, Asn-346-Tyr, Asn-346-Lys, and Asn-346-His substitutions, respectively. These plasmids were transformed into E. coli TOP10 and E. coli HB4.

Table 1. Primers used for site-directed mutagenesis experiments
PrimersSequences
pCMY-2-N346I-15′-GCTAACAAAAACTATCCCATTCCAGCGAGAGTCGCC-3′
pCMY-2-N346I-25′-GGCGACTCTCGCTGGAATGGGATAGTTTTTGTTAGC-3′
pCMY-2-N346G-15′-GCAAACAAAAGCTATCCTGGCCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346G-25′-GGCCGCCTCGACACGGACAGGGCCAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346A-15′-GCAAACAAAAGCTATCCTGCCCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346A-25′-GGCCGCCTCGACACGGACAGGGGCAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346V-15′-GCAAACAAAAGCTATCCTGTCCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346V-25′-GGCCGCCTCGACACGGACAGGGACAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346E-15′-GCAAACAAAAGCTATCCTGAGCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346E-25′-GGCCGCCTCGACACGGACAGGCTCAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346D-15′-GCAAACAAAAGCTATCCTGACCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346D-25′-GGCCGCCTCGACACGGACAGGGTCAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346S-15′-GCAAACAAAAGCTATCCTAGCCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346S-25′-GGCCGCCTCGACACGGACAGGGCTAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346T-15′-GCAAACAAAAGCTATCCTACCCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346T-25′-GGCCGCCTCGACACGGACAGGGGTAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346Q-15′-GCAAACAAAAGCTATCCTCAACCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346Q-25′-GGCCGCCTCGACACGGACAGGTTGAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346Y-15′-GCAAACAAAAGCTATCCTTACCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346Y-25′-GGCCGCCTCGACACGGACAGGGTAAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346K-15′-GCAAACAAAAGCTATCCTAAGCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346K-25′-GGCCGCCTCGACACGGACAGGCTTAGGATAGCTTTTGTTTGC-3′
pCMY-2-N346H-15′-GCAAACAAAAGCTATCCTCACCCTGTCCGTGTCGAGGCGGCC-3′
pCMY-2-N346H-25′-GGCCGCCTCGACACGGACAGGGTGAGGATAGCTTTTGTTTGC-3′

Antimicrobial agents and MICs determination

The antibiotic agents and their sources have been described elsewhere (Bellais et al., 2000). MICs were determined by an agar dilution technique on Mueller–Hinton agar (Sanofi-Diagnostics Pasteur, Paris, France) with an inoculum of 104 CFU per spot and were interpreted according to the guidelines of the Clinical & Laboratory Standards Institute (2010). Escherichia coli strain 25299 was used as quality control strain.

β-Lactamase purification

Recombinant E. coli TOP10 strains were grown overnight at 37 °C in 4 L of trypticase soy (TS) broth containing amoxicillin (100 mg L−1) and kanamycin (30 mg L−1), 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 (Bellais et al., 2000). AmpC β-lactamases were purified as described previously (Mammeri et al., 2007). To assess the purity of the extracts, purified enzymes were subjected to SDS–PAGE analysis (Laemmli & Favre, 1973).

Kinetic measurements

Purified β-lactamases were used to determine the kinetic parameters (Km and kcat) of cephaloridine, ceftazidime, and imipenem 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 (GE Healthcare, Velizy-Villacoublay, France). Km and kcat values for cephaloridine were determined by analyzing the ß-lactam hydrolysis under initial rate conditions using the Eadie–Hofstee linearization of the Michaelis–Menten equation as previously described (Cornish-Bowden, 1995). Because the Km values for imipenem were low, Ki were determined instead of Km using cephaloridine as the substrate, and the kcat values were determined from initial rates at saturating substrate concentrations [(S) = 100 × Km) using 100 μL of the nondiluted enzyme extracts (Cornish-Bowden, 1995).

Protein structure analysis

A structural alignment of CMY-2 β-lactamase (PDB 1ZC2), and the AmpC β-lactamase of E. coli K-12 bound to ceftazidime and imipenem (PDB 1IEL, PDB 1LL5) (Powers et al., 2001; Beadle & Shoichet, 2002), was carried out using the pymol v1.5 software (Delano Scientific), which is available over the internet web site (http://pymol.sourceforge.net/). Virtual models of variant β-lactamases were constructed using the crystal structure of the native CMY-2 enzyme by replacing Asn-346 by Ile, Gly, Ala, Val, Glu, Asp, Ser, Thr, Gln, Tyr, Lys, and His.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Twelve variant β-lactamases, CMY-2-N346I, CMY-2-N346A, CMY-2-N346V, CMY-2-N346G, CMY-2-N346S, CMY-2-N346T, CMY-2-N346Y, CMY-2-N346D, CMY-2-N346E, CMY-2-N346Q, CMY-2-N346K, and CMY-2-N346H, were constructed by site-directed mutagenesis. Structure and chemical properties of the residues at position 346 in the parental and variant β-lactamases are summarized in Table 2. Recombinant plasmids were introduced into E. coli TOP10 and E. coli HB4 giving rise to E. coli TOP10 (pCMY-2-N346X) and E. coli HB4 (pCMY-2-N346X) recombinant clones, X corresponding to the twelve different variants precited.

Table 2. Structure and polarity of amino acids in the native CMY-2 β-lactamase and its variant enzymes
EnzymesAmino acids at position 346Side chainsPolarity
CMY-2Asn image_n/fml12199-gra-0001.pngPolar uncharged
CMY-2-N346IIle image_n/fml12199-gra-0002.png Nonpolar
CMY-2-N346AAla image_n/fml12199-gra-0003.png Nonpolar
CMY-2-N346VVal image_n/fml12199-gra-0004.png Nonpolar
CMY-2-N346GGly image_n/fml12199-gra-0005.png None
CMY-2-N346SSer image_n/fml12199-gra-0006.png Polar uncharged
CMY-2-N346TThr image_n/fml12199-gra-0007.png Polar uncharged
CMY-2-N346YTyr image_n/fml12199-gra-0008.png Polar uncharged
CMY-2-N346DAsp image_n/fml12199-gra-0009.png Negatively charged
CMY-2-N346EGlu image_n/fml12199-gra-0010.png Negatively charged
CMY-2-N346QGln image_n/fml12199-gra-0011.png Polar uncharged
CMY-2-N346KLys image_n/fml12199-gra-0012.png Positively charged
CMY-2-N346HHis image_n/fml12199-gra-0013.png Positively charged

The replacement of Asn-346 by amino acids containing none or short side chain, such as Gly and Ala, reduced drastically the MICs of ESCs and carbapenems (Table 3), which demonstrated the contribution of the side-chain residue at position 346 to the catalytic process. Moreover, the substitution of Asn-346 with residues containing bulky side chain, such as Glu, Gln, Tyr, His, and Lys, decreased the MICs of β-lactams, which could be attributable to steric hindrance in the active site, as confirmed by the results of the molecular modeling study (Fig. 2).

Table 3. MICs of β-lactams for the E. coli TOP10 and the porin-deficient E. coli HB4 recombinant clones
β-lactams (mg L−1)E. coli TOP10 (pCMY-2)E. coli TOP10 (pCMY-2-N346I)E. coli TOP10 (pCMY-2-N346K)E. coli TOP10 (pCMY-2-N346G)E. coli TOP10 (pCMY-2-N346H)E. coli TOP10 (pCMY-2-N346Y)E. coli TOP10 (pCMY-2-N346D)E. coli TOP10 (pCMY-2-N346AE. coli TOP10 (pCMY-2-N346VE. coli TOP10 (pCMY-2-N346EE. coli TOP10 (pCMY-2-N346SE. coli TOP10 (pCMY-2-N346QE. coli TOP10 (pCMY-2-N346TE. coli TOP10
Ceftazidime2565122822441288128321280.125
Cefepime120.0640.50.50.0640.1250.2510.1250.50.250.50.064
β-lactams (mg L−1)E. coli HB4 (pCMY-2)E. coli HB4 (pCMY-2-N346I)E. coli HB4 (pCMY-2-N346K)E. coli HB4 (pCMY-2-N346G)E. coli HB4 (pCMY-2-N346H)E. coli HB4 (pCMY-2-N346Y)E. coli HB4 (pCMY-2-N346D)E. coli HB4 (pCMY-2-N346A)E. coli HB4 (pCMY-2-N346V)E. coli HB4 (pCMY-2-N346E)E. coli HB4 (pCMY-2-N346S)E. coli HB4 (pCMY-2-N346Q)E. coli HB4 (pCMY-2-N346T)E. coli HB4
Imipenem3224220.250.25440.254880.125
Ertapenem2563264641642323223232641
image

Figure 2. Structural representation of the variant enzymes of CMY-2 obtained by changing Asn-346 by Ile, Ala, and Lys from the crystallographic structure 1ZC2 using pyMOL (a–d, respectively). The acyl-enzyme adduct of ceftazidime is represented in the active site by superimposition of the crystal structure of the AmpC enzyme of E. coli K-12 bound to ceftazidime (PDB 1IEL) and the crystal structure of CMY-2 (PDB 1ZC2).

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It is worth mentioning that in almost all known AmpC β-lactamases, a polar Asn or an apolar Ile are found at position 346. Herein, the effects induced by the Asn-346-Ile substitution were of particular interest, as it modified the hydrolysis spectrum of CMY-2. It slightly increased the MICs of ESCs, whereas it distinctly decreased that of imipenem (32->2 mg L−1) (Table 3), suggesting the role of the residue polarity at this position.

The suppression of the phenotypic resistance to β-lactams by negatively charged residues at position 346 further confirmed the contribution of the residue polarity to the catalytic process. For example, the ceftazidime and carbapenems hydrolysis by AmpC variants harboring Glu or Asp at position 346 exhibited a 4- to 128-fold decrease in MIC values as compared to CMY-2-type β-lactamases containing Gln or Asn, respectively (Table 3). It appears that negatively charged side chain at position 346 interplayed negatively with the catalytic process, probably due to repulsive interactions with the C-4/C-3 carboxylate of ESCs and carbapenems (Fig. 1).

To assess the biochemical effects induced by polarity change at position 346, the CMY-2 β-lactamase and the variant enzymes CMY-2-N346I and CMY-2-N346D (Table 2), were purified to near homogeneity (> 95%). The steady-state parameters for the wild-type and mutant enzymes for cephaloridine, ceftazidime, and imipenem were tested. It showed that the Asn-346-Asp replacement reduced the affinity of the enzyme for those compounds, resulting in a decreased catalytic efficiency (kcat/Km), although the deacylation step was slightly improved (kcat) (Table 4). The Asn-346-Ile substitution also led to a loss of affinity for imipenem (Table 4), but the effect of this replacement on the ceftazidime hydrolysis could not be determined because of nonlinearity in this kinetic reaction. Nevertheless, the Asn-346-Ile substitution was previously shown to increase the kcat value of the AmpC enzyme of E. coli against ceftazidime, which was in agreement with an improved deacylation step (Le Turnier et al., 2009).

Table 4. Kinetic parameters for the parental CMY-2 β-lactamase and the CMY-2-N346I and CMY-2-N346D variant enzymes
β-LactamasesCMY-2CMY-2-N346ICMY-2-N346D
kcat (s−1)Km (μM)kcat/Km (mM−1 s−1)kcat (s−1)Km (μM)kcat/Km (mM−1 s−1)kcat (s−1)Km (μM)kcat/Km (mM−1 s−1)
  1. a

    For compounds with a Km values < 5 μM, Ki were determined instead of Km, using cephaloridine as substrate. The values are the mean of three independent experiments.

  2. b

    Nd, not determinable (did not follow first-order reaction).

Cephaloridine412 ± 25125 ± 303 3007 ± 0.9900 ± 308175 ± 28120  ± 321 460
Ceftazidimea0.005 ± 0.0080.15 ± 0.333NDbNDbNDb0.03 ± 0.011.20 ± 0.525
Imipenema0.04 ± 0.011 ± 0.5400.004 ± 0.00743 ± 10.090.2 ± 0.05280 ± 350.7

According to the crystallographic study of Lobkowsky et al., the Asn-346 residue does not interact directly with the putative deacylating water molecule (Lobkovsky et al., 1993). It appears therefore that the Asn-346-Ile replacement plays a role in placing the acyl-enzyme intermediate of ESCs in a position that is more competent for hydrolytic attack. This hypothesis is supported by the crystallographic studies of Heinze-Krauss et al. and Crichlow et al., which suggested that the acyl-enzyme adduct may block the approach of the water molecule to the ester bond in Ambler class C enzymes (Heinze-Krauss et al., 1998; Crichlow et al., 2001).

Although this study demonstrated the contribution of the Asn-346 to the carbapenenemase activity of CMY-2 β-lactamase, it is worth mentioning that other amino acids are probably involved. For example, the chromosome-borne cephalosporinase of E. coli, which harbores an Asn residue at position 346, cannot confer imipenem resistance, thus confirming that the carbapenem-hydrolyzing activity results from the combined effect of several amino acids (Mammeri et al., 2008, 2010). However, it seemed to us worthwhile to ascertain the role of Asn-346 especially as it matched perfectly the ability to confer carbapenem resistance among plasmid-borne AmpC β-lactamases (Mammeri et al., 2010).

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

This study sheds new lights on the structure activity relationship of Ambler class C enzymes. It shows for the first time that the amino acid at position 346 contributes significantly to the β-lactamase activity by assisting substrate binding and turnover, as Arg-244 in Ambler class A enzyme (Lobkovsky et al., 1993). Asparagin and isoleucine residues, which are well conserved among Ambler class C enzymes at position 346, modulate the hydrolysis spectrum of cephalosporinases in an opposing sense. Ile-346 confers higher level of cephalosporins resistance, whereas Asn-346 confers carbapenem resistance in combination with outer membrane impermeability.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. References

We thank Stephanie Trudel for technical assistance in sequencing experiment. The authors declare no conflict of interest in this study.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. Acknowledgements
  8. References
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