Journal of Antimicrobial Chemotherapy (1999) 43, 23-29
© 1999 The British Society for Antimicrobial Chemotherapy
Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 ß-lactamases
a Department of Bacteriology, Hellenic Pasteur Institute; b Laboratory of Antimicrobial Agents, Department of Microbiology, Medical School, University of Athens, M. Asias 75, 11527, Athens, Greece
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Abstract |
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In SHV-type ß-lactamases, position 276 (in Ambler's numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 ß-lactamase and its extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276 was studied. Mutations were introduced by a PCR-based site-directed mutagenesis procedure. Wild-type SHV-1 and -5 ß-lactamases and their respective Asn276
Asp mutants were
expressed under isogenic conditions by cloning the respective bla genes into the
pBCSK(+) plasmid and transforming Escherichia coliDH5
.
Determination of IC50 showed that SHV-1(Asn276Asp), compared with
SHV-1, was inhibited by
8-
and 8.8-fold higher concentrations of clavulanate and tazobactam respectively. Replacement of
Asn276 by Asp in SHV-5 ß-lactamase caused a ten-fold increase in the IC50 of
clavulanate; the increases in the IC50s of tazobactam and sulbactam were 10- and
5.5-fold, respectively. ß-Lactam
susceptibility testing showed that both Asn276Asp mutant enzymes, compared with the
parental ß-lactamases, conferred slightly lower levels of resistance to penicillins
(amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of
the expanded-spectrum oxyimino ß-lactams tested (cefotaxime, ceftriaxone and aztreonam).
The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were
slightly elevated in the clones producing the mutant ß-lactamases. The latter clones were
also less susceptible to penicillin-inhibitor combinations. Asn276Asp mutation was
associated
with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of
TEM-1 ß-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5
are discussed. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Combinations of ß-lactam antibiotics with ß-lactamase inhibitors are useful in treating infections caused by ß-lactamase-producing bacteria.1 Among the clinically important ß-lactamases in enterobacteria are the plasmid-mediated TEM-1/2 and SHV-1 penicillinases (group 2b), and their extended-spectrum derivatives (group 2be).2 These enzymes are susceptible to the inhibitory activity of clavulanic acid and tazobactam. The intensive use of penicillin-inhibitor combinations, however, has facilitated the emergence of inhibitor-resistant ß-lactamase variants (group 2br).2 Most of the mutant enzymes that occur in vivo have been derived from TEM-1 or TEM-2 penicillinases by replacement of Met69 by Ile, Leu or Val,3,4,5,6,7 ,8 Arg244 by Cys or Ser, 7,8,9,10,11,12 and Asn276 by Asp4 ,7,8 (numbering is according to Ambler et al.13). Similar inhibitor- resistant mutants of SHV-type ß-lactamases have not yet been found in clinical strains. Studies with SHV-type laboratory mutant enzymes, obtained either spontaneously or by site-specific mutagenesis, showed that Met69
Ile or Val and
Arg244Ser or Cys substitutions confer resistance to mechanism-based inhibitors, as for
TEM
ß- lactamases.14,15,16
Characterization of an in-vitro constructed mutant of TEM-1 ß-lactamase showed that
Asn276Asp substitution conferred resistance to clavulanate and reduced hydrolytic
activity against penicillins and cephalosporins.17 In
addition, Asn276Gly replacement in OHIO-1 ß- lactamase (an
enzyme of the SHV family) modified inhibitor binding specificity and altered affinity for
penicillins.18 In this work, we examined the effect of
Asn276Asp substitution in
SHV-1 ß-lactamase and its extended-spectrum derivative SHV-5.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Bacterial strains, plasmids and cloning of ß-lactamase genes
The Escherichia coli DH5 strain (deoR, endA1, gyrA96, hsdR17 (rk-mk+), recA1, relA1, supE44, thi-1
(lacIZYA-argFV169), 
80 lacM15,
F-,
-) was used to express the wild-type and mutant ß-lactamases. The
plasmids
used in the study are described in Table I. To achieve isogenic
conditions, 1.4 kb SmaI–ClaI fragments, encompassing the coding and
promoter regions of blaSHV-1 and blaSHV-5, were
purified from low-melting point agarose and ligated into the multicloning
site of pBCSK(+). The resulting plasmids were used to transform E. coli DH5
competent cells. ß-Lactam-resistant clones were selected in Luria– Bertani agar
(Unipath Ltd, Basingstoke, UK) supplemented with ampicillin (50 mg/L) plus chloramphenicol
(20 mg/L).
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PCR-based site-directed mutagenesis
Mutant ß-lactamases were constructed by the `megaprimer' PCR-based site-directed mutagenesis method essentially as described previously.19 A mutagenic and an external primer were used in the first round of PCR to create the `megaprimer'. In the second round of PCR, the `megaprimer' and an external primer were used. The resulting amplicons were digested with SmaI and ClaI and subcloned into pBCSK(+). The mutagenic primer (5'-TGGCCGAGCGAGATCAGCAAAT-3') was 22 nucleotides long and contained a single base mismatch close to the centre of the sequence in order to direct mutagenesis at codon 276 of the mature peptide (with GAT instead of AAT at codon 276). Oligonucleotide primers were prepared in an Applied Biosystems DNA synthesizer according to the instructions of the manufacturer (Applied Biosystems, Foster City, CA, USA). DNA sequencing was performed by the dideoxy chain termination method using a Sequenase 2.0 kit (United States Biochemical Corp., Cleveland, OH, USA). To confirm the lack of unwanted changes, the complete nucleotide sequences of both strands of the mutant genes were determined.
Susceptibility testing
Susceptibility to various ß-lactam antibiotics and penicillin-inhibitor combinations was determined by an agar dilution assay according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).20 Mueller– Hinton agar (Unipath) containing the appropriate antibiotic concentrations was inoculated with 104 cfu/spot and incubated for 18 h at 37°C. The initial screening for clones producing mutant ß-lactamases was performed by a disc diffusion method. Antibiotic discs, ß-lactam antibiotics, clavulanate and sulbactam were purchased from commercial sources. Tazobactam was a gift from Wyeth Hellas S.A. Etest strips detecting the extended-spectrum ß-lactamases (ESBLs) (ceftazidime and ceftazidime– clavulanate) were also used according to the instructions of the manufacturer (Biodisk, Solna, Sweden).
ß-Lactamase assays
To obtain enzyme preparations containing the wild-type and mutant ß-lactamases, the
respective E. coli DH5 clones were grown exponentially at 37°C for 18 h in
tryptone– soya broth (Unipath). Bacterial cells were harvested and washed twice in
phosphate-buffered saline (pH 7.0). ß-Lactamases were released after mild ultrasonic
treatment of cells suspended in phosphate buffer (PB, 100 mM, pH 7.0). The extracts were
clarified by ultracentrifugation and dialysed overnight against PB. The protein content of the
extracts was determined with a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA,
USA). Isoelectric focusing (IEF) was performed in polyacrylamide gels containing ampholytes
which covered a pH range from 3.5 to 9.5 (Pharmacia-LKB Biotechnology, Uppsala, Sweden).
ß-Lactamase bands were visualized with nitrocefin (Unipath). The ß-lactamase activity
of the extracts was quantified using nitrocefin as substrate. Results were expressed as units of
activity. One unit was the amount of enzyme hydrolysing 1 mmol of substrate/ min/mg of protein
at pH 7.0 and 37°C. Inhibition profiles were determined using clavulanate, tazobactam and
sulbactam as described previously.21 Nitrocefin was used
as the reporter substrate at a concentration of 100 mM. The
amount of each ß-lactamase preparation was normalized to give 150 mM nitrocefin
hydrolysed per minute. The IC50 values were determined by incubating the enzyme
preparations with various
concentrations of inhibitor for 5 min before the addition of nitrocefin. The maximum rates of
hydrolysis of various ß-lactam substrates were determined by UV spectrophotometry
(37°C, pH 7.0) as described previously
15 and expressed relative to that of nitrocefin, which was set at 100 (relative Vmax).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The susceptibility to ß-lactams of the E. coli clones producing either a wild-type or a mutant SHV ß-lactamase is presented in Table II. Units of activity for each crude enzyme preparation were as follows: SHV-1, 37.1 U; SHV-1(Asn276
Asp), 30.5 U; SHV-5,
14.3 U; and SHV-5(Asn276Asp), 15.6 U. The inhibition and substrate profiles of
SHV-1, SHV-5 and the mutant enzymes are shown in Tables III and IV respectively. The IEF
experiments (Figure 1) showed that the mutant ß-lactamases were
focused at a lower pH
than the respective parental enzymes, as was expected from the replacement of the neutral
asparagine by the negatively charged aspartic acid.
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Effect of Asn276
Asp on SHV-1
The clone producing SHV-1(Asn276Asp) was slightly less resistant to amoxycillin,
ticarcillin and piperacillin than the clone producing the SHV-1 ß-lactamase. In contrast, the
mutant enzyme, compared with SHV-1, conferred higher levels of resistance to
penicillin-inhibitor combinations. Cephalothin and cefprozil were four-fold more active against
the isogenic clone that expressed the SHV-1 (Asn276Asp) mutant ß-lactamase.
Compared with the SHV-1-producing E. coli clone, its
SHV-1(Asn276Asp)-producing counterpart was found to be more susceptible to all
`third-generation' oxyimino-ß-lactams tested, except ceftazidime. The MICs
of the latter antibiotic remained unaltered. Notably, the MICs of cefpirome and cefepime were
consistently one- to two-dilutions higher in the E. coli clone producing the
SHV-1(Asn276Asp) mutant ß- lactamase (Table II).
As is shown in Table III, Asn276Asp substitution rendered the
SHV-1
ß-lactamase less susceptible to mechanism-based inhibitors, increasing the IC50
values of clavulanic acid and tazobactam by a factor of 8.8 and 8.0 respectively.
The IC50 of sulbactam, when tested against SHV-1(Asn276Asp), was >20
µM and higher concentrations of this inhibitor were not used.
The relative maximum hydrolysis rates are presented in Table IV. The
results for cephalothin
and cefepime were in line with the differences observed in the isogenic MIC determinations; the
Asn276Asp mutant enzyme hydrolysed cefepime more rapidly and cephalothin more
slowly than the SHV-1 ß-lactamase. Despite the evident decrease in efficiency against
penicillins (Table II), the mutant ß-lactamase hydrolysed penicillin
G more quickly than the
parental enzyme.
Effect of Asn276Asp on SHV-5
Like the pair of clones described above, the E. coli strain expressing the
SHV-5(Asn276Asp) mutant enzyme was less resistant to penicillins, cephalothin and
cefprozil than its SHV-5-producing isogenic counterpart. The MICs of penicillin-inhibitor
combinations were higher for the SHV-5(Asn276Asp)-producing clone than for the
strain expressing the SHV-5 ESBL. The most noticeable effect of the Asn276Asp
replacement on the resistance phenotype conferred by SHV-5 was the drastic reduction of
activity against cefotaxime and ceftriaxone. The MICs of ceftazidime and aztreonam were
virtually unaltered. The combination of ceftazidime with clavulanate was active against the
SHV-5-producing E. coli strain. The clone expressing the
SHV-5(Asn276Asp) mutant ß-lactamase was less susceptible to the latter
combination (Table II and Figure 2). As can also
be seen inFigure 2, the
SHV-5(Asn276Asp) mutant did not seem to be an ESBL according to the
requirements of this particular Etest-based method. Cefpirome and cefepime were slightly less
active against the clone that produced the mutant SHV-5 (Table II).
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The concentrations of clavulanate, tazobactam and sulbactam required for a 50% reduction (IC50) of the rate of nitrocefin hydrolysis by SHV-5(Asn276
Asp) were
10.0-, 5.2- and 1.8-fold higher than those for SHV-5 ß- lactamase (Table III).
SHV-5(Asn276Asp) hydrolysed penicillin G and cefepime more quickly and
cefotaxime more slowly than the SHV-5 ß-lactamase. Although the mutant ß-lactamase
conferred a lower level of resistance to cephalothin than did SHV-5 (Table II), the antibiotic was
hydrolysed more quickly by the former enzyme (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Class A ß-lactamases all interact with ß-lactams in a similar mode: a well ordered network of hydrogen bonds and electrostatic interactions aligns the substrate within the active site, facilitating a nucleophilic attack against the ß-lactam ring by Ser70, and the release of the inactivated product.22 Asparagine at position 276 is not conserved among class A enzymes, but is present in both TEM and SHV ß-lactamases, which have extensive homology (67%).23 Previous studies with TEM-1 indicated that Asn276 cannot be substituted by most amino acids, including aspartic acid, without a marked decrease in hydrolytic activity. 17
ß-Lactam susceptibility testing using isogenic systems can demonstrate differences in
hydrolytic efficiencies of related ß-lactamases.
24,25 The MICs of E. coli clones producing SHV-1, SHV-5 and their
respective Asn27Asp mutants were determined under isogenic conditions. Therefore,
the observed changes in the MICs of the ß-lactams and ß-lactam-inhibitor combinations
suggested that replacement of Asn276 by aspartic acid conferred resistance to ß-lactamase
inhibitors and influenced the hydrolytic efficiencies of SHV-1 and SHV-5 ß-lactamases.
Asn276 is on the C-terminal -helix and its side-chain lies far from the active site of TEM
and
SHV enzymes. The carbonyl group of Asn276 accepts two hydrogen bonds from Arg244 and
this bonding contributes to the proper orientation of the guanidium group of Arg244.23 The latter positively charged group is critical for ß-lactam
binding, and for
inactivation by `suicide' inhibitors: one of its NH2 groups forms a
hydrogen bond with the C-3 (C-4) ß-lactam carboxylate, and
the second more exposed amino group holds in place a water molecule (W673) which
participates in the process of irreversible inactivation by clavulanate.22 In TEM-1(Asn276Asp), Asp276 may form a salt bridge with Arg244,17 leading to a
slight change in the orientation of the
guanidium group and altering
the position of W673. Such alterations in the active site cavity may explain why
SHV-1(Asn276Asp) and SHV-5(Asn276Asp) were less susceptible to
inhibitors than were the parental ß-lactamases.
As shown by the MIC determinations, the net result of the Asn276Asp substitution in
SHV-1 and SHV-5 ß-lactamases was to reduce hydrolytic activity against
mostß-lactams. As mentioned above, Arg244 is involved mainly in substrate binding.
Assuming that the consequence of (Asn276Asp mutation is to alter the position of the
Arg244 side-chain, this reduction resulted from lower enzyme– substrate affinity.
Replacement of Asn276 by Asp caused diverse changes in the substrate profiles of SHV-1 and
SHV-5 (Table IV). An increase in the hydrolysis rates of some
ß-lactams has been observed
with the analogous mutant ß-lactamases TEM-1(Asn276Asp)17 and OHIO-1 (Asn276Gly),18
while the respective catalytic efficiencies were lower than those of the parental
ß-lactamases. Such `discrepancies' underline the different interactions of
each particular ß-lactamase with different ß-lactams, and the differences in the active
site of an ESBL with that of its parental penicillinase. Interestingly, the replacement of Asn276
by Asp improved the ability of SHV-1 and SHV-5 to inactivate the
`fourth-generation' cephalosporins cefpirome and cefepime. The presence of
aspartic acid
at position 276 leads to a decrease in the positive potential of the active site,22 and thus may facilitate electrostatically the docking of the latter antibiotics,
which possess a positively charged quaternary ammonium group at C-3.
Several recent studies have reported the emergence of inhibitor-resistant TEM-1/TEM-2
ß-lactamase variants. In some areas (e.g. Clermont-Ferrand, France), these enzymes appear
at a relatively high frequency among enterobacteria.6,7,8 Analogous
inhibitor-resistant SHV variants have not been described.
The inhibitor-resistant SHV-10 has been derived from an SHV-5 variant by replacement of
Ser130 by Gly.26 TEM-1/TEM-2 ß-lactamases
occur more frequently than SHV-1
among enterobacteria (reviewed in references 27 and 28). Therefore the possibility of selection of
inhibitor-resistant SHV variants is expected to be lower. Furthermore, the emergence of some of
these mutants, e.g. SHV-1(Asn276Asp), could pass unnoticed in routine susceptibility
tests. The above hypotheses may explain partly the apparent absence of inhibitor-resistant SHV
ß-lactamases.
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Acknowledgments |
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We thank Dr H. Hachler for providing plasmids encoding SHV ß-lactamases. We also thank Drs V. Miriagou and C. A. Owen for helpful suggestions.
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Notes |
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* Corresponding author. Tel:+33-(1)778-5638; Fax+33-(1)770-9180; Email: Lstbact{at}hotmail.com
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Received 20 May 1998; returned 9 July 1998; revised 3 August 1998; accepted 17 August 1998
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