JAC Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 -lactamases

Journal of Antimicrobial Chemotherapy (1999) 43, 23 29 JAC Aspartic acid for asparagine substitution at position 276 reduces susceptibility to mechanism-based inhibitors in SHV-1 and SHV-5 -lactamases Panagiota Giakkoupi a, Eva Tzelepi a, Nicholas J. Legakis b and Leonidas S. Tzouvelekis b * 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 In SHV-type -lactamases, position 276 (in Ambler s numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 -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 -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 coli DH5. Determination of IC 50 showed that SHV-1(Asn276 Asp), 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 -lactamase caused a ten-fold increase in the IC 50 of clavulanate; the increases in the IC 50 s of tazobactam and sulbactam were 10- and 5.5-fold, respectively. -Lactam susceptibility testing showed that both Asn276 Asp mutant enzymes, compared with the parental -lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino -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 -lactamases. The latter clones were also less susceptible to penicillin-inhibitor combinations. Asn276 Asp mutation was associated with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of TEM-1 -lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed. Introduction Combinations of -lactam antibiotics with -lactamase inhibitors are useful in treating infections caused by -lactamase-producing bacteria. 1 Among the clinically important -lactamases in enterobacteria are the plasmidmediated 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 -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 8 Arg244 by Cys or Ser, 7 12 and Asn276 by Asp 4,7,8 (numbering is according to Ambler et al. 13 ). Similar inhibitorresistant mutants of SHV-type -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 Arg244 Ser or Cys substitutions confer resistance to mechanism-based inhibitors, as for TEM - lactamases. 14 16 Characterization of an in-vitro constructed mutant of TEM-1 -lactamase showed that Asn276 Asp substitution conferred resistance to clavulanate and reduced hydrolytic activity against penicillins and cephalosporins. 17 In addition, Asn276 Gly replacement in OHIO-1 - lactamase (an enzyme of the SHV family) modified *Corresponding author. Tel: 33-(1)778-5638; Fax: 33-(1)770-9180; E-mail: Lstbact@hotmail.com 1999 The British Society for Antimicrobial Chemotherapy 23

P. Giakkoupi et al. inhibitor binding specificity and altered affinity for penicillins. 18 In this work, we examined the effect of Asn276 Asp substitution in SHV-1 -lactamase and its extendedspectrum derivative SHV-5. 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. Materials and methods Bacterial strains, plasmids and cloning of -lactamase genes The Escherichia coli DH5 strain ( deor, enda1, gyra96, hsdr17 (r k m k ), reca1, rela1, supe44, thi-1 (lacizyaargfv169), 80 lacz M15, F, ) was used to express the wild-type and mutant -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 bla SHV-1 and bla SHV-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. -Lactam-resistant clones were selected in Luria Bertani agar (Unipath Ltd, Basingstoke, UK) supplemented with ampicillin (50 mg/l) plus chloramphenicol (20 mg/l). Susceptibility testing Susceptibility to various -lactam antibiotics and penicillininhibitor 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 10 4 cfu/spot and incubated for 18 h at 37 C. The initial screening for clones producing mutant -lactamases was performed by a disc diffusion method. Antibiotic discs, -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 -lactamases (ESBLs) (ceftazidime and ceftazidime clavulanate) were also used according to the instructions of the manufacturer (Biodisk, Solna, Sweden). PCR-based site-directed mutagenesis Mutant -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 -Lactamase assays To obtain enzyme preparations containing the wild-type and mutant -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). -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). -Lactamase bands were visualized with nitrocefin (Unipath). The -lactamase activity of the Table I. Plasmids used in the study Plasmid Characteristics Source or reference pbcsk( ) puc19-derived high-copy plasmid conferring resistance to chloramphenicol Stratagene a pan1 pbcsk( ) derivative containing a SmaI ClaI fragment encoding SHV-1 16 pan2 pan1 derivative encoding SHV-1(Asn276 Asp) mutant -lactamase This study pnf2 pbcsk( ) derivative containing a SmaI ClaI fragment encoding SHV-5 26 pan6 pnf2 derivative encoding SHV-5(Asn276 Asp) mutant -lactamase This study a Stratagene, La Jolla, CA, USA. 24

Asn276 Asp substitution in SHV -lactamases extracts was quantified using nitrocefin as substrate. Results were expressed as units of activity. One unit was the amount of enzyme hydrolysing 1 mol 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 M. The amount of each -lactamase preparation was normalized to give 150 M nitrocefin hydrolysed per minute. The IC 50 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 -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 V max ). Results The susceptibility to -lactams of the E. coli clones producing either a wild-type or a mutant SHV -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(Asn276 Asp), 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 -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. Effect of Asn276 Asp on SHV-1 The clone producing SHV-1(Asn276 Asp) was slightly less resistant to amoxycillin, ticarcillin and piperacillin than the clone producing the SHV-1 -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 Table III. Inhibition profiles of SHV-1, SHV-5 and the respective mutant -lactamases; the values in parentheses show the fold increase of IC 50 values of each inhibitor caused by the replacement of Asn276 by Asp IC 50 ( M) of inhibitor -Lactamase clavulanate tazobactam sulbactam SHV-1 wild type 0.06 0.14 3.10 Asn276 Asp 0.53 (8.8) 1.13 (8.0) 20 SHV-5 wild type 0.02 0.08 1.80 Asn276 Asp 0.20 (10.0) 0.42 (5.2) 3.18 (1.8) Table II. Susceptibility to -lactam antibiotics of E. coli DH5 clones expressing SHV-1, SHV-5 and the respective Asn276 Asp mutant -lactamases MIC (mg/l) for E. coli clones producing: Antibiotic SHV-1 Asn276 Asp SHV-5 Asn276 Asp DH5 Amoxycillin 4096 2048 2048 512 2 Ticarcillin 2048 1024 2048 512 1 Piperacillin 1024 512 1024 512 0.5 Amoxycillin clavulanate a 512 1024 32 64 2 Ticarcillin clavulanate a 256 1024 32 64 1 Piperacillin tazobactam b 128 512 2 64 0.5 Cephalothin 1024 64 1024 512 4 Cefprozil 512 32 2048 512 2 Cefotaxime 0.12 0.06 32 4 0.03 Ceftriaxone 0.12 0.03 16 2 0.01 Ceftazidime 2 2 128 128 0.12 Ceftazidime clavulanate b ND c ND 0.25 2 0.12 Aztreonam 0.25 0.12 256 128 0.06 Cefpirome 0.25 1 4 8 0.03 Cefepime 0.25 0.5 2 4 0.03 a Inhibitor concentration fixed at 2 mg/l. b Inhibitor concentration fixed at 4 mg/l. c Not determined. 25

P. Giakkoupi et al. Table IV. Maximum hydrolysis rates of -lactam antibiotics. The values are relative to the hydrolysis rate of nitrocefin which was set at 100. The percentage changes in relative hydrolysis rates are in parentheses Relative V max a SHV-1 SHV-5 -Lactam wild type Asn276 Asp wild type Asn276 Asp Nitrocefin 100 100 100 100 Penicillin G 189.3 258.8 ( 37%) 5.7 4.4 ( 23%) Cephalothin 3.3 1.9 ( 42%) 1.8 2.5 ( 39%) Cefotaxime ND b ND 1.6 0.9 ( 44%) Cefepime 0.8 1.3 ( 62%) 1.6 2.3 ( 44%) a The values are the means of three determinations not differing by 10%. b Not determined. Hydrolysis rates were too low. Figure 1. Isoelectric focusing of SHV-1, SHV-1(Asn276 Asp), SHV-5 and SHV-5 (Asn276 Asp) -lactamases (lanes 1 4, respectively). A preparation of SHV- -lactamase-free E. coli DH5 cells is in lane 5. -Lactamases with known pis (PSE-2, pi 6.1; SHV-1, pi 7.6; SHV-5, pi 8.2 and LAT-1) are in lane 6. against the isogenic clone that expressed the SHV-1 (Asn276 Asp) mutant -lactamase. Compared with the SHV-1-producing E. coli clone, its SHV-1(Asn276 Asp)- producing counterpart was found to be more susceptible to all third-generation oxyimino- -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(Asn276 Asp) mutant - lactamase (Table II). As is shown in Table III, Asn276 Asp substitution rendered the SHV-1 -lactamase less susceptible to mechanism-based inhibitors, increasing the IC 50 values of clavulanic acid and tazobactam by a factor of 8.8 and 8.0 respectively. The IC 50 of sulbactam, when tested against SHV-1(Asn276 Asp), was 20 M 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 Asn276 Asp mutant enzyme hydrolysed cefepime more rapidly and cephalothin more slowly than the SHV-1 -lactamase. Despite the evident decrease in efficiency against penicillins (Table II), the mutant -lactamase hydrolysed penicillin G more quickly than the parental enzyme. Effect of Asn276 Asp on SHV-5 Like the pair of clones described above, the E. coli strain expressing the SHV-5(Asn276 Asp) 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(Asn276 Asp)-producing clone than for the strain expressing the SHV-5 ESBL. The most noticeable effect of 26

(a) (b) Figure 2. Application of ESBL-detecting Etest in the E. coli strains producing (a) wild-type and (b) Asn276 Asp mutant SHV -lactamases. The upper side of the strip contained ceftazidime; the lower side contained ceftazidime plus a fixed concentration (4 mg/l) of clavulanate. the Asn276 Asp 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(Asn276 Asp) mutant -lactamase was less susceptible to the latter combination (Table II and Figure 2). As can also be seen in Figure 2, the SHV- 5(Asn276 Asp) mutant did not seem to be an ESBL according to the requirements of this particular Etestbased method. Cefpirome and cefepime were slightly less active against the clone that produced the mutant SHV-5 (Table II). The concentrations of clavulanate, tazobactam and sulbactam required for a 50% reduction (IC 50 ) 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 - lactamase (Table III). SHV-5(Asn276 Asp) hydrolysed penicillin G and cefepime more quickly and cefotaxime more slowly than the SHV-5 -lactamase. Although the mutant -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). Discussion Class A -lactamases all interact with -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 -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 -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 -Lactam susceptibility testing using isogenic systems can demonstrate differences in hydrolytic efficiencies of related -lactamases. 24,25 The MICs of E. coli clones producing SHV-1, SHV-5 and their respective Asn276 Asp mutants were determined under isogenic conditions. Therefore, the observed changes in the MICs of the -lactams and -lactam-inhibitor combinations suggested that replacement of Asn276 by aspartic acid conferred resistance to -lactamase inhibitors and influenced the hydrolytic efficiencies of SHV-1 and SHV-5 -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 -lactam binding, and for inactivation by suicide inhibitors: one of its NH 2 groups forms a hydrogen bond with the C-3 (C-4) -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(Asn276 Asp), 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(Asn276 Asp) and SHV-5(Asn276 Asp) were less susceptible to inhibitors than were the parental -lactamases. As shown by the MIC determinations, the net result 27

P. Giakkoupi et al. of the Asn276 Asp substitution in SHV-1 and SHV-5 -lactamases was to reduce hydrolytic activity against most -lactams. As mentioned above, Arg244 is involved mainly in substrate binding. Assuming that the consequence of Asn276 Asp 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 -lactams has been observed with the analogous mutant -lactamases TEM-1(Asn276 A s p ) 17 and OHIO-1 (Asn276 Gly), 18 while the respective catalytic efficiencies were lower than those of the parental -lactamases. Such discrepancies underline the different interactions of each particular -lactamase with different -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 -lactamase variants. In some areas (e.g. Clermont-Ferrand, France), these enzymes appear at a relatively high frequency among enterobacteria. 6 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 - l a c t a m- ases 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(Asn276 Asp), could pass unnoticed in routine susceptibility tests. The above hypotheses may explain partly the apparent absence of inhibitor-resistant SHV -lactamases. Acknowledgements We thank Dr H. Hachler for providing plasmids encoding SHV -lactamases. We also thank Drs V. Miriagou and C. A. Owen for helpful suggestions. References 1. Sutherland, R. (1995). -Lactam/ -lactamase inhibitor combinations: development, antibacterial activity and clinical applications. Infection 23, 191 200. 2. Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for -lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 1211 33. 3. Blazquez, J., Baquero, M.-R., Canton, R., Alos, I. & Baquero, F. (1993). 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