The Journal of Antibiotics (2016) 69, 858–862 & 2016 Japan Antibiotics Research Association All rights reserved 0021-8820/16 www.nature.com/ja ORIGINAL ARTICLE Kinetic characterization of GES-22 β-lactamase harboring the M169L clinical mutation Aysegul Saral1,2, David A Leonard3, Azer Ozad Duzgun4, Aysegul Copur Cicek5, Cynthia M June3 and Cemal Sandalli2 The class A β-lactamase GES-22 has been identified in Acinetobacter baumannii isolates in Turkey, and subsequently shown to differ from GES-11 by a single substitution (M169L). Because M169 is part of the omega loop, a structure that is known to have major effects on substrate selectivity in class A β-lactamases, we expressed, purified and kinetically characterized this novel variant. Our results show that compared with GES-116×His, GES-226×His displays more efficient hydrolysis of penicillins, and aztreonam, but a loss of efficiency against ceftazidime. In addition, the M169L substitution confers on GES-22 more efficient hydrolysis of the mechanistic inhibitors clavulanic acid and sulbactam. These effects are highly similar to other mutations at the homologous position in other class A β-lactamases, suggesting that this methionine has a key structural role in aligning active site residues and in substrate selectivity across the class. The Journal of Antibiotics (2016) 69, 858–862; doi:10.1038/ja.2016.48; published online 11 May 2016 INTRODUCTION The origin of GES-type β-lactamases remains unknown, but β-Lactamases are bacterial enzymes that hydrolyze β-lactam they generally are found in class 1 integron gene cassettes on both antibiotics, rendering these compounds ineffective.1 These enzymes chromosomes and plasmids.7 The first GES β-lactamase, GES-1, are grouped into four classes A, B, C and D, with class A, C and D was described in France in 2000.8 This enzyme confers resistance enzymes making use of a catalytic serine to hydrolyze the β-lactam to penicillins, narrow- and expanded-spectrum cephalosporins ring, and class B enzymes using a metal cofactor (Zn). Penicillins and and ceftazidime,9 and 26 variants have been reported to date cephalosporins have been used to treat bacterial infections, but their (http://lahey.org/studies/other.asp). GES-2 showed better hydrolytic efficacy has been greatly diminished by resistance mechanisms. efficiency against imipenem than GES-1, and its activity was less When these antibiotics began to be inactivated by class A β-lactamases, inhibited by clavulanic acid, tazobactam and imipenem than GES-1. several generations of cephalosporins were developed for use in There is a single amino acid change between GES-1 and GES-2 clinical settings. Inappropriate use of these drugs resulted in the (G170N) at position 170 in the omega loop of Ambler class A extended-spectrum β-lactamases (ESBL).2 ESBLs confer resistance to enzymes.8 GES-3 has two amino acid substitutions compared with penicillins, first-, second- and third-generation cephalosporins, and GES-1 (E104K, M62T).10 Substitution at the G243 residue in GES-9 aztreonam (but not the cephamycins or carbapenems) and they are and GES-11 confers increased activity against aztreonam and inhibited by β-lactamase inhibitors. ESBL type β-lactamases are found ceftazidime.9 GES-4, -5, -6 and -14 possess a substitution at the most commonly in class A, though there are a growing number of G170 residue (N or S) that leads to carbapenemase activity.11 GES-12 examples in class C and D.3 Most class A ESBLs are found in the TEM, differs from GES-11 by a single amino acid (T237A), which causes SHV and CTX-M families,1 though variants in PER, VEB, TLA-1, twofold higher efficiency against aztreonam and ceftazidime. GES-14, GES/IBC, SFO-1 and BES-1 have also been reported.4 with two amino acid substitutions (G243A and G170S), can hydrolyze Acinetobacter baumannii is a Gram-negative, opportunistic pathogen both oxyimino-cephalosporins and carbapenems.9 that causes a range of infections, including bacteraemia, pneumonia, GES-type β-lactamases have been found in Pseudomonas aeruginosa, meningitis, urinary tract infections and wound infections.5 ESBLs Escherichia coli, Klebsiella pneumoniae, Aeromonas media, Aeromonas from the Ambler class A group including VEB-1, PER-1, PER-2, veronii, Klebsiella oxytoca, A. baumannii, Serratia marcescens and TEM-92, TEM-116, CTXM-2, CTX-M-43, GES-11, -12, -14, -22 and Enterobacter cloacae. More recently, GES-22 was found in an -24 have been found in A. baumannii.6 A. baumannii isolate from Turkey.12 GES-22 differs from GES-11 by 1Department of Nutrition and Dietetics, Faculty of Health Sciences, Artvin Coruh University, Artvin, Turkey; 2Microbiology and Molecular Biology Research Laboratory, Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, Rize, Turkey; 3Department of Chemistry, Grand Valley State University, Allendale, MI, USA; 4Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, Gümüşhane University, Gumushane, Turkey and 5Department of Medical Microbiology, Faculty of Medicine, Recep Tayyip Erdogan University, Rize, Turkey Correspondence: Dr C Sandalli, Microbiology and Molecular Biology Research Laboratory, Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, Rize 53100, Turkey. E-mail: [email protected] Received 13 December 2015; revised 16 March 2016; accepted 30 March 2016; published online 11 May 2016 GES-22 β-lactamase in M169L clinical mutation ASaralet al 859 one amino acid substitution M169L, the only known variant at this peptides of GES-22 and GES-11 were determined using SignalP 4.1 server position. GES-22 and its parent GES-11 have been reported in (http://www.cbs.dtu.dk/services/SignalP/). The DNA sequences of GES-11 and previous studies from Turkey.5,13 In another study from Kuwait, GES-22 without the signal sequence (that is, the first 18 residues) were fi GES-type ESBLs were found in A. baumannii.14 Altogether, these ampli ed using iProof High-Fidelity DNA Polymerase (Bio-Rad, Hercules, CA, ′ studies show that the Middle East and Turkey could be a reservoir for USA) and primers with restriction sites for EcoRI and XhoI(GES_EcoRI_F:5- GAATTCTCGGAAAAATTAACCTTCAAGACC-3′ and GES _XhoI_R: 5′- A. baumannii producing GES-type ESBLs.5 Sequencing of the integron CTCGAGCTATTTGTCCGTGCTCAGGATGA-3′). After restriction, the PCR carrying bla and bla in isolates from Turkey show that GES-11 GES-22 amplicons were introduced into pET28a with T4 DNA ligase. Sequencing of the they possess the same genetic structure as GES-11 from France. coding regions was carried out by Macrogen, Seoul, Korea. Sequencing results This suggests that GES-22 evolved from GES-11 under conditions of were analyzed using the alignment search tool BLAST (http://www.ncbi.nlm. 12 antibiotic stress with one amino acid change. nih.gov/BLAST) and the multiple sequence alignment program CLUSTALW Position 169 is located in the omega loop of class A β-lactamases (http:// www.ebi.ac.uk/Tools/msa/clustalw2/). including members of the GES-type β-lactamases (Figure 1). In a previous study of the SHV β-lactamase subfamily, it was shown that Expression and purification of GES-22 and GES-11 the substitution R169L in SHV-57 induced a conformational change pET28a-GES-22 and pET28a-GES-11 were transformed into E. coli BL21 (DE3) in N170. This mutation that causes resistance to ceftazidime, but not for overexpression. E. coli cells harboring pET28a-GES-22 and pET28a-GES-11 to cefazolin is inhibited with clavulanic acid.15 Also, increased vector were grown to an OD at 600 nm of ~ 0.6 in LB medium containing ceftazidime hydrolysis caused by mutations at position 169 have been kanamycin (25 μgml− 1) at 37 °C, and overexpression was induced by the – described in other studies.16 18 Position 169 is most typically addition of 0.1 mM isopropyl β-D-thiogalactopyranoside overnight at 18 °C. occupied by methionine or leucine in class A β-lactamases, though The cells were collected by centrifugation and lysed by sonication. some exceptions exist (TLA-2, SHV-57, CTX-M-93; Figure 2). The After sonication, the lysate was centrifuged at 15 000 r.p.m. in a Sorvall fi neighboring residue at position 170 is important in imipenem SS-34 rotor (Sorvall, Newtown, CT, USA) for 30 min at 4 °C. The clari ed supernatants were loaded onto a HisPur Cobalt Resin (Thermo Fisher hydrolysis in GES-type β-lactamases.11,19 The residue at position 170 Scientific, Rockford, IL, USA) equilibrated with 50 mM Tris-HCl, 0.5 M NaCl, affects the conformation of the active site of the GES-type β-lactamases 10 mM imidazole, pH 7.4. The column was then washed extensively with through interactions with the E166 side chain. The presence of a column equilibration buffer, and then washed with 50 mM Tris-HCl, 0.5 M hydrogen-bonding interaction between S170 and E166 has been NaCl, 25 mM imidazole, pH 7.4. The purified β-lactamase protein was eluted 19 found to be vitally important for carbapenemase activity. with 50 mM Tris-HCl, 0.5 M NaCl, 250 mM imidazole, pH 7.4. The GES-22 and Given the importance of M169 and other proximal residues, we GES-11 containing fractions were pooled and dialyzed against 50 mM Tris-HCl, wished to determine whether position 169 in GES-22 altered 0.2 M NaCl, pH 7.4 overnight. The protein concentrations were determined by the activity of this β-lactamase against pencillins, cephalosporins measuring absorbance at 280 nm, and its purity was shown to be 495% by and carbapenems. Toward this aim, we purified the enzyme and used SDS-PAGE. Protein samples were stored at − 80 °C. steady-state kinetic analysis to measure its activity against a wide variety of these substrates.