Biomedical Research

Abstract

Objective: The aim of this study was to determine the effects of residues at 228th positions of VIM-38 on minimum inhibition concentration (MIC) and molecular docking analysis.

Materials and Methods: blaVIM-38 gene was cloned into expression vector pET100/D-TOPO. Then pET100/D-TOPO-VIM-38 was used to generate the R228S mutation by site-directed mutagenesis. The mutant was transformed into E. coli Dh5α. Changes in the activity of mutation R228S was determined by E-test. Molecular docking analysis of the binding affinity of meropenem and imipenem with VIM-38 and VIM-38 R228S was performed using AutoDock Vina_1_1_2.

Results: According to E-test results, pET100/D-TOPO-VIM-38 and pET100/D-TOPO-VIM-38/ R228S have same MIC value for amoxicillin, ticarcillin/clavulanic acid, ampicillin and ticarcillin. It was determined decrease in pET100/D-TOPO-VIM-38/ R228S of MIC for aztreonam, amikacin and cefixine. There are 3-fold deference in MIC between pET100/D-TOPO-VIM-38 and pET100/D-TOPO-VIM-38/ R228S for cefoxitin. pET100/D-TOPO-VIM-38 has 0.5 μg/ml and pET100/D-TOPO-VIM-38/ R228S has 0.125 μg/m for imipenem, we observed 4-fold deference. Also, there is about 170-fold deference between pET100/D-TOPO-VIM-38 (16 μg/ml) and pET100/D-TOPO-VIM-38/ R228S (0.094 μg/ml) for meropenem. VIM-38 and VIM-38 R228S have very close to the negative low free energies of binding of the meropenem and imipenem.

Conclusion: These results showed that substitution (R228S) slightly have effect on enzyme activity.

Keywords

Docking, MIC, Mutation, VIM

Introduction

Antibiotic resistance in the bacteria is a major problem worldwide. β-Lactam antibiotics are clinical significant in the treatment of bacterial infections, but resistance increasingly compromises their clinical use [1]. β-Lactamases are hydrolysed β-lactam antibiotics. β-Lactamases are the most important mechanism of resistance to β-lactam antibiotics. β- Lactamases are grouped into four classes: those in A, B, C and D. Class A, C and D enzymes making use of a catalytic serine to hydrolyse the β-lactam ring, while Class B comprises the metallo-β-lactamases (MBLs), which employ metal ions in catalysis [1]. MBLs or class B β-lactamases are zinc-dependent enzymes and they have broad hydrolytic activity against all β- lactams except aztreonam [1]. Genes encoding MBLs are located as cassettes in integrons which contribute to their dissemination and potential for expression [2]. To date, there are many type of MBLs identified such as the IMP, VIM, SPM, GIM, NDM, and SIM. The most commonly detected MBLs are IMP and VIM types in the worldwide [2]. Up to now, 46 VIMtype MBL variants have been described in total and each variant has one or several amino acid substitutions from each other (http://www.lahey.org/Studies/). Although VIM-type metallo beta lactamase have been identified in several Enterobacteriaceae [3]. VIM-1 was the first to be reported in 1999 in Italy, and then VIM-2 variant identified in France [4] and Italy [5]. VIM-5 was identified in Turkey in Klebsiella pneumoniae, P. aeruginosa and Enterobacter cloacae isolate [6-8]. VIM-38 has recently been identified in P. aeruginosa isolates in Turkey and, it differs from VIM-5 by a single substitution (Ala316Val) [9]. In a study on the enzymatic property of VIM-38, its steady-state kinetic analyses showed that VIM-38 hydrolysed all tested penicillins, cephalosporins and carbapenems [1].

The VIM-type enzymes belong to subclass B1 of molecular class B and VIM-type MBL has 3 different group (VIM-1, VIM-2 and VIM-7). VIM-38 belong to the VIM-1 cluster, and differ from VIM-1 by 6 residues (Ala130Lys, His224Leu, Glu225Ala, Ser228Arg and Lys291Thr, Ala316Val), respectively. VIM-38 contains His224Leu and Ser228Arg substitutions relative to VIM-1; these residues are positioned on the L10 loop and are proposed to influence the substrate specificity of VIM variants [1,2,10-14].

It was aimed to determine the effects of residues at 228th positions of VIM-38 on minimum inhibition concentration (MIC) and molecular docking analysis in this study.

Material and Methods

Cloning experiments of blaVIM-38 gene

blaVIM-38 gene was detected by PCR. Cloning of the blaVIM-38 gene to expression vector pET100/D-TOPO was amplified using primers to obtain the whole gene sequence. The obtained PCR fragment was purified with a QIAquick column (Qiagen, Courtaboeuf, France) and cloned into the pET100/D-TOPO vector (Invitrogen Life Technologies, Saint Aubin, France). Recombinant plasmids were selected onto Mueller–Hinton agar (MH) plates containing ampicillin (50 mg/ml). The cloned DNA fragment inserted into one of the recombinant plasmids was sequenced by Macrogen. Sequencing results were analysed using alignment search tool, BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and the multiple sequence alignment program CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Site-directed mutagenesis of the R228S

Mutation was completed through blaVIM-38 allele which was cloned into the pET100/D-TOPO expression vector. Arginine amino acid position at 228 will be transformed Serine amino acids by directed mutations out of the pET100/D-TOPOVIM- 38. Primers were designed to generate the mutation. A single reaction mixture contained: 2 μl of plasmid DNA, 20 pM of each primer, 10 μl reaction buffer, 3 μl 25 mM MgCl₂, 200 μM of dNTPs and 1.5 U Pfu Polymerase (Promega, Madison, USA) in a final volume of 50 μl. PCR result was analysed on 1% agarose containing 0.5 μg/ml ethidium bromide, and subsequently visualized under UV light. PCR product was cleaned up by PCR-clean up kit (Promega, USA) and DpnI enzyme digestion were made. After digestion the samples were transformed into E. coli Dh5α. Plasmids were isolated and submitted to DNA sequence analysis. Sequencing results were analysed using alignment search tool, BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and the multiple sequence alignment program CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

E-test

The E-test was performed using E-test strips containing ticarcillin, tazobactam, aztreonam, cefixine, amoxicillin, amikacin, ampicillin, cefoxitin, ticarcillin/clavulanic acid, imipenem, meropenem and ertapenem according to the manufacturer's instructions in plates with MH agar. Plates were inoculated and incubated under the same conditions described for the agar dilution method, and MICs were determined.

Swiss modelling

Since the three-dimensional (3D) structure of VIM-38 and VIM-38 R228S are not known, the 3D structures of these β- lactamases were predicted by Swiss-Model [15-17]. Crystal structure of VIM-5 was used as a template.

Molecular docking

Molecular docking analysis of the modelled protein structures was carried out to identify if substitutions in the 228.amino acid of VIM-38 resulted in variations in their binding affinity to meropenem and imipenem. The structures of meropenem and imipenem were retrieved from the PubChem compound database. PyRx 0.8 was used for energy minimization calculations. Also, ligands and modelled proteins were converted PDBQT format with using PyRx 0.8. Grid box was cantered active site of proteins and enlarged the dimensions of the cubic search space to completely cover the site. Autodock Vina_1_1_2 was used for molecular docking calculations [18].

Results

blaVIM-38 gene was cloned to pET100/D-TOPO vector. Conversion of the arginine amino acid (at position 228) into serine amino acid was confirmed by sequencing analysis using pET100/D-TOPO _VIM-38 vectors. MIC values were determined by E-test method. MIC values are shown in Table 1.

Table 1. MIC values of pET100/D-TOPO-VIM-38 and pET100/DTOPO- VIM-38/ R228S.

According to E-test results, amoxicillin, ticarcillin/clavulanic acid, ampicillin, and ticarcillin MIC values were found same for E. coli containing pET100/D-TOPO-VIM-38 and pET100/D-TOPO-VIM-38/ R228S. On the other hand, it was determined decrease in MIC for imipenem, meropenem, tazobactam, aztreonam, amikacin, cefixine and cefoxitin. Also, R228S led to increase in MIC for ertapenem. pET100/DTOPO- VIM-38 has 0.38 μg/ml while pET100/D-TOPOVIM- 38/ R228S has 0.125 μg/ml MIC for cefixime. pET100/D-TOPO-VIM-38 has 1.5 μg/ml and pET100/DTOPO- VIM-38/ R228S has 0.38 μg/ml MIC for amikasin. pET100/D-TOPO-VIM-38 has 6 μg/ml and pET100/D-TOPOVIM- 38/ R228S has 2 μg/ml, there are 3-fold deference for cefoxitin. There are 4-fold deference MIC of imipenem for pET100/D-TOPO-VIM-38 0.5 μg/ml and pET100/D-TOPOVIM- 38/ R228S 0.125 μg/ml. Also, there is about 170-fold deference between pET100/D-TOPO-VIM-38 (16 μg/ml) and pET100/D-TOPO-VIM-38/ R228S (0.094 μg/ml) for meropenem (Table 1).

Analysis of binding affinity of meropenem and imipenem with VIM-38 and VIM-38 R228S was performed using AutoDock Vina_1_1_2. The negative low free energy of binding of docked complexes indicated high affinity of β-lactamase for these antibiotics [15]. The negative low free energies of binding of the meropenem and imipenem for VIM-38 and VIM-38 R228S were very close to each other (Table 2).

Table 2. Molecular docking analysis results of VIM-38 and VIM-38/ R228S.

Discussion

Creating the basic mechanisms of bacterial resistance to β- lactams are producing hydrolytic enzymes. These hydrolytic enzymes are called β-lactamases and they break the amide bond in the β-lactam ring of β-lactams [19,20]. So far more than 1000 β-lactamase has been reported [21]. These enzymes are encoded chromosomally or genes (that coding these enzymes) are located on mobile genetic elements such as transposons and plasmids. β-lactamases they show too many differences according to their functional, biochemical and similarity of amino acid sequences. According to Ambler molecular classification, β-lactamases can be divided into two super families structurally: serin (classes A, C and D) and metallo-β-lactamases (class B). Both serine β-lactamases and metallo-β-lactamases are able to hydrolyse the β-lactams, although catalytic mechanisms are different. The spread of MBLs among Gram-negative pathogens are a very serious problem [22]. More importantly, MBLs are carried with other resistance genes which restricted treatment options with the formation of multi-resistance [23]. Rapid spread of MBL are because of the mobile genetic elements such as plasmids, transposable elements and ISC relationship between MBL genes [22,23]. Spread of earned MBLs is a very crucial for infection control and the treatment of patients [22]. VIM-type of metallo- beta-lactamases have been reported to be 46 variants and they have one or more amino acid changes between these variants (http://www.lahey.org/Studies/). Each year, some of the new variants are defined in Enterobacteriaceae. Accumulated mutations on the gene take years and the amount of the accumulated mutations on a gene that gives information about the gene has a long history. Sitedirected mutagenesis experiments on metallo-beta-lactamase conducted on the results are of great importance because occurring variants of increasing antibiotic concentrations (MICs) contain mutations selected on their ability to remain alive in the bacterial host. Therefore, experiments with purified recombinant proteins increase the understanding about effects of mutations that confer resistance [24].

At 228th position, VIM-2 has an arginine and VIM-1 has a serine. VIM-2 group enzymes interact directly with the substrate, whereas, VIM-1 group enzymes definitely too short to create any interaction with the substrate. This can be explained VIM-2 group has higher affinity for substrates [10]. Also, VIM-38 has arginine at position 228th.

In a study has been reported comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-5 and VIM-38. The results showed that small differences in the kinetic parameters of the tested VIM variants [1]. In this study, substitution of Serine at the position 228 with Arginine, when compared with VIM-38 and VIM-38/R228S appears to give almost similar MIC values.

According to the results of molecular docking, the mutation at position 228 in VIM-38 showed that it did not affect binding affinity for imipenem and meropenem efficiently. To support this result, the binding energy can also be calculated with using different molecular docking programs. Because different molecular docking programmes can use different algorithms for calculating free binding energy [25]. Since the crystal structure of VIM-38 was not in Protein DataBank, the threedimensional structure of proteins was obtained by using Swiss Model. In one study six different homology modeling programs; Modeller, SegMod/ENCAD, SWISS-MODEL, 3DJIGSAW, nest, and Builder was compared. In this study it was found that three modeling programs, Modeller, nest, and SegMod/ ENCAD, perform better than the others [26]. 3D structures of proteins obtained with using different homology modelling programs can be used as receptors in docking programme. And docking results with these receptors can be compared. Differences between docking results and experimental data was found in many studies. It was showed that one of the reasons for differences between docking results and experimental data might be ignoring multiple binding in docking programmes [27]. In future studies, enzyme kinetics assays will be performed to determine precisely the effect of R228S mutation in VIM-38 on substrate affinity and catalytic efficiency.

Acknowledgements

This study was supported by Gumushane University Research Fund Grants BAP- 17.F5119.02.01.

References

1. Makena A, Düzgün AÖ, Brem J, McDonough MA, Rydzik AM, Abboud MI, Saral A, Cicek AC, Sandalli C, Schofield CJ. Comparison of Verona Integron-Borne Metallo-β-lactamase Variants Reveals Differences in Stability and Inhibition Profiles. Antimicrob Agents Chemother 2015; 60: 1377-13784.
2. Siarkou VI, Vitti D, Protonotariou E, Ikonomidis A, Sofianou D. Molecular epidemiology of outbreak-related pseudomonas aeruginosa strains carrying the novel variant blaVIM-17 metallo-beta-lactamase gene. Antimicrob Agents Chemother 2009; 53: 1325-13230.
3. Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18: 306-325.
4. Poirel L, Naas T, Nicolas D, Collet L, Bellais S, Cavallo JD, Nordmann P. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother 2000; 44: 891-897.
5. Pallecchi L, Riccio ML, Docquier JD, Fontana R, Rossolini GM. Molecular heterogeneity of blaVIM-2-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-β-lactamase. FEMS Microbiol Lett 2001; 195: 145-150.
6. Midilli K, Aygün G, Kuskucu M. A new variant of metallo-β-lactamase detected in a Klebsiella pneumoniae strain: VIM-5. KLIMIK Congress Turkey 2003.
7. Bahar G, Mazzariol A, Koncan R, Mert A, Fontana R, Rossolini GM, Cornaglia G. Detection of VIM-5 metallo-β-lactamase in a Pseudomonas aeruginosa clinical isolate from Turkey. J Antimicrob Chemother 2004; 54: 282-283.
8. Gacar GG, Midilli K, Kolayli F, Ergen K, Gundes S, Hosoglu S, Karadenizli A, Vahaboglu H. Genetic and enzymatic properties of metallo-β-lactamase VIM-5 from a clinical isolate of Enterobacter cloacae. Antimicrob Agents Chemother 2005; 49: 4400-4403.
9. Iraz M, Duzgun AO, Cicek AC, Bonnin RA, Ceylan A, Saral A, Nordmann P, Sandalli C. Characterization of novel VIM carbapenemase, VIM-38, and first detection of GES-5 carbapenem-hydrolyzing β-lactamases in Pseudomonas aeruginosa in Turkey. Diagn Microbiol Infect Dis 2014; 78: 292-294.
10. Docquier JD, Lamotte-Brasseur J, Galleni M, Amicosante G, Frère JM, Rossolini GM. On functional and structural heterogeneity of VIM type metallo-β-lactamases. J Antimicrob Chemother 2003; 51: 257-266.
11. Leiros HK, Edvardsen KS, Bjerga GE, Samuelsen O. Structural and biochemical characterization of VIM-26 shows that Leu224 has implications for the substrate specificity of VIM metallo-β-lactamases. FEBS J 2015; 282: 1031-1042.
12. Merino M, Perez-Llarena FJ, Kerff F, Poza M, Mallo S, Rumbo-Feal S, Beceiro A, Juan C, Oliver A, Bou G. Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo-β-lactamases. J Antimicrob Chemother 2010; 65: 1950-1954.
13. Mojica MF, Mahler SG, Bethel CR, Taracila MA, Kosmopoulou M, Papp-Wallace KM, Llarrull LI, Wilson BM, Marshall SH, Wallace CJ, Villegas MV, Harris ME, Vila AJ, Spencer J, Bonomo RA. Exploring the Role of Residue 228 in Substrate and Inhibitor Recognition by VIM Metallo-β-lactamases. Biochem 2015; 54: 3183-3196.
14. Kupper MB, Herzog K, Bennink S, Schlomer P, Bogaerts P, Glupczynski Y, Fischer R, Bebrone C, Hoffmann KM. The three-dimensional structure of VIM-31, a metallo-β-lactamase from Enterobacter cloacae in its native and oxidized form. FEBS J 2015; 282: 2352-2360.
15. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Gallo Cassarino T, Bertoni M, Bordoli L, Schwede T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 2014; 42: W252-W258.
16. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006; 22: 195-201.
17. Gundogdu-Hizliates C, Alyuruk H, Gocmenturk M, Ergun Y, Cavas L. Synthesis of new ibuprofen derivatives with their in silico and in vitro cyclooxygenase-2 inhibitions. Bioorg Chem 2014; 52: 8-15.
18. Singhal N, Kumar M, Virdi JS. Molecular Analysis of Β-Lactamase Genes to Understand Their Differential Expression in Strains of Yersinia Enterocolitica Biotype 1A. Sci Rep 2014; 4: 5270.
19. Essack SY. The development of β-lactam antibiotics in response to the evolution of β-lactamases. Pharm Res 2001; 18: 1391-1399.
20. Siu LK. Antibiotics: action and resistance in Gram-negative bacteria. J Microbiol Immunol Infect 2002; 35: 1-11.
21. Davies J. Origins and evolution of antibiotic resistance. Microbiologia 2010; 74: 417-433.
22. Cornaglia G, Giamarellou H, Rossolini GM. Metallo b-lactamase: a last frontier for b-lactams? Lancet Infect Dis 2011; 11: 381-393.
23. Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-b-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18: 306-325.
24. Meini MR, Llarrull LI, Vila AJ. Evolution of Metallo-β-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution. Antibiotics (Basel) 2014; 3: 285-316.
25. Azam SS, Abbasi SW. Molecular docking studies for the identification of novel melatoninergic inhibitors for acetylserotonin-O-methyltransferase using different docking routines, heoretical. Theor Biol Med Model 2013; 10: 1-16.
26. Wallner B, Elofsson A. All are not equal: A benchmark of different homology modeling programs. Protein Sci 2005; 14: 1315-1327.
27. Atkovska K, Samsonov SA, Paszkowski-Rogacz M, Pisabarro MT. Multipose Binding in Molecular Docking. Int J Mol Sci 2014; 15: 2622-2645.