Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis
Azam AHMADY1, Toktam POOLAD2, Poorya RAFEE1, Mojtaba TOUSHEH3, Manijeh KAHBAZI1, Mohammad ARJOMANDZADEGAN1
1Arak Üniversitesi Tıp Fakültesi, Tüberküloz ve Pediatrik İnfeksiyon Hastalıkları Araştırma Merkezi, Arak, İran,
2İslami Azad Üniversitesi, Kürdistan Bilim ve Araştırma Şubesi, Mikrobiyoloji Bölümü, Tahran, İran,
3İsfahan Üniversitesi Hücresel ve Moleküler Biyoloji Bölümü, İsfahan, İran.
ÖZET
Pirazinamid dirençli ve duyarlı Mycobacterium tuberculosis izolatlarında pirazinamidaz yapısal değişikliklerinin çalışması
Giriş:Pirazinamid, tüberküloz tedavisindeki ilk basamak ilaçlardan biridir. 359 ve 374 pnc genlerinin iki nükleotidindeki mutasyonun, pirazinamid direnciyle yüksek derecede ilişkili olduğu gösterilmiştir.
Materyal ve Metod:Bu çalışmada, 30 klinik Mycobacterium tuberculosis izolatında sekans analiziyle bu iki kodondaki mutasyonlar araştırıldı. Pirazinamid dirençli ve duyarlı izolatlarda, mutasyonlu ve mutasyonsuz bu gen tarafından kodla- nan protein yapıları araştırıldı.
Bulgular:359 ve 374 pozisyonlarındaki mutasyonun, elektronik yük ve mutasyona uğramış aminoasitlerin aktif enzim du- rumuna uzaklığı gibi bazı parametreleri değiştirdiği saptandı. Bu durumlarda, pirazinamidazın yapı ve fonksiyonu değişti ve antibiyotik etkisizdi ve sonuçta M. tuberculosis’de pirazinamide dirence neden oldu.
Sonuç:Sonuç olarak, bu çalışmada pirazinamide dirençli klinik M. tuberculosis izolatlarında protein değişiklikleri tanım- landı.
Anahtar Kelimeler: Mycobacterium tuberculosis, ilaç direnci, pirazinamidaz, protein yapısı, kodon.
Yazışma Adresi (Address for Correspondence):
Dr. Mohammad ARJOMANDZADEGAN, Tuberculosis and Pediatric Infectious Diseases Research Center, Arak University of Medical Sciences, ARAK - IRAN
e-mail: mmatinam81@yahoo.com
INTRODUCTION
Pyrazinamide (PZA) antibiotic is from the nicotinamide analogues which despite is a strong anti bacterial espe- cially in the fairly acidic environment of body micropha- ges and also in zones with acute inflammation. PZA is an analog of nicotinamide and a prodrug that inhibits the growth of Mycobacterium tuberculosis. PZA diffuses into M. tuberculosis cell, where the enzyme pyrazinami- dase (PZase) converts PZA to the active form pyrazino- ic acid (1). Pyrazinoic acid was thought to inhibit the enzyme fatty acid synthase (FAS) I, which is disrupts membrane potential and interferes with energy produc- tion, necessary for survival of M. tuberculosis it is we- akly bactericidally effective on the M. tuberculosis yet, it is an efficient drug in the first two-month treatment of tuberculosis, when there are acute inflammation diffe- rences in body, and its usage has decreased the treat- ment duration and the possibility of relapse (3-5).
PZA requires conversion to the bactericidal compound pyrazinoic acid by the bacterial PZase activity of nico- tinamidase to show activity against M. tuberculosis.
Mutations leading to a loss of PZase activity cause PZA resistance in M. tuberculosis. Reduction in PZase acti-
vity or deletion because of mutation in pnc gene causes resistance to PZA resistance (5,6).
Accumulation of pyrazinoic acid disrupts membrane potential, necessary for survival of M. tuberculosis at an acidic site of infection. Pyrazinoic acid binds to the ribosomal protein S1 (RpsA) and inhibits trans-transla- tion. This may explain the ability of PZA to kill dormant mycobacterium.
Although mutations in any point of pncA gene causes resistance but frequency of mutations in nucleotides of 359 and 374 has been emphasized in literature (7).
The aim of this work was comparison study of protein structure of PZase enzyme in normal and mutated forms.
MATERIALS and METHODS Bacterial Isolates
In this study, 30 clinical isolates of M. tuberculosis sen- sitive and resistant to PZA, were selected by proportion method.
PCR and Sequencing
Polymerase chain reaction (PCR) by specific primers of pnc-8 (5’-GGTTGGGTGGCCGCCGGTCAG-3’) and SUMMARY
Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis
Azam AHMADY1, Toktam POOLAD2, Poorya RAFEE1, Mojtaba TOUSHEH3, Manijeh KAHBAZI1, Mohammad ARJOMANDZADEGAN1
1Tuberculosis and Pediatric Infectious Diseases Research Center, Faculty of Medicine, Arak University, Arak, Iran,
2Department of Microbiology, Kordestan Science and Research Branch, Islamic Azad University, Arak, Iran,
3 Department of Cellular and Molecular, Isfahan University, Isfahan, Iran.
Introduction:Pyrazinamide is one of the first line four drugs for treatment of tuberculosis. It was proved that mutations in two nucleotides of 359 and 374 pnc genes are highly associated with resistance to pyrazinamide.
Matedials and Methods:In this study, mutations in these two codones in 30 clinical isolates of Mycobacterium tuberculo- sis were detected by means of sequencing. Protein structures encoded by this gene with and without mutation were inves- tigated in resistant and susceptible isolates to pyrazinamide, respectively.
Results:Mutation in the positions 359 and 374 altered some parameters like change in electronic charge, distance change of mutated amino acids to situation of active enzyme and metal connection situation. In these conditions, structure and function of pyrozinamidase enzyme were changed and antibiotic was ineffective and consequently caused resistance to pyrazinamide in M. tuberculosis.
Conclusion:This work was revealed protein changes in resistance to pyrazinamide in clinical isolates of M. tuberculosis.
Key Words: Mycobacterium tuberculosis, drug resistance, pyrazinamidase, protein structure, codon.
Tuberk Toraks 2013; 61(2): 110-114 • doi: 10.5578/tt.3888
pnc-11 (5’-GCTTTGCGGCGAGCGCTCCA-3’) have been accomplished by a thermocycler (Eppendorf 5332) in anealing temperature of 64°C (5).
PCR products has been sequenced by an ABI system for detection of any mutations in two nucleotides of 359 and 374.
Bioinformatics Analysis
The three dimensional (3-D) structure of the protein coded by pnc-A gene has been evaluated by an infor- matics investigation using Molegro Virtual Docker (MVD) software.
RESULTS and DISCUSSION Molecular Study
PCR amplification on purified DNA of 30 isolates reve- aled 774 bp (base pair) including the entire ORF of pncA gene (400 bp) plus a part of downstream and upstream of this gene. This confirmed correct conditi- ons and performance of PCR reaction by pnc-8 and pnc-11 primers for amplification (Figure 1).
Results from sequencing of 744-bp fragment in all isolates revealed that susceptible isolates to PZA ha- ve any mutations in nucleotides of 359 and 374. So- me of resistant isolates to PZA without any mutations in these nucleotides had mutations in other nucleoti- des.
Protein Study
Comparing wild-type and minimized mutant structures with two codons of 120 and 125, which have respecti- vely been created by changing T to C in 359 nucleoti- de and T to G in 374 nucleotide, confirms the differen- ces in two predicted structures (Figure 2-4).
Mutated protein structures of changed amino acids show the importance of mutation in studied nucleotide.
In nucleotide 359, after T changing to C, the Hydrop- hobic amino acid No. 120 (Leu) changes to amino acid with special side chain that named proline, and in nuc- leotide 374, after T changing to G, amino acids No.
125 [Val] changes to Gly.
Software analysis by Molegro virtual docker showed that 3-D structure of pyrazinamidase enzyme (PZAase) consists of four alpha helix and four B-sheets (Figure 5). The special arrangement of these motifs creates a hole in the molecule that, along with Fe2+ion, is neces- sary for the activity of the enzyme. This ion plays a di- rect, catalytic role in hydrolyze of the medicine. The important amino acid residues in performance of PZA- ase include residues in the enzyme active situation (D8, A134, and C138) and amino acid residues in the metal connection situation (MCS) (D49, H51, H58, and H71) (1,8). Mutation in these amino acids and other amino acids present near these situations has effect on the physicochemical activity of this enzyme. So far, in different studies, after mutation, different parameters have been attended like change in electronic charge and volume of mutated amino acids, distance change of mutated amino acids compared to situation of acti- ve enzyme and metal connection situation, and directi- on of side-chain of mutated amino acid (8,9).
Figure 1. Amplification of PCR product of 744 bp by pnc-8 and pnc-11 primers.
Figure 2. Comparison of the predicted two structures wild-type and mutant (L120P) by using molegro virtual docker software:
these images showed that changing in situation of codon 120 created another small cavity and distance of proline to active enzyme situation (18.1044 nm) and metal connection situation (20.7969 nm) changed. These parameters at wild-type struc- tures are 17.098 nm and 19.5774 nm.
In this paper, situation of codon 120 of three amino acids was found to be at the end of one of the helixes, and af- ter the mutation in this protein, another small hole is cre- ated. This last one cause a distance change of proline compared to active enzyme situation and metal connec- tion situation and direction of side-chain of mutated ami- no acid. But in situation of codon 125, direction of side- chain of valine changes compared to wild-type, after this amino acid changes to glycine. With relation between structure and performance of biological macromolecu- les, this change causes decrease or elimination of enzy- me activity, and it can consequently cause resistance to pyrazinomidase (1,10). For example distance between wild type amino acid at 120 (leucine) to active site (ca- vity) from 17.098 nm changed to 18.1044 nm in mutant amino acid in this situation and so on.
CONCLUSION
In this study, the structures of wild and mutated pyra- zinamidases from clinical isolates M. tuberculosis we- re analyzed and single amino acid replacement was Figure 3. Comparison of the predicted two structures wild-type and mutant (V125G) by molegro virtual docker software: with occurrance a mutation at amino acid No. 125, distance to enzyme active site from 19.9544 nm in wild-type structure is con- verted to 23.0768 nm in mutant (mut) structure and distance the situation 125 to the metal ion position from the 24.1225 nm in wild-type is converted to 26.6282 nm in mut structure.
Figure 4. Comparison of the predicted two structures wild-type and mutant (L 120P and V125G) by molegro virtual docker soft- ware: in mutant structure (L120P and V125G) of this protein is created a extended new cavity.
Figure 5. Schematic view of three-dimensional structure of pyrazinamidase: as shown the most of superficial regions of the protein are acidic areas (gray colour in the image) and sections marked with an arrow represents the cavity.
studied. Mutations in nucleotides 359 and/or 374 from clinical isolates sensitive and resistant to pyra- zinamidase were proved by sequencing. Bioinforma- tics analysis revealed that in the mutant protein structure of the pyrazinamidase enzyme was chan- ged some parameters such as electrical charge of the mutated amino acid, the volume of the mutated ami- no acid, the distance of the mutated amino acid to the active site, the distance of the mutated amino acid to the metal-coordination site, and the orientati- on of the side-chain of the mutated amino acid. In si- tuation of codon 125 or 120, direction of side-chain of mutated amino acid and distances to active site changes compared to wild-type. As for relation bet- ween structure and performance of enzyme, these changes causes decrease or elimination of enzyme activity, and it can consequently cause resistance to pyrazinamidase. Therefore the antibiotic will be inef- fective on the bacterium.
CONFLICT of INTEREST None declared.
REFERENCES
1. Sun Z, Zhng Y. Reduced pyrazinamidase activity and the na- tural resistance of Mycobacterium kansasii to the antitubercu- losis drug pyrazinamide. Antimicrob Agents Chemother 1999;
43: 537-42.
2. Boshoff HI, Mizrahi V, Barry CE. Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purifi- ed fatty acid synthase I. J Bacteriol 2002; 184: 2167-72.
3. Ngo SC, Zimhony O, Chung WJ, Sayahi H, Jacobs WR, Welchpi JT. Inhibition of isolated Mycobacterium tuberculosis fatty acid synthase I by pyrazinamide analogs. Antimicrob Agents Chemother 2007; 1: 2430-5.
4. Zimhony O, Cox JS, Welch JT, Vilcheze C, Jacobs WR. Pyrazi- namide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis. Nature Medicine 2000;
6: 1043-7.
5. Perdiga JO, Macedo R, Malaquias A, Ferreira A, Brum L, Por- tugal I. Genetic analysis of extensively drug-resistant Myco- bacterium tuberculosis strains in Lisbon, Portugal. Antimicrob Chemother 2010; 65: 224-7.
6. Zhang H, Bi LJ, Li CY, Sun ZJ, Deng JY, Zhang ZE. Mutation found in the pncA gene of Mycobacterium tuberculosis in cli- nical pyrozinamide-resistant isolates from a local region of China. J Int Med Res 2009; 37: 1430-5.
7. Mphahlele M, Syre H, Valvatne H, Stavrum R, Mannsaker T, Muthivhi T, et al. Pyrazinamide resistance among South Afri- can multidrug-resistant Mycobacterium tuberculosis isolates.
J Clin Microbiol 2008; 46: 3459-64
8. Quiliano M, Gutierrez AH, Gilman RH, Lopez C, Evangelista W, Sotelo J, et al. Structure-activity relationship in mutated pyrazinamidases from Mycobacterium tuberculosis. Bioinfor- mation 2011; 6: 335-9.
9. Clatchy JK, Tsang AY, Cernich MS. Use of pyrazinamidase ac- tivity on Mycobacterium tuberculosis as a rapid method for determination of pyrazinamide susceptibility. Antimicrob Agents Chemother 1981; 20: 556-7.
10. Zhang Y, Mitchison D. The curious characteristics of pyrazina- mide. Int J Tuberc Lung Dis 2003; 7: 6-21.
