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The GMP Synthase ( EC 6.3.5.2) from Mycobacterium tuberculosis as a Target for the Development of an Attenuated Strain V Mostra de Pesquisa da Pós- Graduação

Franco, T. M. A1, 2, Basso, L. A1, 2, Santos, D. S1, 2 (orientador)

1Programa de Pós Graduação em Medicina e Ciências da Saúde, Faculdade de Medicina, PUCRS; 2Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, PUCRS.

Introduction

Human tuberculosis (TB) is a contagious-infectious disease mainly caused by Mycobacterium tuberculosis (MENDONÇA, 2007; DUCATI, 2006). The main form of TB transmission occurs by infective particles expelled by a patient when he/she coughs, sneezes and even through the speech. The incidence of TB has increased nowadays due to several factors, such as co-infection with HIV, resistance of microorganisms to drugs used in therapy [emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains], patient's non-adherence to treatment, and low efficacy of BCG vaccine in adults. Epidemiological data of 2007 indicated overall incidence of 139 TB cases per 100.000 inhabitants, which corresponds to 9.27 million cases of TB worldwide, taking 1.18 million of these patients to death (WHO: Report WHO/HTM/TB/2009.411, 2009). These factors point to the urgent need for developing new anti-TB drugs, which would be ideally more efficient, less toxic, and that would reduce the current treatment course. Among possible new therapies against TB, the study of in M. tuberculosis might lead to the discovery of novel selective inhibitors and vaccines (MENDONÇA, 2007; SHAH, 2007). Synthase (GMPS) is a key enzyme in the purine biosynthetic pathway, where it catalyzes the conversion of to Guanosine Monophosphate (KAMALAKANNAN, 2002). This enzyme is extremely important to guanine synthesis from de novo, and its inhibition could result in depletion of guanine nucleotides. In M. tuberculosis this fact takes importance since these nucleotides are crucial for the viability of the bacillus, since its genome is composed of

V Mostra de Pesquisa da Pós-Graduação – PUCRS, 2010 1035 approximately 65-70% of the guanine and cytosine nucleotides. Furthermore, it has been described for Shigella flexneri and Francisella tularensis that the concomitant deletion of the genes encoding the enzymes GMPS (guaA) and IMPDH (guaB) resulted in samples with significantly attenuated mieloprotective effects (SANTIAGO, 2009). The guaA (Rv3396c) gene encoding GMPS (EC 6.3.5.2) was identified by in the M. tuberculosis H37Rv genome. In this work, we present the initial characterization of a probable GMP synthase enzyme from M. tuberculosis.

Metodology

Two oligonucleotides were designed complementary to the amino-terminal and the carboxy-terminal ends of the M. tuberculosis Rv03396c coding sequence containing restriction sites for NdeI and BamHI, respectively. These primers were used in PCR amplifications of the M. tuberculosis guaA gene using genomic DNA and Pfu DNA polymerase. The PCR (1.578 bp) was purified by electrophoresis in agarose gel, cleaved with NdeI and BamHI, and ligated into the pET-23a(+) expression vector previously digested with the same restriction enzymes. The recombinant plasmid was sequenced to both to confirm the identity of the gene and the absence of undesired mutations. Different E. coli host strains were used for expression tests in LB medium containing the required antibiotics, with or without induction with isopropyl-β-D-tiogalactopiranoside (IPTG). Different temperatures of incubation (30 or 37 °C) were also tested. During incubation, samples (1.0 mL) were collected at various time points, centrifuged, the cells were disrupted by sonication. Soluble and insoluble protein fractions were analyzed by SDS-PAGE and Comassie Brilliant Blue staining. The soluble fraction was subjected to different protein purification protocols. Protein concentration was estimated by the method of Bradford. The molecular mass was confirmed by Mass Spectometry and the oligomeric state determination by size exclusion liquid chromatography.

Results and Discussion

The M. tuberculosis guaA gene was successfully amplified using standard PCR conditions and genomic M. tuberculosis DNA as the template. In addition, it was cloned into the pCR-Blunt vector and subcloned into the pET-23a(+) expression vector. Automated DNA sequencing of the cloned gene showed that the M. tuberculosis guaA sequence does not contain any mutation. Furthermore, the M. tuberculosis GMPS enzyme was successfully

V Mostra de Pesquisa da Pós-Graduação – PUCRS, 2010 1036 expressed in the soluble fraction of E. coli BL21 (DE3) grown at 37ºC in LB medium without IPTG induction. The GMPS protein was successfully purified using a Q-Sepharose Fast Flow column, followed by Superdex-200 size-exclusion chromatography and Butyl HP hydrophobic interaction chromatography, dialysed against 50mM Tris/HCl (pH 7.5), 20 mM

MgCl2, and stored at -80ºC. Molecular mass analysis shows that the purified protein has approximately 55927,6 Da, which corresponds to the probable GMPS from Mycobacterium tuberculosis. The oligomeric state determination by size exclusion liquid chromatography showed that M. tuberculosis GMPS is formed by two identical subunits.

Conclusion

In summary, the guaA gene from M. tuberculosis was successfully amplified, cloned and the encoded protein was expressed in the soluble fraction. The purification protocol was established and yielded approximately 9 mg of homogeneous protein. The molecular mass was in accordance with the theorical value and the protein sowed a dimer in solution. The next steps of this work are focused on the biochemical and kinetic characterization of GMPS, aiming at generating of attenuated samples and the development of new effective vaccines to prevent TB.

Referências MENDONÇA, J. D; ELY, F. PALMA, M. S. FRAZZON, J.; BASSO, L. A.; SANTOS, D. S. Functional Characterization by Genetic Complamentation of aro B-Encoded Dehydroquinate Synthase from Mycobacterium tuberculosis H37Rv and its Heterologous Expression and Purification. Journal of Bacteriology, Sept. 2007, p. 6246-6252.

DUCATI, R.G; NETTO, A. R; BASSO, L. A; SANTOS, D. S. The resumpition of consumption- A review on tuberculosis. Mem. Instit. Oswaldo Cruz, 2006. Vol. 101, N° 7, pp. 697-714.

WHO: Report WHO/HTM/TB/2009.411, (2009). Página da organização Mundial da Saúde. Disponível em http://www.who.int/tb/publications/global_report/2009/pdf/chapter1.pdf. Acesso em: Agosto de 2009.

SHAH, N. S.; WRIGHT, A.; BAI, G. H.; BARRERA, L.; BOULAHBAL, F.; CASABONA, N. M.; DROBNIEWSKI, F.; GILPIN, C.; HAVELKOVÁ, M.; LEPE, R.; LUMB, R.; METCHOCK, B.; PORTAELS, F.; RODRIGUES, M. F.; GERDES, S. R.; DEUN, A. V.; VINCENT, V.; LASERSON, K.; WELLS, C.; CEGIELSKI, P. Worldwide Emergence of Extensively Drug-resistant Tuberculosis. Emerging Infectious Diseases- www.cdc.gov/eid- 2007, vol. 13, No. 3.

KAMALAKANNAN, V.; RAMACHANDRAN, G.; NARAYANAN, S.; VASAN, S.K.; NARAYANAN, P.R. Identification of a novel mycobacterial transcriptional regulator and its involvement in growth rate dependence and stringent control. FEMS Mycrobiology Letters 2002, 209: 261-266.

ZALKIN, H., AND P. NYGAARD. 1996. Biosyntesis of purine nucleotides, p. 561-579. In F. C. Neidhard et al. (ed.), escherichia coli and Salmonela: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.

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