Sains Malaysiana  37(1): 101-106(2008)

Penyaringan Domain Adp-Ribosilasi Gen Toksin

Daripada Burkholderia pseudomallei Virulen

(Screening Adp-Ribosilation Domain of Toxin Gene From

Burkholderia pseudomallei Virulent)

Shahrul Hisham Zainal Ariffin, Rahmah Mohamed,

Zulkeflie Zamrod & Mohammad Noor Embi

Pusat Pengajian Biosains Dan Bioteknologi

Fakulti Sains Dan Teknologi, Universiti Kebangsaan Malaysia

43600 Bangi, Selangor, D.E., Malaysia

Diserahkan:  6 Jun 2007 / Diterima :  26 Jun 2007

ABSTRAK

Bahagian aktif bagi enzim toksin bakteria daripada Burkholderia pseudomallei, Pseudomonas aeruginosa dan difteria merupakan domain ADP-ribosilasi. Domain ini didapati terpelihara di antara ketiga-tiga mikroorganisme. Di dalam kajian ini, domain ADP-ribosilasi Burkholderia pseudomallei telah diamplifikasi daripada genom B.  pseudomallei virulen dengan menggunakan pencetus-pencetus yang dibina berdasarkan kepada jujukan domain ADP ribosilasi Pseudomonas aeruginosa. Hasil DNA amplifikasi ditulenkan dan digunakan sebagai prob (HPCR2) untuk menyaring DNA selitan daripada B. pseudomallei yang diklonkan ke dalam vektor pengekspresan pSport-I. Objektif kajian ini adalah untuk menyaring lapan klon yang positif hasil daripada penyaringan awal melalui pendekatan immunoblot menggunakan antitoksin daripada arnab. Penyaringan ini juga melibatkan tiga klon yang tidak memberikan isyarat positif semasa penyaringan secara immunoblot. Keputusan menunjukkan hanya satu klon (L31) daripada lapan klon immunoblot positif mempunyai domain ADP-ribosilasi. Penjujukan DNA separa klon L31 secara manual melibatkan dua pencetus menghasilkan jujukan sepanjang 450pb. Analisis selanjutnya mendapati daripada enam kemungkinan translasi kepada polipeptida hanya satu polipeptida wujud yang tidak mempunyai sebarang kodon penamat pada jujukan kodonnya.

Kata kunci: Burkholderia pseudomallei; penjujukan manual; domain ADP ribosilasi

ABSTRACT

The active part of bacterial toxin enzymes from Burkholderia pseudomallei, Pseudomonas aeruginosa and difteria is ADP-ribosilation domain. This particular domain is conserved among all three microorganisms. The ADP-ribosilation domain from B. pseudomallei was PCR amplified using B. pseudomallei virulent genome as a template and primers that were generated based on Pseudomonas aeruginosa ADP-ribosilation sequences. The amplification product was purified and used as a probe (HPCR2) to screen the DNA inserts from B. pseudomallei that have been cloned into expression vector pSport-I. The objective of this study was to screen eight positive clones obtained from early screening using immunoblot and antitoxin generated from rabbit.  We also screened three clones that produced negative results during immunoblot screening. The results showed that only one clone (L31) from eight positive clones carried the ADP-ribosilation domain. Manual DNA sequencing of L31 using two primers was able to generate 450bp. Further analysis showed that only one polypeptide from six posibilities was able to be translated without existence of any stop codon.

Keywords: Burkholderia pseudomallei; manual sequencing; ADP-ribosilation domain

RUJUKAN/REFERENCES 

Cheng, A.C. & Currie, V.J. 2005. Melioidosis: epidemiology, pathophysiology and management. Clin. Microbiol Rev. 18(2): 383-416.

Chaowagul, W., White, N.J., Dance, D.A.B., Wattanagon, T., Naigowit, P., Davies, T.W. Looaresuwan, E.S. & Pitakwatchara, N. 1989. Melioidosis: a major cause of community acquired septicemia in Northern Thailand. J. Infect. Dis. 159: 890-899.

Dance, D.A.B.  1991. Melioidosis: the tip of iceberg? Clin. Microbial. Rev. 4: 52-60.

Dance, D.A. 2000. Melioidosis as an emerging global problem. Acta Trop. 74: 115-119.

Dannenberg, A.M. & Scott, E.M. 1958. Melioidosis: pathogenesis and immunity in mice and hamster. A studies of with virulent strains. Pseudomonas pseudomallei. J. Exp. Med. 170: 153-166.

Domenighini, M., Montecucco, C., Ripka, W.C & Rappuolli, R. 1991. Computer modeling of the NAD binding site of ADP-ribosylating toxins: Active site structure an d mechanism of NAD-binding. Mol. Microbial. 5(1): 23-31.

Everett, E.D. & Nelson, R.A. 1975. Pulmonary melioidosis observation on thirty-nine cases. Am. Rev. Respir. Dis. 112: 331-340.

Gray, B.L., Smith, D.H., Baldridge, J. S., Harkons, R.N., Vasil M.L., Chen, . Y. & Heynexer, H. L. 1984. Cloning, nucleotides sequence and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc.  Natl. Acad. Sci USA. 81: 2645-2649.

Jones, M. 1991. Application enzyme linked immunosorbent assay for the diagnosis of melioidosis. Tesis Sarjana Sains. North East Wales Institutes.

Lertpatanasuwan, N., Sermsri, K., Petkaseam, A., Trakulsomboon, S., Thamlikutkul V. & Suputtamongkol, Y. 1999. Arabinose-positive B. pseudomallei infectious in humans. Case Report Cli. Infect. Dis. 28: 927-928.

Levine, H.B. & Wolochow, H. 1960. Occurance of poly-b-hydroxybutyrate in pseudomonas pseudomallei. J. Bacteriol. 79:  305-306.

Mohamed, R., Nathan, S., Noor Embi, M., Rajah, N. & Ismail, G. 1989. Inhibition of macromolecular synthesis in cultured macrophage by P. pseudomallei exotoxin. Microbiol. Immnol. 33: 811-819.

Mohamed, R., Ismail, G., Osma, A., Idris, S.R., Chandrasakaran, S., Suhaima, A., Sivanandan S.P., Ahmad, H., Karuppiah, A & Noor Embi, M. 1991. Melioidosis: Ke arah pengwujudan diagnosis yang berkesan. Penyelidikan Dua Dekad UKM: 23-24.

Mohamed, R., Lavin, M.F. & Noor Embi, M. 1994. Identification of an ADP-ribosyl domain in Pseudomonas pseudomallei genome by PCR. Dlm. Melioidosis. Prevailing problems and future direction. Puthucheary, S. D. dan Yasmin A. M. (pnyt.) pp102. Melioidosis.

Nyonya, R., Ghazally, I., Noor Embi, M & Othman, O. 1985b. Biological activity of ammonium sulfate precipitated toxic material(s) of P. pseudomallei. Proceeding of National Biology Conference II.

Puthucheary, S.D., Lim, H.P. & Yap, P.K. 1981. Acute septicaemic melioidosis: a report of seven cases. Troop. Geogr. Med. 33: 19-22.

Smith, M.D., Angus, B.J., Wuthiekanun, V. & White, N.J. 1997. Arabinose assimilation defines a nonvirulent biotype of B. pseudomallei. Infect. Immun. 65: 4319-4321.

Thomas, J.E. & Ashdown, L.R. 1989. Melioidosis and latent infection. Med. J. Austral. 151: 298-299.

Van Ness, B.G., Howard, J.B. & Bradley, J.W. 1980. ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-dipthamide and its hydrolysis product. J. Biol. Chem. 255: 10710-10716.

Zhang, P., Nelson, S., Simmer, W.R. & Spitzer, J.A. 1997. Acute ethanol intoxification suppresses the pulmonary inflammatory response in rats challenged with intrapulmonary endotoxin. Alcohol. Clin. Exp. Res. 21: 773-775.