Sensitivty of kinetoplastids to aminoglycoside: Correlation with the 3' region of the small subunit rRNA gene

Kinetoplastidlerin bir seri aminoglikozidlerce büyümesinin engellenmesi için in-vitro kültür sistemleri kullanılmıştır. İlacın çeşitli derişimlerinin varlığında parazitler durgun faza kadar geliştirilmişlerdir. Her ilacın IC-50 değeri kontrolle karşılaştırılarak hesaplanmıştır. Çeşitli leishmanial suşların ilaca olan duyarlılığı sırasıyla G418> Hygromycin> Paromomycin> Neomycin şeklindedir. Buna rağmen bizim koşularımızda Gentamycin ve Kanamycin nonleishmanicidal bulunmuşlardır. İlacın parazitin makrofaj hücre hattındaki hücre içi formuna olan etkisi de denenmiştir. Crithidia spp., ve Blastocrithida culicis gibi kinetoplastidler de denenmiş ve tüm bu ilaçlara dayanıklı bulunmuştur. Bu organizmalar için SSUr-RNA genlerinin 3' bölgeleri için ikincil yapılar oluşturulmuştur. Leishmania’ da ikincil yapıda diğer organizmalarda Paromomycin duyarlılığından sorumlu olduğu bildirilen 1409-1491 (E. coli) pozisyonunda C-6 yerine T-A baz eşleşmesi bulunur. Higromisin duyarlılığı için görevli kök G(1494) olarak kalmıştır. Bu ailenin organizmaları için 3’ loop-stem U-yapıları farklıdır ve bu aminoglikozidlere tüm duyarlılığı belirlemede önemli olabilirler. Bu ise leishmania’ ya özgü ilaçların geliştirilmesinde gerçekçi yaklaşımlar sağlayabilir. Memeli hücrelerinin bu ilaca olan duyarlılıkları yüzünden leishmaniasisin test edilmesinde paramomisinin kullanılmasını önerebiliriz.

Kinetoplastidlerin aminoglikozide duyarlılıkları: rRNA geninin küçük alt birinin 3' bölgesi ile ilgisi

In vitro culture systems were used to assess the growth inhibition of kinetoplastids by a variety of aminoglycosides. The parasites were allowed to grow to the stationary phase in the presence of varying concentrations of the drugs. The IC-50 for every drug was calculated by comparison with the control. The sensitivity of various leishmanial strains to these drugs was in the order of: G418 > hygromycin > paromomycin > neomycin; however, under our assay conditions gentamycin and kanamycin were nonleishmanicidal. The effects of the drugs on the intracellular form of the parasite in the macrophage cell line were also tested. Other kinetoplastids, such as Crithidia spp., and Blastocrithidia culicis, were tested and showed resistance to all these drugs. Secondary structures for the 3’ region of the SSUrRNA genes for these organisms were constructed, and correlations between drug sensitivity and the secondary structures are presented. In Leishmania it is a T-A pair in the secondary structure instead of a C-G pair at position 1409-1491 (E. coli), as reported in other organisms, which is responsible for paromomycin sensitivity. The residue responsible for hygromycin sensitivity remained G(1494). The 3’ loop-stem U-structures are different for organisms in this family, which might be of significance in determining the overall sensitivity to these aminoglycosides. This might provide rational approaches to the development of drugs specific for Leishmania. Because of the sensitivity of mammalian cells to this drug, we suggest that paromomycin may be used for testing against leishmaniasis.

PDF

___

1. Croft SL, Urbina JA, Brun R. Chemotherapy of human leishmaniasis and trypanosomiasis. In: Trypanosomiasis and Leishmaniasis (eds. G Hide, JC Mottram, GH Coombs & PH Holmes) CAB International, Wallingford, pp. 245-257, 1997.
2. Thakur CP, Kanyok TP, Pandey AK et al. Treatment of visceral leishmaniasis with injectable paromomycin (aminosidine). An open-label randomized phase-II clinical study. Transactions of the Royal Society of Tropical Medicine and Hygiene 94: 432- 433, 2000.
3. Berman JD, Badaro R, Thakur CP et al. Efficacy and safety of liposomal amphotericin B (AmBisome) for visceral leishmaniasis in endemic developing countries. Bulletin of the World Health Organization 76: 25-32, 1998.
4. Meyerhoff A. US Food and Drug Administration approval of AmBisome (liposomal amphotericin B) for treatment of visceral leishmaniasis. Clinical Infectious Diseases 28: 42-48, 1999.
5. Jha TK, Sundar S, Thakur CP et al. Miltefosine, an oral agent, for the treatment of Indian visceral leishmaniasis. New England Journal of Medicine 341: 1795-1800, 1999.
6. Sherwood JA, Gachihi GS, Muigai RK et al. Phase 2 efficacy trial of an 8-aminoquinoline (WR6026) for treatment of visceral leishmaniasis. Clinical Infectious Diseases 19: 1034-1039,1994.
7. El-On J, Halvey S, Grunwald MH et al. Topical treatment of Old World cutaneous leishmaniasis caused by Leishmania major: a double-blind study. Journal of the American Academy of Dermatology 27: 227-231, 1992.
8. Asilian A, Jalayer T, Whitworth JAG et al. A randomized, placebo-controlled trial of a two-week regimen of aminosidine (paromomycin) ointment for treatment of cutaneous leishmaniasis in Iran. American Journal of Tropical Medicine and Hygiene 53: 648-651, 1995.
9. Grogl M, Schuster BG, Ellis WY et al. Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (aminosidine) and gentamicin. Journal of Parasitology 85: 354-359, 1999.
10. Soto J, Toledo J, Gutierrez P et al. New approaches to cutaneous leishmaniasis treatment in Latin America. Abstracts XV International Congress for Tropical Medicine and Malaria, Cartagena, Colombia 20-25 August 2000, vol. 1, p.60.
11. Moazed D, Noller HF. Interaction of antibiotics with functional sites in 16s ribosomal RNA. Nature 327: 389-394, 1987.
12. El-On J, Halvey S, Grunwald MH et al. Topical treatment of Old World cutaneous leishmaniasis caused by Leishmania major: A double-blind study. Journal of the American Academy of Dermatology 27: 227-231, 1992.
13. El-On J, Hamburger AD. Topical treatment of new and old world leishmaniasis in experimental animals. Transactions of the Royal Society of Tropical Medicine and Hygiene 81: 734-737, 1987.
14. Croft SL. Monitoring drug resistance in leishmaniasis, Tropical Medicine and International Health 6(II): 899-905, 2001.
15. Ogata K, Kurahashi A, Kenmochi N et al. Role of 5SrRNA as positive effectors of some aminoacyl-tRNA synthetases in macromolecular complexes, with specific reference to methionyl-tRNA synthetase. J Biochem 110: 1037-44, 1991.
16. Ogata K, Ohno R, Morioka S et al. Further study on association of 5SrRNA-L5 protein complex and methionyl-tRNA to methionyl-tRNA synthetase in the macromolecular aminoacyltRNA synthetase complex. J Biochem 120: 869-80, 1996.
17. Ogata K, Kurahashi A, Ohno R et al. Interaction of 5SrRNA-L5 protein complex, methionyl-tRNA, and methionyl-tRNA synthetase in the macromolecular ARS complex. J Biochem 117: 750-7, 1995.
18. Ogata K, Kurahashi A, Tanaka S et al. Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant. J Biochem (Tokyo), 110, 1030-1036,1991.
19. Ogata K, Kurahashi A, Nishiyama C et al. Presence of role of the 5SrRNA-L5 protein complex (5SRNP) in the threonyl- and histidyl-tRNA synthetase complex in rat liver cytosol. Biochim Biophys Acta 1218: 388-400, 1994.
20. Schlegal M. Protist evolution and phylogeny as discerned from small subunit ribosomal RNA sequence comparisons. Eur J Protistol 27: 207-219, 1991.
21. Neefs JM, Van de Peer Y, Hendricks L et al. Compilation of small ribosomal subunit RNA sequences, Nucleic Acids Res 18: 2237,1990.
22. Hutchin T. A molecular basis for human hypersensitivity to amino glycoside antibiotics nucleic acids. Res 21: 4174-4179,1993.
23. Chance ML. New developments in the chemotherapy of leishmaniasis. Ann Trop Med Parasitol 89 (Suppl. 1): 37-43,1995.
24. Spangler EA, Blackburn EH. The nucleotide sequence of the 17S ribosomal genes of tetrahymena termophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin. J Biol Chem 260: 6334-6340, 1985.
25. Edlind TD. Susceptibility of Giardia lamblia to aminoglycoside protein synthesis inhibitors; Correlation with rRNA structure. Antimicrobial agents’ chemotherapy 33: 484-488, 1989.
26. Sweeney R, Chen L, Yao MC. Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila. Mol Cell Biol 13: 4814-4825, 1993.
27. Fong D, Man-Ying C, Rodriguez R, et al. Paromomycin resistance in L. tropica: Lack of correlation with mutation in the small subunit ribosomal RNA gene. Am J Trop Med Hyg 51: 758-766, 1991.
28. Maarouf M, Lawrence F, Brown S et al. Biochemical alterations in paromomycin-treated Leishmania donovani promastigotes. J Parasitology Research, 83: 198-202, 1997.
29. Hermann T, Schmid B, Heumann H, et al. A three-dimensional working model for a guide RNA from Trypanosoma brucei. Nucleic Acids Res 25: 2311-2318, 1997.
30. Moazed D, Stem S, Noller HFJ. Mol Biol 187: 399-416, 1986.
31. Li M, Tzagoloff A, Underblink-Lyon K et al. Identification of the Paromomycin resistance mutation in the ssrRNA gene of yeast mitochondria. J Biol Chem 257: 5921-5928, 1982.