George C. Hill, Ph.D.
Associate Dean for Diversity
Vanderbilt University Medical Center
301 Light Hall
Nashville, TN 37232
Molecular and biochemical basis of differentiation and characterization of the trypanosome alternative oxidase in African trypanosomes, the causative agent of African trypanosomiasis.
Trypanosoma brucei is the causative agent of African trypanosomiasis and is a re-emerging infection in sub-Sahara Africa. In both the bloodstream and insect vector stages, there is a unique cyanide insensitive, salicylhydroxamic acid (SHAM) sensitive alternative oxidase that is expressed. This trypanosome alternative oxidase (TAO) is the only oxidase present in the bloodstream forms. However, in addition to the TAO, the procyclic trypanosomes contain an established cytochrome system. Our laboratory has characterized the electron transport systems in both systems.
We have identified the TAO as a potential target for trypanocidal drugs and begun to synthesize compounds that inhibit the TAO in vitro and in vivo. In the bloodstream stages, this unique cyanide insensitive, salicylhydroxamic acid (SHAM) sensitive alternative oxidase is expressed as the only oxidase present in the bloodstream forms. RNA interference (RNAi) studies have been performed that demonstrate that the TAO is essential for survival. RNAi plasmid constructs using p2T7ti_B/GFP were made, linearized and transfected into T. brucei 427 bloodstream trypomastigotes. After transfection, selection, and induction, the TAO message in bloodstream transfectants was markedly reduced in the presence of tetracycline after 48 hours. Importantly, immunoblot analysis revealed that there was also approximately a 1.5 fold decrease in the TAO protein level in these bloodstream cells after RNAi induction. In vivo studies revealed that there was a five-fold reduction in the parasitemia levels in RNAi induced mice and rats infected with T. brucei RNAi-transfected cells and their life was extended, clearly demonstrating the proof of principal that the TAO can serve as an effective target for trypanocidal drugs. There was also a five-fold reduction in the TAO protein level isolated from parasites infected in RNAi induced mice as compared to the control. Additionally, respiration assays of RNAi-transfected T. brucei isolated from rats revealed that there was 100% SHAM inhibition in cells induced with tetracycline.
The prenylated salicylic acid derived substructural motif is the key chemical and/or structural feature important for inhibition of TAO within the limited series of exploratory compounds we have investigated. It has also been demonstrated that synthesizing carbohydrate-linked analogs improves solubility properties. Further testing of compounds in this category is being pursued as well as studies testing the effectiveness of these compounds in laboratory models of trypanosomiasis.
Current efforts are also being devoted to the purification of the TAO and subsequent crystallization of this essential enzyme in African trypanosomes, the causative agent of African sleeping sickness.
Minu Chaudhuri, Ph.D. - Meharry Medical College, Department of Microbiology, Nashville, TN
Kelly Chibale, Ph.D. - University of Cape Town, Department of Chemistry, Cape Town, South Africa
- Lepesheva, GI, Ott, RD, Hargrove, TY, Kleshchenko, YY, Schuster, I, Nes, WD, Hill, GC, Villalta, F, Waterman, MR Sterol 14alpha-demethylase as a potential target for antitrypanosomal therapy: enzyme inhibition and parasite cell growth. Chem Biol, 14(11), 1283-93, 2007.
- Lepesheva, GI, Zaitseva, NG, Nes, WD, Zhou, W, Arase, M, Liu, J, Hill, GC, Waterman, MR CYP51 from Trypanosoma cruzi: a phyla-specific residue in the B'' helix defines substrate preferences of sterol 14alpha-demethylase. J Biol Chem, 281(6), 3577-85, 2006.
- Ott, R, Chibale, K, Anderson, S, Chipeleme, A, Chaudhuri, M, Guerrah, A, Colowick, N, Hill, GC Novel inhibitors of the trypanosome alternative oxidase inhibit Trypanosoma brucei brucei growth and respiration. Acta Trop, 100(3), 172-84, 2006.
- Walker, Robert, Saha, Lipi, Hill, George C, Chaudhuri, Minu The effect of over-expression of the alternative oxidase in the procyclic forms of Trypanosoma brucei. Mol Biochem Parasitol, 139(2), 153-62, 2005.
- Lepesheva, Galina I, Nes, W David, Zhou, Wenxu, Hill, George C, Waterman, Michael R CYP51 from Trypanosoma brucei is obtusifoliol-specific. Biochemistry, 43(33), 10789-99, 2004.
- Ajayi, Wilfred U, Chaudhuri, Minu, Hill, George C Site-directed mutagenesis reveals the essentiality of the conserved residues in the putative diiron active site of the trypanosome alternative oxidase. J Biol Chem, 277(10), 8187-93, 2002.
- Chaudhuri, Minu, Sharan, Rita, Hill, George C Trypanosome alternative oxidase is regulated post-transcriptionally at the level of RNA stability. J Eukaryot Microbiol, 49(4), 263-9, 2002.
- Chaudhuri, M, Ajayi, W, Hill, G C Biochemical and molecular properties of the Trypanosoma brucei alternative oxidase. Mol Biochem Parasitol, 95(1), 53-68, 1998.
- Chaudhuri, M, Hill, G C Cloning, sequencing, and functional activity of the Trypanosoma brucei brucei alternative oxidase. Mol Biochem Parasitol, 83(1), 125-9, 1996.
- Chaudhuri, M, Ajayi, W, Temple, S, Hill, G C Identification and partial purification of a stage-specific 33 kDa mitochondrial protein as the alternative oxidase of the Trypanosoma brucei brucei bloodstream trypomastigotes. J Eukaryot Microbiol, 42(5), 467-72, 1995.
- Wirtz, E, Sylvester, D, Hill, G C Characterization of a novel developmentally regulated gene from Trypanosoma brucei encoding a potential phosphoprotein. Mol Biochem Parasitol, 47(1), 119-28, 1991.
- Sylvester, D A, Hill, G C A comparison of the amino acid sequences of Crithidia fasciculata and Trypanosoma rhodesiense cytochromes c. J Parasitol, 76(3), 445-7, 1990.
- Bass, H. S. and Hill, G. C "Approaches to the solubilization and partial purification of glycerol-l-3- phosphate oxidase from mitochondria from trypanosomes." Zoological Science , 735-42, 1990.
- Bass, H S, Njogu, R M, Hill, G C Solubilization and partial purification of glycerol-3-phosphate oxidase from mitochondria of Trypanosoma brucei. Exp Parasitol, 70(4), 486-9, 1990.
- Hill, G. C. Benavides, G., Chaudhuri, S. and Sylvester, D "Expression of mitochondrial and nuclear genes during differentiation of African trypanosomes." Molecular Strategies of Parasitic Invasion (ed. Agabian. N. Goodman H. and Noguiera: NY) Anal Liss, Inc., New York, NY, 1987.
- LeFebvre, R. B. and Hill, G. C "The effects of Trypanosoma brucei differentiating bloodstream trypomastigotes and established procyclic trypomastigotes when grown in the presence of respiratory inhibitors." J. Parasitol, 72481-483, 1986.
- LeFebvre, R. B. and Hill, G. C "Trypanosoma brucei rhodesiense: Mitochondrial proteins of bloodstream and procyclic trypomastigotes." Exper Parasitol., 6285-91, 1986.
- Tielens, A. G. M. and Hill, G. C "The solubilization of a SHAM sensitive, cyanide insensitive ubiquinol oxidase from Trypanosoma brucei." J. Parasitol., 71384-386, 1985.
- Johnson, B. J. B., Hill, G. C., and Donelson, J. E "The maxicircle of Trypanosoma brucei kinetoplast DNA encodes apocytochome c." Mol. Biochem. Parasitol. , 13135-146, 1984.
- Bienen, E. J., Hill, G. C. and Shin, K "Elaboration of mitochondrial function during Trypanosoma brucei differentiation." Mol. Biochem. Parasitol., 775-86, 1983.
- Folkers, K. Vadhanavikit, S., Bueding, E., Hill, G. and Whittaker, C "Identification of forms of coenzymes Q in organisms including some causing tropical diseases." Chemiker. Zatung, 07131-133, 1983.
- Njogu, R. M. Whittaker C. and Hill, G. C "Evidence for a branched electron transport chain Trypanosoma brucei." Biochem. Parasitol., 113-39, 1980.
- Hill, G. C., Shimer, S., B. Caughey and Sauer "Growth of infective forms of Trypanosoma rhodesiense in vitro, the causative agent of African trypanosomiasis." Science, 202763-765, 1978.
- Hill, G. C "Electron transport systems in Kinetoplastida." Biochem. Biophys. Acta , 456149-193, 1976.