In Vitro Trypanocidal Activity of Antibodies to Bacterially Expressed Trypanosoma brucei Tubulin
-
DP Kateete
,
C Alezuyo
,
A Nanteza
,
C Asiimwe
,
GW Lubega
Abstract
Background: There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth.
Methods: T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450μg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera.
Results: Low antisera dilutions (25%) from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions.
Conclusion: Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes.
Matovu E, Enyaru JCK, Legros D, Schmid C, Seebeck T, et al. Melarsoprol refractory T. b. gambiense from Omugo, north-western Uganda. Tropical Medicine & International Health. 2001; 6: 407-411.
Matovu E, Stewart ML, Geiser F, Brun R, Maser P, et al. Mechanisms of Arsenical and Diamidine Uptake and Resistance in Trypanosoma brucei. Eukaryotic Cell. 2003; 2: 1003-1008.
Fairlamb AH. Chemotherapy of human African trypanosomiasis: current and future prospects. Trends in Parasitology. 2003; 19: 488-494.
Steverding D. The development of drugs for treatment of sleeping sickness: a historical review. Parasites & Vectors. 2010; 3: 15.
Donelson JE. Antigenic variation and the African trypanosome genome. Acta Tropica. 2003; 85: 391-404.
Taylor KA, Mertens B. Immune Response of Cattle Infected with African Trypanosomes. Memórias do Instituto Oswaldo Cruz. 1999; 94: 239-244.
Cross GAM. African trypanosomes in the 21st century: what is their future in science and in health? Int J Parasitol. 2001; 31: 427-433.
Magez S, Caljon G, Tran T, Stijlemans B, Radwanska M. Current status of vaccination against African trypanosomiasis. Parasitology. 2010; 137: 2017-2027.
Giles LN. Exploitation of the Protein Tubulin For Controlling African trypanosomiasis. [PhD Dissertation]. Murdoch University; USA, 2005.
Khonde N, Pepin J, Niyonsenga T, Milord F, De Wals P. Epidemiological evidence for immunity following Trypanosoma brucei gambiense sleeping sickness. Trans R Soc Trop Med Hyg. 1995; 89: 607-611.
Fischer A, Simarro PP, Franco JR, Becher H, Steverding D. Increased trypanolytic activity in sera of sleeping sickness patients after chemotherapy. Tropical Medicine & International Health. 2001; 6: 1070-1074.
Lalmanach G, Boulange A, Serveau C, Lecaille F, Scharfstein J, et al. Congopain from Trypanosoma congolense: drug target and vaccine candidate. Biol Chem. 2002; 383: 739-749.
Mkunza F, Olaho WM, Powell CN. Partial protection against natural trypanosomiasis after vaccination with a flagellar pocket antigen from Trypanosoma brucei rhodesiense. Vaccine. 1995; 13: 151-154.
Rasooly R, Balaban N. Structure of p15 trypanosome microtubule associated protein. Parasitol Res. 2002; 88: 1034-1039.
Lubega GW, Byarugaba DK, Prichard RK. Immunization with a tubulin-rich preparation from Trypanosoma brucei confers broad protection against African trypanosomosis. Experimental Parasitology. 2002; 102: 9-22.
Lubega GW, Ochola DO, Prichard RK. Trypanosoma brucei: anti-tubulin antibodies specifically inhibit trypanosome growth in culture. Exp Parasitol. 2002; 102: 134-142.
Balaban N, Waithaka HK, Njogu AR, Goldman R. Intracellular antigens (microtubule- associated protein copurified with glycosomal enzymes) possible vaccines against trypanosomiasis. J Infect Dis. 1995; 172: 845-850.
Li SQ, Yang WB, Lun ZR, Ma LJ, Xi SM, et al. Immunization with recombinant actin from induces protective immunity against equiperdum and T. b. brucei infection. Parasitology Research. 2009; 104: 429-435.
Kimmel BE, Samson S, Wu J, Hirschberg R, Yarbrough LR. Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense: nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins. Gene. 1985; 35: 237-248.
Seebeck T, Whittaker PA, Imboden MA, Hardman N, Braun R. Tubulin genes of Trypanosoma brucei: a tightly clustered family of alternating genes. Proc Natl Acad Sci U S A. 1983; 80: 4634-4638.
Yansura DG, Bass SH. Application of the E. coli trp promoter. Methods Mol Biol. 1997; 62: 55-62.
Lubega GW, Geary TG, Klein RD, Prichard RK. Expression of cloned beta-tubulin genes of Haemonchus contortus in Escherichia coli: interaction of recombinant beta-tubulin with native tubulin and mebendazole. Mol Biochem Parasitol. 1993; 62: 281-292.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, a laboratory manual. 2nd ed. New York; Cold Harbor Springs Press: 1989.
Nakata Y, Tang X, Yokoyama KK. Preparation of competent cells for high-efficiency plasmid transformation of Escherichia coli. Methods Mol Biol. 1997; 69: 129-137.
MacDonald LM, Armson A, Thompson RCA, Reynoldson JA. Expression of Giardia duodenalis [beta]-Tubulin as a Soluble Protein in Escherichia coli. Protein Expression and Purification. 2001; 22: 25-30.
Wampande EM, Richard McIntosh J, Lubega GW. Classical ligands interact with native and recombinant tubulin from Onchocerca volvulus with similar rank order of magnitude. Protein Expr Purif. 2007; 55: 236-245.
Bollag DM, Rozycki MD, Edelstein SJ. Protein Methods. 2nd ed. New York; Wiley- Liss: 1996.
Harlow E, Lane D. Antibodies, a laboratory manual, 2nd ed. New York; Cold Springs Harbor laboratory press: 1988.
Baltz T, Baltz D, Giroud C, Crockett J. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO J. 1985; 4: 1273-1277.
Buckner FS, Kateete DP, Lubega GW, Van Voorhis WC, Yokoyama K. Trypanosoma brucei prenylated-protein carboxyl methyltransferase prefers farnesylated substrates. Biochem J. 2002; 367: 809-816.
MacRae TH, Gull K. Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei. Biochem J. 1990; 265: 87-93.
Vanhamme L, Pays E. The trypanosome lytic factor of human serum and the molecular basis of sleeping sickness. Int J Parasitol. 2004; 34: 887-898.
Radwanska M, Guirnalda P, De Trez C, Ryffel B, Black S, et al. Trypanosomiasis-induced B cell apoptosis results in loss of protective anti-parasite antibody responses and abolishment of vaccine-induced memory responses. PLoS Pathog. 2008; 4: e1000078.
Jackson AP, Vaughan S, Gull K. Evolution of tubulin gene arrays in Trypanosomatid parasites: genomic restructuring in Leishmania. BMC Genomics. 2006; 7: 261.
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| Issue | Vol 7 No 3 (2012) | |
| Section | Articles | |
| Keywords | ||
| Alpha tubulin Beta tubulin Trypanosoma brucei Protein expression Gene cloning | ||
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