Browsing by Author "Lubega, George W."
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Item Anti-Paraflagellar Rodc Antibodies Inhibit the In-Vitro Growth of Trypanosoma Brucei Brucei(American Academic Scientific Research Journal for Engineering, Technology, and Sciences, 2018) Mukisa, Ambrose; Aguttu, Claire; Lubega, George W.; Kyambadde, Joseph; Alibu, Vincent P.; Vuzi, Peter C.Paraflagellar rod (PFR), a conserved structure expressed in all lifecycle stages of the order kinetoplasida except in the amastigotes is vital for the parasites survival. In T.b.brucei, the PFR protein has two major components, PFRc and PFRa with molecular mass 73kDa and 68kDa respectively. Experimental evidences implicate the PFR protein as a highly immunogenic and protective antigen. However, its immunogenic properties underlying its suitability as vaccine candidate has not been adequately investigated in-vitro. This study aimed to demonstrate the growth inhibitory potential of PFR protein against T.b.brucei parasites in–vitro. Antibodies against a recombinant form of the PFRc protein were produced and used to generate immune response. A deoxyribonucleotide (DNA) segment of approximate 672bp encoding the PFRc protein component was amplified using polymerase chain reaction (PCR), cloned and expressed in E.coli (BL21) cells. A 200 μg portion of the purified PFRc protein mixed with 100μl Freund's complete adjuvant (FCA) was used to immunize rabbits. An antibody titre of 2.5 x 104 reciprocal dilutions was obtained following three immunisation boosts, spaced two weeks apart. Western blot analysis showed that rabbit anti-PFRc antibodies recognised specifically a 25kDa protein corresponding to the estimated size of the expressed PFRc protein. 25% of purified anti-rabbit IgG antibodies were able to inhibit ~70% T.b.brucei parasite in vitroItem Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south‐western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci(Transboundary and emerging diseases, 2020) Nanteza, Anne; Obara, Isaiah; Kasaija, Paul; Mwega, Elisa; Kabi, Fredrick; Salih, Diaeldin A.; Njahira, Moses; Njuguna, Joyce; Odongo, David; Bishop, Richard P.; Skilton, Rob A.; Ahmed, Jabbar; Clausen, Peter‐Henning; Lubega, George W.Theileria parva is a tick‐transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti‐transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south‐western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen‐encoding genes that are targets of CD8+T‐cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south‐western Uganda. Cattle‐derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.Item Identification of coding sequences from a freshly prepared Trypanosoma brucei brucei expression library by polymerase chain reaction(International Journal of Biochemistry and Molecular Biology, 2013) Okalang, Uthman; Nanteza, Ann; Matovu, Enock; Lubega, George W.Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries. This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei) expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism. Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under conditions of limited resources among developing countries.Item Preliminary evaluation of a Trypanosoma brucei FG-GAP repeat containing protein of mitochondrial localization [version 1; peer review: 1 approved, 1 approved with reservations](AAS Open Research, 2019) Namyanja, Monica; Xu, Zhi-Shen; Mugasa, Claire Mack; Lun, Zhao-Rong; Matovu, Enock; Chen, Zhengjun; Lubega, George W.Trypanosoma brucei, a causative agent of African Trypanosomiasis, is known to cross the blood brain barrier during the second stage of the disease. It was previously suggested that this parasite crosses the blood brain barrier in a manner similar to that of lymphocytes. This would imply that trypanosomes possess integrins that are required to interact with adhesion molecules located on the blood brain barrier microvascular endothelial cells, as a first step in traversal. To date, no T. brucei integrin has been described. However, one T. brucei putative FG-GAP repeat containing protein (typical of integrins) encoded by the Tb927.11.720 gene, was predicted to be involved in cell-cell/cell-matrix adhesion. Therefore, this study sought to characterize a putative FG-GAP repeat containing protein (FG-GAP RCP) and to determine its cellular localization as a basis for further exploration of its potential role in cell-cell or cell-matrix adhesion. Methods: In this study, we successfully cloned, characterized, expressed and localized this protein using antibodies we produced against its VCBS domain in T. brucei. Results: Contrary to what we initially suspected, our data showed that this protein is localized to the mitochondria but not the plasma membrane. Our data showed that it contains putative calcium binding motifs within the FG-GAP repeats suggesting it could be involved in calcium signaling/binding in the mitochondrion of T. brucei. Conclusion: Based on its localization we conclude that this protein is unlikely to be a trypanosomal integrin and thus that it may not be involved in traversal of the blood brain barrier. However, it could be involved in calcium signaling in the mitochondrion.