Browsing by Author "Kangethe, Richard Thiga."

Now showing 1 - 1 of 1

Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes.
(2011) Kangethe, Richard Thiga.; Coetzer, Theresa Helen Taillefer.
African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. T. congolense, T. vivax and T. brucei brucei cause nagana in cattle. The variable nature of the parasite surface coat has hindered the development of an effective vaccine. An option for developing vaccines and chemotherapeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an “anti-disease” approach. Two pathogenic factors released during infection are oligopeptidase B (OPB) and TcoCATL (congopain). TcoCATL, a major lysosomal cysteine peptidase, is a member of the papain family C1 cysteine peptidases. RNA interference (RNAi) was used to down-regulate the expression of TcoCATL in T. congolense IL3000 TRUM183:29-13 parasites in vivo during mouse infections. TcoCATL RNAi was monitored in infected mouse blood by comparing the hydrolysis of Z-Phe-Arg-AMC and parasitaemia between mice in which RNAi was induced and control mice. Mice infected with parasites induced for TcoCATL RNAi had lower parasitaemia when compared to control mice. An attempt was also made at deleting the entire CATL gene array in both T. congolense IL3000 and T. brucei 427 Lister strains. The second pathogenic factor studied, OPB, is a cytosolic trypanosomal peptidase that hydrolyses peptides smaller than 30 amino acid residues, C-terminal to basic residues. In order to evaluate the role that OPB play during disease, RNAi was also applied to knock-down the expression levels of OPB in T. brucei T7T and T. congolense IL3000 TRUM183:29-13 strains (TbOPB and TcoOPB respectively). Oligopeptidase B null mutant strains (Δopb) were also generated in T. brucei brucei Lister 427. An attempt was also made to generate OPB null mutants in T. congolense IL3000 parasites. Western blot analysis of the knock-down experiments using chicken anti-TcoOPB peptide IgY showed that only TbOPB levels were reduced in T. brucei T7T parasites induced for RNAi when compared to TcoOPB RNAi induced cultures. Quantitative assessment of a fourteen day induction experiment for OPB RNAi in T. brucei showed an 87% reduction in TbOPB levels when compared to levels on day one. There was no growth effect observed in T. brucei parasites cultured in vitro and induced for TbOPB RNAi. It was concluded that TbOPB is not necessary for the in vitro survival of T. brucei parasites, thus making the generation of OPB null mutants possible. Δopb T. brucei parasites were successfully generated and grew normally in vitro and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed Δopb parasites in extra vascular regions showing that T. brucei OPB (TbOPB) is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatin gel analysis of Δopb null mutants and wild type strains showed an increase in cysteine peptidase activity. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by knocking out TbOPB, and a significant increase of T. brucei prolyl oligopeptidase (TbPOP) activity was observed. However, western blot analysis did not show any increase of TbPOP protein levels in Δopb parasites, suggesting that either TbOPB is responsible for generating an endogenous inhibitor for TbPOP or that another POP-like enzyme might compensate for a loss in OPB activity in Δopb null mutants. This study made a significant contribution to an understanding of the interplay between different trypanosomal peptidases that are important pathogenic factors in trypanosomosis. It highlights the need to simultaneously target several trypanosomal peptidases to develop an effective vaccine or chemotherapeutic agents for African animal trypanosomosis.