Investigation into the major surface proteases of African trypanosomes.
Delport, Alexandré Marie Chaplin.
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The unicellular parasite of the genus Trypanosoma infects a number of mammalian species including livestock and humans. In sub-Saharan Africa three main parasitic species cause disease: Trypanosoma brucei, T. congolense and T. vivax. The lack of sensitive diagnosis and increased drug resistance leaves an avenue in trypanosome research for exploring novel virulence factors as diagnostic and chemotherapeutic agents. The work reported in this dissertation involved investigation of the identified virulence factor, the Major Surface Protease (MSP), of African trypanosomes. The MSPs comprise a group of metalloproteases which have been found in over 13 Leishmanian species, T. cruzi, T. brucei and T. congolense as well as other Kinetoplastids. In this study, putative M8 metalloprotease sequences were also identified in the T. vivax genome. These putative sequences were grouped into four classes of protein, TvMSP-A, -C, -D and -E by phylogenetic comparison with other MSPs. Three-dimensional modelling showed high structural identity with leishmanolysin from Leishmania major. The T. vivax MSP sequences were used, in conjunction with T. brucei and T. congolense sequences, to select immunogenic peptide regions to produce anti-peptide antibodies. Three peptides were selected with the intention to 1) detect both TbMSP-B and TcoMSP-B (peptide Tb/TcoMSP:303-314, cross-species), 2) detect only TbMSP-C (peptide TbMSP:400-412) and 3) detect only TvMSP-C (peptide TvMSP:686-697). They were used to generate two types of detection molecules: complete IgY anti-peptide antibodies and single chained fragments (scFvs), with the capability to detect the peptide in an ELISA format. Single scFv expressing E. coli colonies were successfully selected and shown to detect two (Tb/TcoMSP:303-314 and TbMSP:400-412) of the three peptides. The anti-peptide antibodies, produced in chickens, were used to successfully detect native MSP within T. brucei and T. congolense parasite lysates; however, cross-reactivity between species was seen. The T. brucei MSP-C class of protease was successfully cloned, expressed and purified, although, numerous truncated proteins and gene mutations occurred [truncated (t)TbMSP-C]. The expression constructs rTbMSP-C and rTcoMSP-C were hence synthesised. These two enzymes were successfully expressed and purified and were shown to form high molecular weight multimers. Furthermore, the enzymes were able to cleave the peptide substrate H-Suc-Leu-Tyr-AMC with acidic pH optima and activity was inhibited with metalloprotease inhibitors, EDTA and 1,10 phenanthroline. The successful detection of rTcoMSP-C by T. congolense infected cattle sera was also observed. tTbMSP-C, rTbMSP-C and rTcoMSP-C were detected with the chicken anti-peptide antibodies and it was again found that these antibodies cross-reacted with different species MSPs. The high identity shared between MSPs from all Trypanosoma species made selecting species-specific antibodies difficult. Further work to detect native MSP-C protease within infected sera or blood would give a definitive conclusion of its use in diagnostics. Moreover, antibodies that detect just T. brucei and T. congolense still need to be produced. Preliminary activity assays were performed on rTbMSP-C and rTcoMSP-C but, additional research on the kinetics of these proteases is still needed. In summary, it was shown that MSPs have the potential to be novel diagnostic markers especially for cross-trypanosomal species detection. Furthermore, activity of rTbMSP-C and rTcoMSP-C has substrate cleavage specificity and pH optima that are comparable with leishmanolysin.