Characterization and Functional analysis of tricorn interacting factor 3 in Trypanosoma brucei brucei as potential drug targets against African Trypanosomiasis

Trypanosomes are protozoans causing African trypanosomiasis, a neglected tropical disease in Africa affecting humans and animals. Despite advancement in African Trypanosomiasis research, the disease continues to threaten millions of people and animals in Sub-Saharan Africa. Control methods have focused on the use of drugs which have adverse effects and vector control methods which have proved to be ecologically unsustainable while vaccines are still not available due to antigenic variation. Studies have shown that the pathogenesis of Trypananosoma brucei brucei infections is parasite driven and various classes of Trypananosoma brucei brucei peptidases have been studied and implicated as virulence factors. Proteasomal degradative pathway is the core non-lysosomal protein degradation machinery in Trypanosoma brucei brucei and together with its accessory proteins, they are responsible for cell quality control. Tricorn protease and its interacting factors, F1, F2,and F3 act in vivo downstream of the proteasome and have been implicated in cell maintenance, defense, growth and development in many pathogenic organisms. The aim of this study was to determine the presence and investigate role of tricorn interacting factor 3 orthologs in Trypanosoma brucei brucei endocytic system. A combination of bioinformatics programs including PSI-BLAST, Hidden Markov Models and orthology clustering were used to identify tricorn interacting factor 3 orthologs. Molecular characterization involved generation of transcripts through PCR and mRNA detection by reverse transcription with gene specific primers. Real time PCR using SYBR green detection method was used to determine the expression levels of genes encoding tricorn interacting factor 3 orthologs in the bloodstream form of Trypanosoma brucei brucei.Cloning and expression of tricorn interacting factor 3 orthologs was done and expressed proteins were analyzed through SDS-PAGE. Functional analysis through RNAi involved ligation of the dsRNA targeting Tb927.11.3570 and Tb927.3.4750/90 between the two T7 promoters of linearized p2T7-177 vector to generate recombinant plasmids for transfection. Transfection was done through electroporation and RNAi was induced by tetracycline disodium. Relative expression levels of Tb927.11.3570 and Tb927.3.4750/90 in transgenic parasites was determined through qRT-PCR and parasite densities were determined by Neubuaer chamber. A total of 24 hits were identified and were categorized as proteins with isolated tricorn-like domains (21) and tricorn interacting factor 3 orthologs (3). Two (2) of the tricorn interacting factor 3 orthologs appeared to represent a gene duplication process. The 21 proteins contained isolated tricorn protease domains mainly; tricorn protease N-terminal domain, tricorn protease domain 2 and PDZ domains which are known to have no catalytic activity. Further analysis of the tricorn interacting factor 3 orthologs revealed conservation of the zinc binding motif ({HEXXH (18X) E}, the GXMEN motif specific for M1 family as well as the N-terminal substrate anchoring residue, glutamate and the proton acceptor, tyrosine. The protein-protein interactions network as predicted by STRING database revealed interactions with proteasome core complex. The two (2) monomer models built through SWISS model showed 91.1% and 88.1% of residues in most favoured regions