Discussion

A cysteine peptidase of the Clostripain family (Clan CD, family C11) has been identified and characterized in Trypanosoma brucei [i] PNT1 (PMID: 26940875). Phylogenetic analysis showed that kinetoplastid PNT1 proteins are very divergent from orthologues of other organisms. Knockdown of PNT1 was lethal in the bloodstream form by RNAi and the induced population accumulated akinetoplastic (1N0K) cells.  Electron microscopy data confirmed that PNT1 is localized on the kinetoplast. Downregulation of PNT1 in an akinetoplastic cell line resulted in viable parasites, suggesting an essential function in the mitochondrion.  Mislocated kinetoplasts were observed in cells overexpressing PNT1. Data from the recoded wild type and mutated add backs of PNT1 confirm that cysteine peptidase activity is essential for PNT1 function. Moreover, preliminary data in Leishmania mexicana [i] using the DiCRE and CRISPR CAS9 mediated knockdown of the PNT1 orthologue suggests that PNT1 is essential for the survival of this parasite. In summary, our data suggest that PNT1 cysteine peptidase is essential for cell viability in both Trypanosoma brucei [i] and Leishmania mexicana [i]. Importantly, as there are no homologs of PNT1 in mammalian cells, PNT1 is a potential drug target across the trypanosomatids. This work is supported by the MRC.