SLS Inducers and Pathways leading to Programmed Cell Death in Trypanosoma brucei

The Trypanosomatids are unicellular eukaryotic parasites in which Trypanosoma brucei causes the Human African sleeping sickness. This parasite possesses a unique trans splicing mechanism in which a Spliced leader (SL) exon is added to all 5’ end of mRNA transcription in order to generate a mature mRNA from the polysictronic units. SL promotor is controlled by different transcription factors including the TATA Binding protein (TRF4). Perturbations of protein translocation across the ER, low pH, depletion of the essential ER resident chaperones, proteins that involved in disulfide bond formation and protein involved in mitochondrial protein import induce the spliced leader RNA silencing (SLS) pathway (Uthman et al., mBio 2021). Anti-parasitic drugs also induce SLS. SLS induction involves the activation of a serine-threonine kinase PK3 that undergoes auto-phosphorylation and translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates TRF4, leading to the shut-off of SL RNA transcription and programmed cell death (PCD). To date, no caspases have been found in T. brucei. To identify factors that execute the SLS death pathway, we performed an RNAi screen for drug resistance with the assumption that losing the death pathway will result in drug tolerance. We purified proteins that interact with PK3 BioID under SLS induction by low pH, oxidative stress and drugs. We also determined the phosphor-proteome under SLS. Two proteins the mitochondrial transmembrane protein ATOM 69 and Calpain protease 1.1 were identified in all three screens and evidence will be provided for their role in PCD.