Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes.

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, TbFOPL and TbOFD1, in the evolutionarily divergent sleeping sickness parasite Trypanosoma brucei, showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, TbFOPL, affects neither axoneme nor flagellum elongation. Instead, TbFOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar TbFOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, TbFOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for TbFOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of TbFOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.