Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum, the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo. These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro. Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we did not observe evidence for mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro. Our data hence does not support scenarios in which currently applied therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.

Plasmodium falciparumis the most virulent malaria parasite and accounts for the majority of over 600'000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged, posing a serious threat to disease control efforts. The need for drugs with alternative modes of action is thus undoubted. The parasite's FKBP protein (PfFKBP35) has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506. Whilst there is considerable interest in targetingPfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing. As a consequence, the function ofPfFKBP35 in live parasites remains elusive. Here, we report the generation of inducible knock-out, knock-down and over-expression cell lines and show that blood stage parasites requirePfFKBP35 for maintaining ribosome function. LimitingPfFKBP35 levels are lethal toP. falciparumand result in a delayed-death phenotype that is characterized by defective ribosome homeostasis and protein translation. We furthermore show that, while FK506 is indeed binding toPfFKBP35, the drug exerts it parasiticidal activity in aPfFKBP35-independent manner and, using cellular thermal shift assays, we identify FK506-targets beyondPfFKBP35. In addition to revealing first functional insights on the role ofPfFKBP35 inP. falciparumparasites, our results show that current efforts to developPfFKBP35-targeting drugs need to be reconsidered urgently.

Plasmodium falciparum accounts for the majority of over 600’000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein PfFKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting PfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting PfFKBP35 levels are lethal to P. falciparum and result in a delayed-death phenotype that is characterized by defective ribosome homeostasis and stalled protein translation. We furthermore show that FK506, unlike the role of this drug in model organisms, exerts its anti-proliferative activity in a PfFKBP35-independent manner and, using cellular thermal shift assays, we identify FK506-targets beyond PfFKBP35. In addition to revealing first insights into the function of PfFKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action – with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.