New findings provide insight on the mechanisms of malarial resistance, demonstrating that inactivation or mutation of Kelch13 compartment proteins reduces the parasite’s uptake of hemoglobin.
Growing parasitic resistance is a central challenge to malaria treatment efforts. It has been established that resistance to the frontline antimalarial artemisinin and its derivatives is mediated by mutations in the Plasmodium falciparum parasite’s Kelch13 protein; however the precise role of Kelch13 in resistance and cellular function has previously been poorly understood.
Results of a study published in Science provide new insights on the mechanisms of malarial resistance, demonstrating that inactivation or mutation of Kelch13 compartment proteins reduce the parasite’s uptake of hemoglobin. P falciparum’s hemoglobin consumption is what activates artemisinin and derivatives, therefore the reduction results in drug resistance.
Previous research by the study authors established the focus of Kelch13 activity within the parasite in close proximity to its food vacuole, a lysosome-esque compartment where hemoglobin endocytosed from the host cell is digested. The recently published study confirmed that Kelch13 activity congregates around the food vacuole, and that the protein is located at a previously unknown cellular structure or compartment.
From there, various observations about the Kelch13 compartment, such as the presence of epidermal growth factor receptor substrate 15 (EPS15) and colocation with adaptor protein complex 2 (AP-2), further established the likelihood of Kelch13 compartment involvement in endocytosis.
Inactivation of Kelch13 related proteins significantly reduced the transport of hemoglobin to the food vacuole, yet inactivation of Kelch13 itself did not. In fact, inactivation of Kelch13 significantly increased parasite size relative to the control.
Investigators reasoned that Kelch13 may instead be involved in ring stage endocytosis. However, it was not initially clear whether young ring stages endocytose hemoglobin at all, and investigators used experiments involving fluorescent dextrans to demonstrate the uptake of host cell material in young ring stages.
Subsequent experimentation on young ring stage parasites showed that inactivation of Kelch13 did significantly reduce endocytic uptake. To assess whether this could drive resistance, investigators tested hemoglobin uptake into rings of resistant P falciparum containing mutated genomic Kelch13C580Y.
The resistant parasites displayed reduced endocytic uptake. To further confirm their hypothesis, the investigators then inactivated Kelch13 to measure whether doing so could render parasites resistant. These parasites tested showed levels of resistance similar to the resistant Kelch13C580Y parasites.
“Thus, Kelch13 inactivation reduces endocytosis of host cell material and increases resistance. We conclude that early ring stages already endocytose hemoglobin, and that this process is reduced in parasites with a resistance-conferring mutation in Kelch13 or in which Kelch13 is inactivated, and that this correlates with ART [artemisinin and derivatives] resistance,” the study authors wrote.
While it was previously well known that artemisinin and derivatives are activated by parasitic endocytosis of hemoglobin, it was not realized that this was involved in resistance in parasites with Kelch13 mutations.
Other research into the area has tended to emphasize potential resistance roles of Kelch13 downstream of artemisinin activation, for example in mitigating cellular stress response. The study authors proposed instead that Kelch13 and Kelch13 compartment proteins control the level of endocytosis, influencing the amount of hemoglobin available and the concentration of active artemisinin.
“We envisage that the mechanism of ART resistance indicated by this work will aid in finding ways to antagonize it. It may also inform the choice of ART partner drugs, particularly as hemoglobin digestive processes are the target of existing drugs,” study authors concluded.