The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection

Infection with Plasmodium falciparum enhances extracellular vesicle (EV) production in parasitized red blood cells (pRBCs), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular body formation and membrane shedding, both reported mechanisms of EVs production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy and dot blot assays. Finally, disruption of the Pfvps60 gene led to a reduction in the number of the produced EVs in the KO strain and affected the distribution of other ESCRT-III components. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EV production. Author summary Malaria is a disease caused by Plasmodium parasites that is still a leading cause of death in many low-income countries, and for which currently available therapeutic strategies are not succeeding in its control, let alone eradication. An interesting feature observed after Plasmodium invasion is the increase of extracellular vesicles (EVs) generated by parasitized red blood cells (pRBCs), which lack a vesicular trafficking that would explain EV production. Here, by combining different approaches, we demonstrated the participation of the endosomal sorting complex required for transport (ESCRT) machinery from Plasmodium falciparum in the production of EVs in pRBCs. Moreover, we were able to detect ESCRT-III proteins adjacent to the membrane of the host and in EVs purified from a pRBC culture, which shows the export of these proteins and their participation in EV production. Finally, the disruption of an ESCRT-III associated gene, Pfvps60, led to a significant reduction in the amount of EVs. Altogether, these results confirm ESCRT-III participation in EV production and provide novel information on the P. falciparum protein export mechanisms, which can be used for the development of new therapeutic strategies against malaria, based on the disruption of EV formation and trafficking.