Zygote morphogenesis but not the establishment of cell polarity in Plasmodium berghei is controlled by the small GTPase, RAB11A

Abstract

Plasmodium species are apicomplexan parasites whose zoites are polarized cells with a marked apical organisation where the organelles associated with host cell invasion and colonization reside. Plasmodium gametes mate in the mosquito midgut to form the spherical and presumed apolar zygote that morphs during the following 24 hours into a polarized, elongated and motile zoite form, the ookinete. Endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and the small GTPase RAB11A is an important regulator of protein transport via recycling endosomes. PbRAB11A is essential in blood stage asexual of Plasmodium. Therefore, a promoter swap strategy was employed to down-regulate PbRAB11A expression in gametocytes and zygotes of the rodent malaria parasite, P. berghei which demonstrated the essential role of RAB11A in ookinete development. The approach revealed that lack of PbRAB11A had no effect on gamete production and fertility rates however, the zygote to ookinete transition was almost totally inhibited and transmission through the mosquito was prevented. Lack of PbRAB11A did not prevent meiosis and mitosis, nor the establishment of polarity as indicated by the correct formation and positioning of the Inner Membrane Complex (IMC) and apical complex. However, morphological maturation was prevented and parasites remained spherical and immotile and furthermore, they were impaired in the secretion and distribution of microneme cargo. The data are consistent with the previously proposed model of RAB11A endosome mediated delivery of plasma membrane in T. gondii and implicate it in microneme function. Author Summary According to the WHO there was estimated to be over 200 million cases of malaria in 2017 and nearly half a million deaths. The disease is caused by the Plasmodium parasite which is passed between human hosts by a mosquito vector. In order to transmit through the mosquito the single-celled parasite undergoes many developmental changes as it morphs from apolar and non-motile blood forms to polarised and motile ookinetes in the mosquito midgut. Transport of proteins within the cell during these critical transitions relies on specific endosome vesicles to correctly target proteins within the parasite. We investigated the role of the RAB11A protein which is known to be involved in endosomal vesicle targeting to generate cellular polarity in other organisms. Because RAB11A is also essential for parasite growth in the mammalian host we used a promoter swap system to specifically switch off the protein in the sexual transmission stages. In the absence of RAB11A parasites were unable to form elongated, motile ookinetes and were unable to pass through the mosquito. Interestingly the parasites were able to form some of the (polarising) structures specific to ookinetes however full morphological transformation did not occur and the parasites were not motile. We show that although proteins are still delivered to the parasite surface, secretion is impaired and that the mutant parasites are smaller despite obvious microtubule formation implying that there is a deficit in delivery of membrane to the surface.