Characterization of the ATG8-conjugation system in 2 <i>Plasmodium</i> species with special focus on the liver stage

<i>Plasmodium</i> parasites successfully colonize different habitats within mammals and mosquitoes, and adaptation to various environments is accompanied by changes in their organelle composition and size. Previously, we observed that during hepatocyte infection, <i>Plasmodium</i> discards organelles involved in invasion and expands those implicated in biosynthetic pathways. We hypothesized that this process is regulated by autophagy. <i>Plasmodium</i> spp. possess a rudimentary set of known autophagy-related proteins that includes the ortholog of yeast Atg8. In this study, we analyzed the activity of the ATG8-conjugation pathway over the course of the lifecycle of <i>Plasmodium falciparum</i> and during the liver stage of <i>Plasmodium berghei</i>. We engineered a transgenic <i>P. falciparum</i> strain expressing mCherry-PfATG8. These transgenic parasites expressed mCherry-PfATG8 in human hepatocytes and erythrocytes, and in the midgut and salivary glands of <i>Anopheles</i> mosquitoes. In all observed stages, mCherry-PfATG8 was localized to tubular structures. Our EM and colocalization studies done in <i>P. berghei</i> showed the association of PbATG8 on the limiting membranes of the endosymbiont-derived plastid-like organelle known as the apicoplast. Interestingly, during parasite replication in hepatocytes, the association of PbATG8 with the apicoplast increases as this organelle expands in size. <i>PbATG3</i>, <i>PbATG7</i> and <i>PbATG8</i> are cotranscribed in all parasitic stages. Molecular analysis of PbATG8 and PbATG3 revealed a novel mechanism of interaction compared with that observed for other orthologs. This is further supported by the inability of <i>Plasmodium</i> ATG8 to functionally complement <i>atg8Δ</i> yeast or localize to autophagosomes in starved mammalian cells. Altogether, these data suggests a unique role for the ATG8-conjugation system in <i>Plasmodium</i> parasites.