Bamini Jayabalasingham

Autophagy in protists.

Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles, and defense against parasitic invaders. During the last 10-20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.

Metamorphosis of the malaria parasite in the liver is associated with organelle clearance.

Malaria parasites encounter diverse conditions as they cycle between their vertebrate host and mosquito vector. Within these distinct environments, the parasite undergoes drastic transformations, changing both its morphology and metabolism. Plasmodium species that infect mammals must first take up residence in the liver before initiating red blood cell infection. Following penetration into hepatocytes, the parasite converts from an invasion-competent, motile, elongated sporozoite to a metabolically active, round trophozoite. Relatively little is known about the cellular events involved in sporozoite metamorphosis. Our data uncover the early cellular events associated with these transformations. We illustrate that the beginning of metamorphosis is marked by the disruption of the membrane cytoskeleton beneath the plasma membrane, which results in a protruding area around the nucleus. As this bulbous region expands, the two distal ends of the sporozoite gradually retract and disappear, leading to cell sphericalization. This shape change is associated with major interior renovations and clearance of superfluous organelles, e.g. micronemes involved in invasion. The membrane cytoskeleton is reorganized into dense lamellar arrays within the cytoplasm and is partially expulsed by converting parasites. Simultaneously, micronemes are compartmentalized into large exocytic vesicles and are then discharged into the environment. At the completion of metamorphosis, the parasites only retain organelles necessary for replication. These observations lay the groundwork for further investigations on the developmental pathways implicated in the metamorphosis of the malaria parasite.

Structural characterization and inhibition of the Plasmodium Atg8-Atg3 interaction

The autophagy-related proteins are thought to serve multiple functions in Plasmodium and are considered essential to parasite survival and development. We have studied two key interacting proteins, Atg8 and Atg3, of the autophagy pathway in Plasmodium falciparum. These proteins are vital for the formation and elongation of the autophagosome and essential to the process of macroautophagy. Autophagy may be required for conversion of the sporozoite into erythrocytic-infective merozoites and may be crucial for other functions during asexual blood stages. Here we describe the identification of an Atg8 family interacting motif (AIM) in Plasmodium Atg3, which binds Plasmodium Atg8. We determined the co-crystal structure of PfAtg8 with a short Atg3¹⁰³⁻¹¹⁰ peptide, corresponding to this motif, to 2.2 Å resolution. Our in vitro interaction studies are in agreement with our X-ray crystal structure. Furthermore they suggest an important role for a unique Apicomplexan loop absent from human Atg8 homologues. Prevention of the protein-protein interaction of full length PfAtg8 with PfAtg3 was achieved at low micromolar concentrations with a small molecule, 1,2,3-trihydroxybenzene. Together our structural and interaction studies represent a starting point for future antimalarial drug discovery and design for this novel protein-protein interaction.

Characterization of the ATG8-conjugation system in 2 Plasmodium species with special focus on the liver stage: Possible linkage between the apicoplastic and autophagic systems?

Plasmodium 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, Plasmodium discards organelles involved in invasion and expands those implicated in biosynthetic pathways. We hypothesized that this process is regulated by autophagy. Plasmodium 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 Plasmodium falciparum and during the liver stage of Plasmodium berghei. We engineered a transgenic P. falciparum strain expressing mCherry-PfATG8. These transgenic parasites expressed mCherry-PfATG8 in human hepatocytes and erythrocytes, and in the midgut and salivary glands of Anopheles mosquitoes. In all observed stages, mCherry-PfATG8 was localized to tubular structures. Our EM and colocalization studies done in P. berghei 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. PbATG3, PbATG7 and PbATG8 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 Plasmodium ATG8 to functionally complement atg8Δ yeast or localize to autophagosomes in starved mammalian cells. Altogether, these data suggests a unique role for the ATG8-conjugation system in Plasmodium parasites.

Role of an Ancestral D-Bifunctional Protein Containing Two Sterol-Carrier Protein-2 Domains in Lipid Uptake and Trafficking in Toxoplasma

The inability to synthesize cholesterol is universal among protozoa. The intracellular pathogen Toxoplasma depends on host lipoprotein-derived cholesterol to replicate in mammalian cells. Mechanisms of cholesterol trafficking in this parasite must be important for delivery to proper organelles. We characterized a unique d-bifunctional protein variant expressed by Toxoplasma consisting of one N-terminal d-3-hydroxyacyl-CoA dehydrogenase domain fused to two tandem sterol carrier protein-2 (SCP-2) domains. This multidomain protein undergoes multiple cleavage steps to release free SCP-2. The most C-terminal SCP-2 carries a PTS1 that directs the protein to vesicles before processing. Abrogation of this signal results in SCP-2 accumulation in the cytoplasm. Cholesterol specifically binds to parasite SCP-2 but with 10-fold lower affinity than phosphatidylcholine. In mammalian cells and Toxoplasma, the two parasite SCP-2 domains promote the circulation of various lipids between organelles and to the surface. Compared with wild-type parasites, TgHAD-2SCP-2-transfected parasites replicate faster and show enhanced uptake of cholesterol and oleate, which are incorporated into neutral lipids that accumulate at the basal end of Toxoplasma. This work provides the first evidence that the lipid transfer capability of an ancestral eukaryotic SCP-2 domain can influence the lipid metabolism of an intracellular pathogen to promote its multiplication in mammalian cells.

Recent insights into fatty acid acquisition and metabolism in malarial parasites.

The malarial parasite has a tremendous requirement for fatty acids during the replicative stages that take place in the mammalian host. A series of recent papers, discussed below, have revealed some of the mechanisms employed by the parasite to meet these demands.