Structure-Function Relationship for a Divergent Atg8 Protein Required for a Nonautophagic Function in Apicomplexan Parasites.

Atg8 family proteins are highly conserved eukaryotic proteins with diverse autophagy and nonautophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well established, little is known about the molecular changes that facilitated acquisition of divergent, nonautophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study nonautophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here, we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of the Atg8 protein family, is required for P. falciparum Atg8 (PfAtg8) apicoplast localization and function, likely via Atg8 lipidation. On the other hand, another region previously implicated in canonical Atg8 interactions, the N-terminal helix, is not required for apicoplast-specific PfAtg8 function. Finally, our investigations at the cellular level demonstrate that the unique apicomplexan-specific loop, previously implicated in interaction with membrane conjugation machinery in recombinant protein-based in vitro assays, is not required for membrane conjugation nor for the apicoplast-specific effector function of Atg8 in both P. falciparum and related Apicomplexa member Toxoplasma gondii. These results suggest that the effector function of apicomplexan Atg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions. IMPORTANCE The most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other nonautophagic functions critical to cell function and disease pathogenesis that are so far understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their nonautophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and nonautophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, nonautophagic function of Atg8 separate from its role in autophagy: they have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of PfAtg8's function in apicoplast biogenesis will have important implications for the evolution of diverse nonautophagic functions of the Atg8 protein family.

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts.

Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one-third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein, we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.

Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions.

Toxoplasma gondii is a parasitic protist possessing a limited set of proteins involved in the autophagy pathway, a self-degradative machinery for protein and organelle recycling. This distant eukaryote has even repurposed part of this machinery, centered on protein ATG8, for a non-degradative function related to the maintenance of the apicoplast, a parasite-specific organelle. However, some evidence also suggest Toxoplasma is able to generate autophagic vesicles upon stress, and that some autophagy-related proteins, such as ATG9, might be involved solely in the canonical autophagy function. Here, we have characterised TgPROP1 and TgPROP2, two Toxoplasma proteins containing WD-40 repeat that can bind lipids for their recruitment to vesicular structures upon stress. They belong to the PROPPIN family and are homologues to ATG18/WIPI, which are known to be important for the autophagic process. We conducted a functional analysis of these two Toxoplasma PROPPINs. One of them is dispensable for normal in vitro growth, although it may play a role for parasite survival in specific stress conditions or for parasite fitness in the host, through a canonical autophagy-related function. The other, however, seems important for parasite viability in normal growth conditions and could be primarily involved in a non-canonical function. These divergent roles for two proteins from the same family illustrate the functional versatility of the autophagy-related machinery in Toxoplasma.

Autophagy participates in the unfolded protein response in Toxoplasma gondii.

Environmental and genetic perturbations of endoplasmic reticulum (ER) function can lead to the accumulation of unfolded proteins. In these conditions, eukaryotic cells can activate a complex signaling network called the unfolded protein response (UPR) to reduce ER stress and restore cellular homeostasis. Autophagy, a degradation and recycling process, is part of this response. The parasitic protist Toxoplasma gondii is known to be able to activate the UPR upon ER stress, and we now show that this pathway leads to autophagy activation, supporting the idea of a regulated function for canonical autophagy as part of an integrated stress response in the parasites.

TgPL2, a patatin-like phospholipase domain-containing protein, is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma.

Patatin-like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin-like catalytic domain, whose phospholipase activity relies on a serine-aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin-like phospholipase domain-containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin-like domain. Using immunofluorescence assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast-generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite.

Toxoplasma gondii autophagy-related protein ATG9 is crucial for the survival of parasites in their host.

Autophagy is a conserved, life-promoting, catabolic process involved in the recycling of nonessential cellular components in response to stress. The parasite Toxoplasma gondii is an early-diverging eukaryote in which part of the autophagy machinery is not exclusively involved in a catabolic process but instead has been repurposed for an original function in organelle inheritance during cell division. This function, depending essentially on protein TgATG8 and its membrane conjugation system, is crucial for parasite survival and prevented an in depth study of autophagy in the mutants generated so far in Toxoplasma. Thus, in order to decipher the primary function of canonical autophagy in the parasites, we generated a cell line deficient for TgATG9, a protein thought to be involved in the early steps of the autophagy process. Although the protein proved to be dispensable for the development of these obligate intracellular parasites in vitro, the absence of TgATG9 led to a reduced ability to sustain prolonged extracellular stress. Importantly, depletion of the protein significantly reduced parasites survival in macrophages and markedly attenuated their virulence in mice. Altogether, this shows TgATG9 is important for the fate of Toxoplasma in immune cells and contributes to the overall virulence of the parasite, possibly through an involvement in a canonical autophagy pathway.

Repurposing of conserved autophagy-related protein ATG8 in a divergent eukaryote.

Toxoplasma gondii and other apicomplexan parasites contain a peculiar non-photosynthetic plastid called the apicoplast, which is essential for their survival. The localization of autophagy-related protein ATG8 to the apicoplast in several apicomplexan species and life stages has recently been described, and we have shown this protein is essential for proper inheritance of this complex plastid into daughter cells during cell division. Although the mechanism behind ATG8 association to the apicoplast in T. gondii is related to the canonical conjugation system leading to autophagosome formation, its singular role seems independent from the initial catabolic purpose of autophagy. Here we also discuss further the functional evolution and innovative adaptations of the autophagy machinery to maintain this organelle during parasite division.

Whole Genome Re-Sequencing Identifies a Quantitative Trait Locus Repressing Carbon Reserve Accumulation during Optimal Growth in Chlamydomonas reinhardtii.

Microalgae have emerged as a promising source for biofuel production. Massive oil and starch accumulation in microalgae is possible, but occurs mostly when biomass growth is impaired. The molecular networks underlying the negative correlation between growth and reserve formation are not known. Thus isolation of strains capable of accumulating carbon reserves during optimal growth would be highly desirable. To this end, we screened an insertional mutant library of Chlamydomonas reinhardtii for alterations in oil content. A mutant accumulating five times more oil and twice more starch than wild-type during optimal growth was isolated and named constitutive oil accumulator 1 (coa1). Growth in photobioreactors under highly controlled conditions revealed that the increase in oil and starch content in coa1 was dependent on light intensity. Genetic analysis and DNA hybridization pointed to a single insertional event responsible for the phenotype. Whole genome re-sequencing identified in coa1 a >200 kb deletion on chromosome 14 containing 41 genes. This study demonstrates that, 1), the generation of algal strains accumulating higher reserve amount without compromising biomass accumulation is feasible; 2), light is an important parameter in phenotypic analysis; and 3), a chromosomal region (Quantitative Trait Locus) acts as suppressor of carbon reserve accumulation during optimal growth.

Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii.

UNLABELLED: Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE: By definition, autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for autophagy-related protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unrelated to the catabolic autophagy process, TgATG8 associates with the outer membrane of the nonphotosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an autophagy-related protein but also sheds new light on the division process of an organelle that is vital to a group of important human and animal pathogens.

Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii.

BACKGROUND: Oils produced by microalgae are precursors to biodiesel. To achieve a profitable production of biodiesel from microalgae, identification of factors governing oil synthesis and turnover is desirable. The green microalga Chlamydomonas reinhardtii is amenable to genetic analyses and has recently emerged as a model to study oil metabolism. However, a detailed method to isolate various types of oil mutants that is adapted to Chlamydomonas has not been reported. RESULTS: We describe here a forward genetic approach to isolate mutants altered in oil synthesis and turnover from C. reinhardtii. It consists of a three-step screening procedure: a primary screen by flow cytometry of Nile red stained transformants grown in 96-deep-well plates under three sequential conditions (presence of nitrogen, then absence of nitrogen, followed by oil remobilization); a confirmation step using Nile red stained biological triplicates; and a validation step consisting of the quantification by thin layer chromatography of oil content of selected strains. Thirty-one mutants were isolated by screening 1,800 transformants generated by random insertional mutagenesis (1.7%). Five showed increased oil accumulation under the nitrogen-replete condition and 13 had altered oil content under nitrogen-depletion. All mutants were affected in oil remobilization. CONCLUSION: This study demonstrates that various types of oil mutants can be isolated in Chlamydomonas based on the method set-up here, including mutants accumulating oil under optimal biomass growth. The strategy conceived and the protocol set-up should be applicable to other microalgal species such as Nannochloropsis and Chlorella, thus serving as a useful tool in Chlamydomonas oil research and algal biotechnology.

The green microalga Chlamydomonas reinhardtii has a single ω-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids.

The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids.

Results of a community-university partnership to reduce deadly hazards in hardwood floor finishing.

A community-university partnership used community-based participatory research (CBPR) to design, implement, and evaluate a multi-cultural public health campaign to eliminate flammable products and reduce use of products high in volatile organic compounds (VOCs) in hardwood floor finishing in Massachusetts. Leading participants were Vietnamese-American organizations and businesses. Following the public health campaign, a multi-lingual survey of self-reported experiences with fires, product use, exposure to outreach activities, and changes made, was conducted with floor finishers. One hundred nine floor finishers responded. Over 40% reported fires at their companies' jobs, mostly caused by lacquer sealers. Over one third had heard radio or TV shows about health and safety in floor finishing, and over half reported making changes as a result of outreach. Exposure to various outreach activities was associated with reducing use of flammable products, increasing use of low-VOC products, and greater knowledge about product flammability. However, most respondents still reported using flammable products. Outreach led by community partners reached large proportions of floor finishers, was associated with use of safer products, and adds to recent work on CBPR with immigrant workers. Continued use of flammable products supports the belief that an enforceable ban was ultimately necessary to eradicate them.