Lilach Sheiner

A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.

Dual Targeting of Antioxidant and Metabolic Enzymes to the Mitochondrion and the Apicoplast of Toxoplasma gondii

Toxoplasma gondii is an aerobic protozoan parasite that possesses mitochondrial antioxidant enzymes to safely dispose of oxygen radicals generated by cellular respiration and metabolism. As with most Apicomplexans, it also harbors a chloroplast-like organelle, the apicoplast, which hosts various biosynthetic pathways and requires antioxidant protection. Most apicoplast-resident proteins are encoded in the nuclear genome and are targeted to the organelle via a bipartite N-terminal targeting sequence. We show here that two antioxidant enzymes—a superoxide dismutase (TgSOD2) and a thioredoxin-dependent peroxidase (TgTPX1/2)—and an aconitase are dually targeted to both the apicoplast and the mitochondrion of T. gondii. In the case of TgSOD2, our results indicate that a single gene product is bimodally targeted due to an inconspicuous variation within the putative signal peptide of the organellar protein, which significantly alters its subcellular localization. Dual organellar targeting of proteins might occur frequently in Apicomplexans to serve important biological functions such as antioxidant protection and carbon metabolism.

Cell Division in Apicomplexan Parasites Is Organized by a Homolog of the Striated Rootlet Fiber of Algal Flagella

Apicomplexan parasites undergo cell division using an evolutionarily conserved mechanism first described in the positioning and assembly of flagella in algae.

Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNAMet formylation in Apicomplexa

Apicomplexans possess three translationally active compartments: the cytosol, a single tubular mitochondrion, and a vestigial plastid organelle called apicoplast. Mitochondrion and apicoplast are of bacterial evolutionary origin and therefore depend on a bacterial‐like translation machinery. The minimal mitochondrial genome contains only three ORFs, and in Toxoplasma gondii the absence of mitochondrial tRNA genes is compensated for by the import of cytosolic eukaryotic tRNAs. Although all compartments require a complete set of charged tRNAs, the apicomplexan nuclear genomes do not hold sufficient aminoacyl‐tRNA synthetase (aaRSs) genes to be targeted individually to each compartment. This study reveals that aaRSs are either cytosolic, apicoplastic or shared between the two compartments by dual targeting but are absent from the mitochondrion. Consequently, tRNAs are very likely imported in their aminoacylated form. Furthermore, the unexpected absence of tRNAMet formyltransferase and peptide deformylase implies that the requirement for a specialized formylmethionyl‐tRNAMet for translation initiation is bypassed in the mitochondrion of Apicomplexa.

A Toxoplasma MORN1 null mutant undergoes repeated divisions but is defective in basal assembly, apicoplast division and cytokinesis.

The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mothers cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.

Toxoplasma gondii transmembrane microneme proteins and their modular design

Host cell invasion by the Apicomplexa critically relies on regulated secretion of transmembrane micronemal proteins (TM‐MICs). Toxoplasma gondii possesses functionally non‐redundant MIC complexes that participate in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. The TM‐MICs are released onto the parasites surface as complexes capable of interacting with host cell receptors. Additionally, TgMIC2 simultaneously connects to the actomyosin system via binding to aldolase. During invasion these adhesive complexes are shed from the surface notably via intramembrane cleavage of the TM‐MICs by a rhomboid protease. Some TM‐MICs act as escorters and assure trafficking of the complexes to the micronemes. We have investigated the properties of TgMIC6, TgMIC8, TgMIC8.2, TgAMA1 and the new micronemal protein TgMIC16 with respect to interaction with aldolase, susceptibility to rhomboid cleavage and presence of trafficking signals. We conclude that several TM‐MICs lack targeting information within their C‐terminal domains, indicating that trafficking depends on yet unidentified proteins interacting with their ectodomains. Most TM‐MICs serve as substrates for a rhomboid protease and some of them are able to bind to aldolase. We also show that the residues responsible for binding to aldolase are essential for TgAMA1 but dispensable for TgMIC6 function during invasion.

Spliced‐leader RNA silencing: a novel stress‐induced mechanism in Trypanosoma brucei

The signal‐recognition particle (SRP) mediates the translocation of membrane and secretory proteins across the endoplasmic reticulum upon interaction with the SRP receptor. In trypanosomes, the main RNA molecule is the spliced‐leader (SL) RNA, which donates the SL sequence to all messenger RNA through trans‐splicing. Here, we show that RNA interference silencing of the SRP receptor (SRα) in Trypanosoma brucei caused the accumulation of SRP on ribosomes and triggered silencing of SL RNA (SLS). SLS was elicited due to the failure of the SL RNA‐specific transcription factor tSNAP42 to bind to its promoter. SL RNA reduction, in turn, eliminated mRNA processing and resulted in a significant reduction of all mRNA tested. SLS was also induced under pH stress and might function as a master regulator in trypanosomes. SLS is reminiscent of, but distinct from, the unfolded protein response and can potentially act as a new target for parasite eradication.

The metabolic roles of the endosymbiotic organelles of Toxoplasma and Plasmodium spp

The apicoplast and the mitochondrion of Apicomplexa cooperate in providing essential metabolites. Their co-evolution during the ancestral acquisition of a plastid and subsequent loss of photosynthesis resulted in divergent metabolic pathways compared with mammals and plants. This is most evident in their chimerical haem synthesis pathway. Toxoplasma and Plasmodium mitochondria operate canonical tricarboxylic acid (TCA) cycles and electron transport chains, although the roles differ between Toxoplasma tachyzoites and Plasmodium erythrocytic stages. Glutamine catabolism provides TCA intermediates in both parasites. Isoprenoid precursor synthesis is the only essential role of the apicoplast in Plasmodium erythrocytic stages. An apicoplast-located fatty acid synthesis is dispensable in these stages, which instead predominantly salvage fatty acids, while in Plasmodium liver stages and in Toxoplasma tachyzoites fatty acid synthesis is an essential role of the plastid.

Protein Trafficking inside Toxoplasma gondii

The accurate targeting of proteins to their final destination is an essential process in all living cells. Apicomplexans are obligate intracellular protozoan parasites that possess a compartmental organization similar to that of free‐living eukaryotes but can be viewed as professional secretory cells. Establishment of parasitism involves the sequential secretion from highly specialized secretory organelles, including micronemes, rhoptries and dense granules. Additionally, apicomplexans harbor a tubular mitochondrion, a nonphotosynthetic plastid organelle termed the apicoplast, acidocalcisomes and an elaborated inner membrane complex composed of flattened membrane cisternae that are derived from the secretory pathway. Given the multitude of destinations both inside and outside the parasite, the endoplasmic reticulum/Golgi of the apicomplexans constitutes one of the most busy roads intersections in eukaryotic traffic.

Genetic Manipulation of Toxoplasma gondii

Abstract The first genetic manipulations applied to Toxoplasma gondii were performed in the 1970s using chemical mutagenesis. These studies were pioneered by developing protocols to reproducibly cultivate tachyzoites in a tissue culture to then mutagenize, select, and finally clone the parasites. These studies were also critical for the establishment of protocols for genetic crosses in the cat. The reverse genetics approach, which introduces foreign DNA into parasites using electroporation, was achieved in 1993. Plasmids containing reporter genes rapidly allowed the identification of selectable marker genes that opened an avenue for stable transformation and the development of an invaluable panoply of tools associated with DNA transfection. Several positive and negative selectable markers have been tailored for homologous recombination leading to allelic replacement and gene knockouts. More recently, the CRISPR/Cas9 genome editing successfully implemented in T. gondii, streamlined the functional analysis of parasite genes, and enabled high-throughput loss-of-function screens. The access to the sequence of genomes from several T. gondii and closely related apicomplexans constitutes a formidable source of information. In this postgenomics area, the accessibility of T. gondii to multiple genetic manipulations strategies and to high-throughput studies continues to rank it as a very attractive and powerful system to improve our understanding of the basic biology of the apicomplexan parasites. The purpose of this chapter is to recapitulate and describe the strategies associated with DNA transfection and genetic manipulations including the most recent technological advances.