Multiple functionally redundant signals mediate targeting to the apicoplast in the apicomplexan parasite Toxoplasma gondii

Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle--the apicoplast--acquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP+ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.     

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.     

Targeting and processing of nuclear-encoded apicoplast proteins in plastid segregation mutants of Toxoplasma gondii

The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.     

Toxoplasma gondii myosin F,an essential motor for centrosomes positioning and apicoplast inheritance

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so‐called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin‐based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.     

Virulence factors of Toxoplasma gondii

Toxoplasma gondii virulence is dependent on factors involved in either parasite-host cell interaction, or in host immune response. It is essentially defined in the mouse and little is known concerning human infection. The genetic dependence of virulence is a growing field, benefiting from the recent development of research of the population structure of T. gondii.     

Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein-attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii-infected cells in lipoate-deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate-deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.     

Identification of potential apicoplast associated therapeutic targets in human and animal pathogen Toxoplasma gondii ME49

Toxoplasma gondii ME49 is an obligatory intracellular apicomplexa parasite that causes toxoplasmosis in humans, domesticated and wild animals. Waterborne outbreaks of acute toxoplasmosis worldwide reinforce the transmission of Toxoplasma gondii ME49 to humans through contaminated water and may have a greater epidemiological impact than previously believed. In the quest for drug and vaccine target identification subtractive genomics involving subtraction between the host and pathogen genome has been implemented for enlisting essential pathogen specific proteins. Using this approach, our analysis on both human and Toxoplasma gondii ME49 reveals that out of 7987 protein coding sequences of the pathogen, 950 represent essential non human-homologous proteins. Subcellular localization prediction & comparative-biochemical pathway analysis of these essential proteins gives a list of apicoplast-associated proteins having unique pathogen-specific metabolic pathway. These apicoplast-associated enzymes involved in fatty acid biosynthesis pathway of Toxoplasma gondii ME49, may be used as potential drug targets, as the pathway is vital for the protozoan's survival. Structure prediction of drug target proteins was done using fold based recognition method. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against Toxoplasma gondii ME49. ABBREVIATIONS: DEG - Database of Essential Gene, KEGG - Kyoto Encyclopaedia of Genes and Genomes, KAAS - KEGG Automated Annotation Server, PFP - Protein Function Prediction, COG - Cluster of Orthologous Genes.     

A thioredoxin family protein of the apicoplast periphery identifies abundant candidate transport vesicles in Toxoplasma gondii

Toxoplasma gondii, which causes toxoplasmic encephalitis and birth defects, contains an essential chloroplast-related organelle to which proteins are trafficked via the secretory system. This organelle, the apicoplast, is bounded by multiple membranes. In this report we identify a novel apicoplast-associated thioredoxin family protein, ATrx1, which is predominantly soluble or peripherally associated with membranes, and which localizes primarily to the outer compartments of the organelle. As such, it represents the first protein to be identified as residing in the apicoplast intermembrane spaces. ATrx1 lacks the apicoplast targeting sequences typical of luminal proteins. However, sequences near the N terminus are required for proper targeting of ATrx1, which is proteolytically processed from a larger precursor to multiple smaller forms. This protein reveals a population of vesicles, hitherto unrecognized as being highly abundant in the cell, which may serve to transport proteins to the apicoplast.     

Maternal inheritance and stage-specific variation of the apicoplast in Toxoplasma gondii during development in the intermediate and definitive host

The structure and location of Toxoplasma gondii apicoplasts were examined in intermediate and definitive hosts and shown to vary in a stage-specific manner. Immunocytochemistry and electron microscopy studies were used to identify changes in the morphology of apicoplasts and in their enoyl reductase (ENR) content during asexual and sexual development. Apicoplasts in tachyzoites were small, multimembraned organelles anterior to nuclei that divided and segregated with the nuclei during endodyogeny. In nonproliferating bradyzoites within mature tissue cysts (1 to 24 months), apicoplasts had high levels of ENR. During coccidian development, asexual multiplication (endopolygeny), resulting in simultaneous formation of up to 30 daughters (merozoites), involved an initial growth phase associated with repeated nuclear divisions during which apicoplasts appeared as single, elongated, branched structures with increased levels of ENR. At initiation of merozoite formation, enlarged apicoplasts divided simultaneously, with constrictions, into portions that segregated to developing daughters. In sexual stages, apicoplast division did not occur during microgametogony, and apicoplasts were absent from the microgametes that were formed. In contrast, during macrogametogony, the apicoplast appeared as a large, branched, perinuclear structure that had very high levels of ENR in the absence of nuclear division. Marked increases in the size of apicoplasts and levels of ENR may be related to requirements of the macrogametocytes to synthesize and store all components necessary for oocyst formation and subsequent extracellular sporulation. Thus, it is shown that apicoplasts are present and contain ENR in all T. gondii life cycle stages except microgametes, which will result in maternal inheritance of the organelle.     

A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion

We previously reported a cytosolic pyruvate kinase (EC 2.7.1.40) from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. Whereas TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K+, and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP, or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggests that a translation product from 1st Met is responsible for the localization to the apicoplast, whereas one from 3rd Met is for the mitochondrion. This is the first study of a potential mitochondrial pyruvate kinase in any system.     

Cis and trans mechanisms of DNA repair.

DNA repair is essential for genetic stability and variability. Remarkable advances in the understanding of DNA repair by the molecular analysis of the substrate (gene repair) or the enzyme (repair genes), emphasize evolutionary conservation. Recent progress also stresses the interaction(s) between DNA repair and numerous other cellular metabolic processes, including non-nuclear and/or non-genetic responses.     

The carboxyltransferase activity of the apicoplast acetyl-CoA carboxylase of Toxoplasma gondii is the target of aryloxyphenoxypropionate inhibitors

Inhibition of growth of the apicomplexan parasite Toxoplasma gondii by aryloxyphenoxypropionate herbicides has been correlated with the inhibition of its acetyl-CoA carboxylase (ACC) by these compounds. Here, full-length and C-terminal fragments of T. gondii apicoplast ACC as well as C-terminal fragments of the cytosolic ACC were expressed in Escherichia coli. The recombinant proteins that were soluble showed the expected enzymatic activities. Yeast gene-replacement strains depending for growth on the expressed T. gondii ACC were derived by complementation of a yeast ACC1 null mutation. In vitro and in vivo tests with aryloxyphenoxypropionates showed that the carboxyltransferase domain of the apicoplast T. gondii ACC is the target for this class of inhibitors. The cytosolic T. gondii ACC is resistant to aryloxyphenoxypropionates. Both T. gondii isozymes are resistant to cyclohexanediones, another class of inhibitors targeting the ACC of grass plastids.     

Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.     

Types of cells involved in antigen-stimulated and spontaneous rosette formation with Toxoplasma gondii.

Antigen-binding cells were identified by using rosette formation of Toxoplasma gondii and defined lymphoid populations under different experimental conditions. Treatment of immunized spleen cell suspensions with anti-Thy 1 serum plus complement inhibited 5 to 29% of the rosette-forming cells (RFC). Higher numbers of thymus-derived lymphocyte-RFC were obtained after incubation at 4 degrees C and by the centrifugation method than by simple incubation at 20 degrees C. RFC were also observed with thymocytes. Combined treatment with anti-Thy 1 serum plus complement and depletion of adherent cells indicated that the major proportion, 46 to 70%, of RFC were B cells. Spleenocytes of nu/nu mice formed similarly high numbers of rosettes. Spontaneous RFC were observed in nonimmunized mice with both spleen and thymus populations. Numbers of rosettes varied considerably depending on the method and the source of cell population used. Removal of adherent cells from spleen suspensions resulted in RFC reduction of 14 to 25% in immunized and 14 to 33% in nonimmunized animals. Pretreatment with anti-mouse immunoglobulin inhibited completely the spleen and spontaneous thymus RFC and partially the thymus RFC in immunized animals.