Rapid control of protein level in the apicomplexan Toxoplasma gondii.
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ABSTRACT:
SUBMITTER: Herm-Gotz A
PROVIDER: S-EPMC2601725 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The obligate intracellular parasite Toxoplasma gondii critically relies on host cell invasion during infection. Proteins secreted from the apical micronemes are central components for host cell recognition, invasion, egress, and virulence. Although previous work established that the sporozoite protein with an altered thrombospondin repeat (SPATR) is a micronemal protein conserved in other apicomplexan parasites, including Plasmodium, Neospora, and Eimeria, no genetic evidence of its contribution to invasion has been reported. SPATR contains a predicted epidermal growth factor domain and two thrombospondin type 1 repeats, implying a role in host cell recognition. In this study, we assess the contribution of T. gondii SPATR (TgSPATR) to T. gondii invasion by genetically ablating it and restoring its expression by genetic complementation. ?spatr parasites were ~50% reduced in invasion compared to parental strains, a defect that was reversed in the complemented strain. In mouse virulence assays, ?spatr parasites were significantly attenuated, with ~20% of mice surviving infection. Given the conservation of this protein among the Apicomplexa, we assessed whether the Plasmodium falciparum SPATR ortholog (PfSPATR) could complement the absence of the TgSPATR. Although PfSPATR showed correct micronemal localization, it did not reverse the invasion deficiency of ?spatr parasites, because of an apparent failure in secretion. Overall, the results suggest that TgSPATR contributes to invasion and virulence, findings that have implications for the many genera and life stages of apicomplexans that express SPATR.
| S-EPMC4187870 | biostudies-other
Project description:Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.
| S-EPMC3351351 | biostudies-literature
Project description:The unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication, and are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division. We determined that Toxoplasma gondii encodes seven atypical P-, H-, Y- and L- type cyclins and ten Crks to regulate cellular processes. We generated and analyzed conditional tet-OFF mutants for seven TgCrks and four TgCyclins that are expressed in the tachyzoite stage. These experiments demonstrated that TgCrk1, TgCrk2, TgCrk4 and TgCrk6, were required or essential for tachyzoite growth revealing a remarkable number of Crk factors that are necessary for parasite replication. G1 phase arrest resulted from the loss of cytoplasmic TgCrk2 that interacted with a P-type cyclin demonstrating that an atypical mechanism controls half the T. gondii cell cycle. We showed that T. gondii employs at least three TgCrks to complete mitosis. Novel kinases, TgCrk6 and TgCrk4 were required for spindle function and centrosome duplication, respectively, while TgCrk1 and its partner TgCycL were essential for daughter bud assembly. Intriguingly, mitotic kinases TgCrk4 and TgCrk6 did not interact with any cyclin tested and were instead dynamically expressed during mitosis indicating they may not require a cyclin timing mechanism. Altogether, our findings demonstrate that apicomplexan parasites utilize distinctive and complex mechanisms to coordinate their novel replicative cycles.
| S-EPMC5510908 | biostudies-literature
Project description:Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.
| S-EPMC549326 | biostudies-literature
Project description:Sphingolipids are essential components of eukaryotic cell membranes, particularly the plasma membrane, and are involved in a diverse array of signal transduction pathways. Mammals produce sphingomyelin (SM) as the primary complex sphingolipid via the well characterised SM synthase. In contrast yeast, plants and some protozoa utilise an evolutionarily related inositol phosphorylceramide (IPC) synthase to synthesise IPC. This activity has no mammalian equivalent and IPC synthase has been proposed as a target for anti-fungals and anti-protozoals. However, detailed knowledge of the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites was lacking. In this study bioinformatic analyses indicated a single copy orthologue of the putative SM synthase from the apicomplexan Plasmodium falciparum (the causative agent of malaria) was a bona fide sphingolipid synthase in the related model parasite, Toxoplasma gondii (TgSLS). Subsequently, TgSLS was indicated, by complementation of a mutant cell line, to be a functional orthologue of the yeast IPC synthase (AUR1p), demonstrating resistance to the well characterised AUR1p inhibitor aureobasidin A. In vitro, recombinant TgSLS exhibited IPC synthase activity and, for the first time, the presence of IPC was demonstrated in T. gondii lipid extracts by mass spectrometry. Furthermore, host sphingolipid biosynthesis was indicated to influence, but be non-essential for, T. gondii proliferation, suggesting that whilst scavenging does take place de novo sphingolipid synthesis may be important for parasitism.
| S-EPMC3629565 | biostudies-literature
Project description:DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host. The spontaneous loss of DNA bases due to hydrolysis and oxidative DNA damage in intracellular parasites is great, but little is known about the type of DNA repair machineries that exist in these early-branching eukaryotes. However, it is clear, processes similar to DNA base excision repair (BER) must exist to rectify spontaneous and host-mediated damage in Toxoplasma gondii. Here we report that T. gondii, an opportunistic protozoan pathogen, possesses two apurinic/apyrimidinic (AP) endonucleases that function in DNA BER. We characterize the enzymatic activities of Toxoplasma exonuclease III (ExoIII, or Ape1) and endonuclease IV (EndoIV, or Apn1), designated TgAPE and TgAPN, respectively. Over-expression of TgAPN in Toxoplasma conferred protection from DNA damage, and viable knockouts of TgAPN were not obtainable. We generated an inducible TgAPN knockdown mutant using a ligand-controlled destabilization domain to establish that TgAPN is critical for Toxoplasma to recover from DNA damage. The importance of TgAPN and the fact that humans lack any observable APN family activity highlights TgAPN as a promising candidate for drug development to treat toxoplasmosis.
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Project description:Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites' ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.
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Project description:Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii. In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.
| S-EPMC30206 | biostudies-literature
Project description:Apicomplexa are obligate intracellular pathogens that have fine-tuned their proliferative strategies to match a large variety of host cells. A critical aspect of this adaptation is a flexible cell cycle that remains poorly understood at the mechanistic level. Here we describe a forward genetic dissection of the apicomplexan cell cycle using the Toxoplasma model. By high-throughput screening, we have isolated 165 temperature sensitive parasite growth mutants. Phenotypic analysis of these mutants suggests regulated progression through the parasite cell cycle with defined phases and checkpoints. These analyses also highlight the critical importance of the peculiar intranuclear spindle as the physical hub of cell cycle regulation. To link these phenotypes to parasite genes, we have developed a robust complementation system based on a genomic cosmid library. Using this approach, we have so far complemented 22 temperature sensitive mutants and identified 18 candidate loci, eight of which were independently confirmed using a set of sequenced and arrayed cosmids. For three of these loci we have identified the mutant allele. The genes identified include regulators of spindle formation, nuclear trafficking, and protein degradation. The genetic approach described here should be widely applicable to numerous essential aspects of parasite biology.
| S-EPMC2242837 | biostudies-literature
Project description:The micronemal protein 2 (MIC2) of Toxoplasma gondii shares sequence and structural similarities with a series of adhesive molecules of different apicomplexan parasites. These molecules accumulate, through a yet unknown mechanism, in secretory vesicles (micronemes), which together with tubular and membrane structures form the locomotion and invasion machinery of apicomplexan parasites. Our findings indicated that two conserved motifs placed within the cytoplasmic domain of MIC2 are both necessary and sufficient for targeting proteins to T. gondii micronemes. The first motif is based around the amino acid sequence SYHYY. Database analysis revealed that a similar sequence is present in the cytoplasmic tail of all transmembrane micronemal proteins identified so far in different apicomplexan species. The second signal consists of a stretch of acidic residues, EIEYE. The creation of an artificial tail containing only the two motifs SYHYY and EIEYE in a preserved spacing configuration is sufficient to target the surface protein SAG1 to the micronemes of T. gondii. These findings shed new light on the molecular mechanisms that control the formation of the microneme content and the functional relationship that links these organelles with the endoplasmic reticulum of the parasite.
| S-EPMC86287 | biostudies-literature