Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii.
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ABSTRACT:
SUBMITTER: Rajendran E
PROVIDER: S-EPMC8370653 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma. Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma-infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication.
| S-EPMC6564765 | biostudies-literature
Project description:Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii-previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite's metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.
| S-EPMC8399914 | biostudies-literature
Project description:Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii. Here, we assess the role of VIT and iron storage in T. gondii. By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.
| S-EPMC10281983 | biostudies-literature
Project description:Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the antimalarial drug chloroquine. PfCRT localizes to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chloroquine resistance transporter family. CRT family proteins are found throughout the Apicomplexa, in some protists, and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) colocalizes with markers for the vacuolar (VAC) compartment in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing its morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT family proteins in apicomplexan parasites.
| S-EPMC4248696 | biostudies-literature
Project description:Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome.
| S-EPMC8513686 | biostudies-literature
Project description:Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+ tolerance of T. gondii extracellular tachyzoites.IMPORTANCEToxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+ We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.
| S-EPMC6531880 | biostudies-literature
Project description:Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.
| S-EPMC8776092 | biostudies-literature
Project description:PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. We generated T. gondii mutants lacking PRMT1 (∆prmt1) by deletion of the PRMT1 gene. ∆prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the complemented strain ∆prmt::PRMT1mRFP. PRMT1 localizes primarily in the cytoplasm with enrichment at the centrosome, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ∆prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole genome expression profiling demonstrated differences in expression of cell cycle regulated genes in ∆prmt1 relative to the complemented ∆prmt1::PRMT1mRFP and parental wild-type strains, but these changes did not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our genetic studies suggest that the most critical function of PRMT1 is within the centrosome as a regulator of daughter cell counting to assure the proper replication of the parasite. RNA samples were isolated in triplicates from RH-hxgprt parent strain (W), PRMT1 knockout (K) strain and PRMT1 knockout strain complemented with RFP-tagged PRMT1 protein (C). Parasites were grown for 32h at 37C. Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies).
2015-09-18 | E-GEOD-73177 | biostudies-arrayexpress
Project description:UNLABELLED:The arginine methyltransferase family (PRMT) has been implicated in a variety of cellular processes, including signal transduction, epigenetic regulation, and DNA repair pathways. PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. To further define the biological function of the PRMT family, we generated T. gondii mutants lacking PRMT1 (?prmt1) by deletion of the PRMT1 gene. ?prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the ?prmt::PRMT1mRFP complemented strain. Tagged PRMT1 localizes primarily in the cytoplasm with enrichment at the pericentriolar material, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ?prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole-genome expression profiling demonstrated differences in expression of cell-cycle-regulated genes in the ?prmt1 strain relative to the complemented ?prmt1::PRMT1mRFP and parental wild-type strains, but these changes do not correlate with a specific block in cell cycle. Although PRMT1's primary biological function was previously proposed to be methylation of histones, our studies suggest that PRMT1 plays an important role within the centrosome to ensure the proper replication of the parasite. IMPORTANCE:Apicomplexan parasites include several important pathogens, including Toxoplasma gondii, a major cause of opportunistic infections and congenital birth defects. These parasites divide using a unique form of cell division called endodyogeny that is different from those of most eukaryotes. PRMT1 is a conserved arginine methyltransferase that was thought to regulate gene expression of T. gondii by modifying histone methylation. Using genetic techniques, we show that disruption of PRMT1 affects the parasite's ability to perform accurate cell division. Our studies reveal an unexpected role for arginine methylation in centrosome biology and regulation of parasite replication.
| S-EPMC4742710 | biostudies-literature
Project description:An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-? activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-? defenses by interfering with the transcription of IFN-?-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-?-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.
| S-EPMC4995087 | biostudies-literature