Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike ⌬tgst2, the modest impairment in ⌬tggt1 and ⌬tggt1/⌬tgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The ⌬tggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in ⌬tggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of ⌬tggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.glucose transport ͉ glutamine metabolism ͉ genetic manipulation

Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites' viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.Up to 20 to 30% of the world population is estimated to be chronically infected with the apicomplexan parasite Toxoplasma gondii. The parasite differentiates within the human host between tachyzoites and bradyzoites, which display distinct physiological features. Mature bradyzoites are adapted for lifelong persistence in their hosts and display an extreme reduction in growth rate, up to a complete arrest of the cell cycle (7). In contrast, tachyzoites, which are present during the acute phase of infection, are characterized by a fast duplication time of 6 to 8 h, indicating that this stage possesses effective pathways for nutrient acquisition and energy metabolism.T. gondii possesses, like many other apicomplexan parasites, a nonphotosynthetic plastid, the so-called apicoplast. This organelle contains a 35-kb circular genome with similarities to plastid genomes from algae and is surrounded by four membranes. These features support the secondary endosymbiosis of a photosynthetic alga by the apicomplexan ancestor as the phylogenetic origin of the apicoplast (21,32,43,51). Numerous nucleus-encoded proteins are imported into the apicoplast, aided by a bipartite presequence which is composed of a signal peptide and an adjacent transit peptide (23,24,39,49).The apicoplast is the location of several anabolic pathways, such as type I...

No abstract

No abstract

BackgroundNewborn screening (NBS) programs for treatable metabolic disorders have been enormously successful, but molecular-based screening has not been broadly implemented so far.MethodsThis prospective pilot study was performed within the German NBS framework. DNA, extracted from dried blood cards was collected as part of the regular NBS program. As cystinosis has a prevalence of only 1:100,000–1:200,000, a molecular genetic assay for detection of the SMN1 gene mutation with a higher prevalence was also included in the screening process, a genetic defect that leads to spinal muscular atrophy (SMA). First tier multiplex PCR was employed for both diseases. The cystinosis screening employed assays for the three most common CTNS mutations covering 75% of German patients; in case of heterozygosity for one of these mutations, samples were screened by next generation sequencing (NGS) of the CTNS exons for 101 CTNS mutations. A detection rate of 98.5% is predicted using this approach.ResultsBetween January 15, 2018 and May 31, 2019, 257,734 newborns were screened in Germany for cystinosis. One neonate was diagnosed with cystinosis, consistent with the known incidence of the disease. No false positive or false negatives were detected so far. Screening, communication of findings to parents, and confirmation of diagnosis were accomplished in a multi-disciplinary setting. This program was accomplished with the cooperation of hospitals, physicians, and parents. In the neonate diagnosed with cystinosis, oral cysteamine treatment began on day 18. After 16 months of treatment the child has no clinical signs of renal tubular Fanconi syndrome.ConclusionsThis pilot study demonstrates the efficacy of a molecular-based neonatal screening program for cystinosis using an existing national screening framework.