Iron depletion proteomics of Toxoplasma tachyzoites
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Toxoplasma Gondii Gt1
TISSUE(S): Cell Culture
DISEASE(S): Toxoplasmosis
SUBMITTER: 
Jack Hanna
LAB HEAD: Clare R Harding
PROVIDER: PXD066828 | Pride | 2026-03-18
REPOSITORIES: Pride
Dataset's files
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| 228_2023-JH-R1-Ctl1-24-Rep.raw | Raw | |||
| 228_2023-JH-R1-DFO-24h-Rep.raw | Raw | |||
| 228_2023-JH-R2-Ctl1-24h.raw | Raw | |||
| 228_2023-JH-R2-DFO-24h.raw | Raw | |||
| 228_2023-JH-R3-Ctl-24.raw | Raw |
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Project description:Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in recovering from alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. The TgGCN5-A knockout is incapable of up-regulating key marker genes expressed during development of the latent cyst form. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. We also describe a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that TgGCN5-A knockout parasites fail to up-regulate 68% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure, including developmentally regulated genes. Wild-type and GCN5-A- Knockout organisms were subjected to control and stress treatments
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Project description:Toxoplasma gondii is a parasitic protist that is the agent of toxoplasmosis. It is capable of infecting a wide variety of vertebrates, including humans. The infection is mainly asymptomatic in immunocompetent patients, but in case of immunosuppression or for the congenital form of toxoplasmosis it can lead to severe pathologies with a possible fatal outcome. Like for other eukaryotes, many key cellular functions in T. gondii involve proteins containing an iron-sulfur cluster as a cofactor. Cytosolic and nuclear iron-sulfur proteins depend on a specific pathway for assembling their iron-sulfur cofactor. We have investigated the T. gondii homolog of the HCF101 protein, initially characterized in plants as a chloroplast-based iron-sulfur transfer protein, by co-immunoprecipitating associated protein partners and identifying them by mass spectrometry. It confirmed that T. gondii HCF101 is not involved in plastid-based iron-sulfur metabolism, but in the biogenesis of cytosolic and nuclear iron-sulfur proteins instead.
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Project description:Toxoplasma gondii is a ubiquitous parasite of animals. We have previously shown that T. gondii possesses an AGC kinase, SPARK, that is necessary for calcium mobilization, invasion, and egress from host cells. Interaction studies have revealed that SPARK complexes with an elongin-like protein, which we term SPARKEL. Here, we performed proximity labeling of parasites expressing SPARKEL-TurboID endogenously.
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Project description:The control of gene expression in Toxoplasma is not well understood. As we and others have shown, this organism does contain bipartite promoters that are sufficient to induce gene expression. This information can be used in downstream experiments to identify proteins that bind to experimentally confirmed cis elements. One of these methods, yeast one-hybrid is currently being employed to identify proteins which bind to the BAG1 and B-NTPase promoter elements. Another powerful method that can be used to identify genomic regions containing controllers of gene expression takes advantage of genetic linkage mapping provided by genetic crosses, called expression quantitative trait locus (eQTL) mapping. In the eQTL approach, one uses gene expression as quantitative traits, or phenotypes, in QTL mapping. QTL mapping is a proven method in Toxoplasma that has recently identified apicomplexan-specific virulence factors in both the Type II X III and Type I X III crosses (Saeij et al., 2006, Taylor et al., 2006, Su et al., 2002). With the recent design of the Affymetrix Toxoplasma GeneChip one can gather gene expression profiles for all predicted genes in the Toxoplasma genome in a rapid and reproducible way. Combining the power of Affymetrix gene expression technology and QTL mapping can lead to the identification of genes with differential expression in the parents and progeny of a genetic cross and then to the identification of the genomic regions that contain mechanisms that have an effect on expression. The first description of combining global gene expression profiles with QTL mapping was published by Brem et al. using the model organism Saccharomyces cerevisiae (Brem et al., 2002). Since that time, eQTL projects have been described for several other organisms including human, mouse, rat, maize, Arabidopsis, barley, eucalyptus, and wheat (Gibson & Weir, 2005). There are two mapping location types expected when using gene expression profiles as phenotypes. Mapping of gene expression profiles can lead to linkage of a locus (region) containing the gene being mapped, thus cis to the gene. Although the boundaries of the locus will contain many genes, it is presumed that for most cis QTLs sequence polymorphisms in the regulatory region(s) of the gene being mapped are the cause of the observed expression differences. The other mapping location type expected is one where the expression profile maps to a locus not containing the gene being mapped, or trans to the gene (Ronald et al., 2005). The most interesting trans loci are those that have multiple genes mapping to the same locus in trans, or to a trans hotspot. These hotspots likely contain factor(s) that regulate many genes, such as transcription factors or factors involved in upstream signal transduction pathways that can eventually lead to a number of transcriptional changes (Yvert et al., 2003). Here we describe the results of an eQTL project using the gene expression profiles for the parents and 34 informative progeny of the type I-GT1-Fudr X type III-CTGara cross for both the tachyzoite and bradyzoite stages. This is the first time global gene expression profiles have been used to determine linkage to regions presumed to be controllers of transcription using the Affymetrix Toxoplasma GeneChip. The dominant genomic associations found were those mapping cis and there were several regions, or hotspots, where multiple genes map in trans. In addition, we observed an unexpected finding suggesting there are several duplication events in the parasites of the Type I X III cross which may also have consequences on gene expression levels. The ToxoGeneChip microarray (http://ancillary.toxodb.org/docs/Array-Tutorial.html) was used to measure both tachyzoite and bradyzoite mRNA expression in the parents and progeny of the I X III cross. For each of the two developmental expression sets, genome-wide eQTL scans were run for each gene specific probeset on the microarray. The parents of the cross are CTGara and GT1-Fudr. The rest comprise the informative progeny.
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