Project description:Background: Considerable work has been carried out to understand the biology of the intermediate stages, the tachyzoite and bradyzoite, of Toxoplasma gondii in large part due to the accessible culturing methods for these stages. However, culturing methods for stages beyond the bradyzoite, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. We begin to unravel the molecular aspects of the merozoite stage focusing on gene expression. Results: To initiate this, we harvested merozoite parasites and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by combining it with a previously published study that generated array data for the oocyst, tachyzoite, and bradyzoite stages (Fritz HM et al. PLoS One, 2012). Principal component analysis highlights the unique profile of the merozoite samples, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen p30 (SAG1) and many dense granule proteins do not label merozoites, and our microarray data confirms that these genes are not expressed at this stage. Also, the expression for many rhoptry and microneme proteins is drastically reduced while the expression for many surface antigens is increased at the merozoite stage. Gene Ontology and KEGG analysis reveals that genes involved in transcription/translation and many metabolic pathways are upregulated at the merozoite stage, highlighting unique growth requirements of this stage. We also show that an upstream promoter region of a merozoite specific gene is sufficient to control stage specific expression at the merozoite stage. Conclusion: The merozoite represents the first developmental stage within the gut of the definitive host. Determining the correct conditions that coax the parasite into the merozoite stage in vitro may allow the parasite to complete sexual development. The data presented here describe the global gene expression profile of merozoite stage and the creation of transgenic parasite strains that will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate coccidian development. The ToxoGeneChip microarray was used to measure both tachyzoite and merozoite mRNA expression in the type II TgNmBr1 strain.

Project description:Background: Considerable work has been carried out to understand the biology of the intermediate stages, the tachyzoite and bradyzoite, of Toxoplasma gondii in large part due to the accessible culturing methods for these stages. However, culturing methods for stages beyond the bradyzoite, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. We begin to unravel the molecular aspects of the merozoite stage focusing on gene expression. Results: To initiate this, we harvested merozoite parasites and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by combining it with a previously published study that generated array data for the oocyst, tachyzoite, and bradyzoite stages (Fritz HM et al. PLoS One, 2012). Principal component analysis highlights the unique profile of the merozoite samples, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen p30 (SAG1) and many dense granule proteins do not label merozoites, and our microarray data confirms that these genes are not expressed at this stage. Also, the expression for many rhoptry and microneme proteins is drastically reduced while the expression for many surface antigens is increased at the merozoite stage. Gene Ontology and KEGG analysis reveals that genes involved in transcription/translation and many metabolic pathways are upregulated at the merozoite stage, highlighting unique growth requirements of this stage. We also show that an upstream promoter region of a merozoite specific gene is sufficient to control stage specific expression at the merozoite stage. Conclusion: The merozoite represents the first developmental stage within the gut of the definitive host. Determining the correct conditions that coax the parasite into the merozoite stage in vitro may allow the parasite to complete sexual development. The data presented here describe the global gene expression profile of merozoite stage and the creation of transgenic parasite strains that will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate coccidian development.

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.

Dataset Information

Types I GT1, II Me49, III CTGara strains - tachyzoite and Compound 1

Publications

The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements.

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