Gene Set Enrichment Analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program

BACKGROUND: Large amounts of microarray expression data have been generated for the Apicomplexan parasite Toxoplasma gondii in an effort to identify genes critical for virulence or developmental transitions. However, researchers’ ability to analyze this data is limited by the large number of unannotated genes, including many that appear to be conserved hypothetical proteins restricted to Apicomplexa. Further, differential expression of individual genes is not always informative and often relies on investigators to draw big-picture inferences without the benefit of context. We hypothesized that customization of gene set enrichment analysis (GSEA) to T. gondii would enable us to rigorously test whether groups of genes serving a common biological function are co-regulated during the developmental transition to the latent bradyzoite form. RESULTS: Using publicly available T. gondii expression microarray data, we created Toxoplasma gene sets related to bradyzoite differentiation, oocyst sporulation, and the cell cycle. We supplemented these with lists of genes derived from community annotation efforts that identified contents of the parasite-specific organelles, rhoptries, micronemes, dense granules, and the apicoplast. Finally, we created gene sets based on metabolic pathways annotated in the KEGG database and Gene Ontology terms associated with gene annotations available at http://www.toxodb.org. These gene sets were used to perform GSEA analysis using two sets of published T. gondii expression data that characterized T. gondii stress response and differentiation to the latent bradyzoite form. CONCLUSIONS: GSEA provides evidence that cell cycle regulation and bradyzoite differentiation are coupled. Δgcn5A mutants unable to induce bradyzoite-associated genes in response to alkaline stress have different patterns of cell cycle and bradyzoite gene expression from stressed wild-type parasites. Extracellular tachyzoites resemble a transitional state that differs in gene expression from both replicating intracellular tachyzoites and in vitro bradyzoites by expressing genes that are enriched in bradyzoites as well as genes that are associated with the G1 phase of the cell cycle. The gene sets we have created are readily modified to reflect ongoing research and will aid researchers’ ability to use a knowledge-based approach to data analysis facilitating the development of new insights into the intricate biology of Toxoplasma gondii. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-515) contains supplementary material, which is available to authorized users

The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation

Distinct Strains of <i>Toxoplasma gondii</i> Feature Divergent Transcriptomes Regardless of Developmental Stage

<div><p>Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of <i>Toxoplasma gondii</i> representing three common genotypes under both <i>in vitro</i> tachyzoite and <i>in vitro</i> bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed <i>in vitro</i> bradyzoites. With an FDR of 10%, we identified 241 genes differentially expressed between CTG tachyzoites and <i>in vitro</i> bradyzoites, including 5 putative AP2 transcription factors. We also observed a close association between cell cycle regulated genes and differentiation. By Gene Set Enrichment Analysis (GSEA), there are a number of KEGG pathways associated with the <i>in vitro</i> bradyzoite transcriptomes of PLK and CTG, including pyrimidine metabolism and DNA replication. These functions are likely associated with cell-cycle arrest. When comparing mRNA levels between strains, we identified 1,526 genes that were differentially expressed regardless of culture-condition as well as 846 differentially expressed only in bradyzoites and 542 differentially expressed only in tachyzoites between at least two strains. Using GSEA, we identified that ribosomal proteins were expressed at significantly higher levels in the CTG strain than in either the RH or PLK strains. This association holds true regardless of life cycle stage.</p></div

Discovery of a splicing regulator required for cell cycle progression.

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms

Hundreds of genes differentially expressed between strains including potential AP2 regulators.

<p>A) Using edgeR, we identify genes that are differentially expressed between strains (FDR <10%), generating three gene lists for tachyzoite condition and three for the bradyzoite condition. We generated Venn diagrams showing the overlap between the lists in the tachyzoite condition and bradyzoite condition. We also compared tachyzoite and bradyzoite gene lists to each other. Venn diagrams shows comparison of genes that appear in both the tachyzoite and the bradyzoite lists and therefore are “stage independent”. AP2 containing genes appearing on more than one list (any intersection) are indicated. The complete set of genes that are differentially expressed are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111297#pone.0111297.s002" target="_blank">Table S2</a> and RPKM values for all replicates are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111297#pone.0111297.s001" target="_blank">Table S1</a>. B) Heat map of genes differentially expressed following CTG bradyzoite differentiation in all six conditions. Red indicates up regulation compared to that gene's expression level under other conditions, whereas green indicates down regulation. Conditions (columns) are clustered based on similarity of expression levels. The five AP2 genes that are differentially expressed are indicated on the right. Heat map was generated using the 'heatmap.2' function in the 'gplots' package for R. Hierarchical clustering of both the rows (genes) and columns (conditions) computed by the 'hclust' function in the R 'stats' package. Based on mean of replicate RPKM values.</p

Patterns of gene expression vary more by strain than by developmental stage.

<p>A) A representative strain from <i>T. gondii</i> lineages Types I (RH), II (PLK), and III (CTG) was selected and grown in tissue culture in either pH neutral conditions, conducive to tachyzoite growth or alkaline conditions, inducing bradyzoite differentiation. B) Multi-dimensional scaling (MDS) plot based on pairwise comparisons for each of the six experimental conditions. This is calculated as the root mean square deviation for the 500 most differentially expressed genes between any two conditions. The distance between any two points represents the average difference in expression levels (RPKM) of the most dissimilar genes, relative to differences observed between other conditions. In effect, the MDS plot provides an overview of the total amount of variation between samples. The axes show arbitrary distances. Experimental conditions include parasite strain (red  =  RH, green  =  PLK, blue  =  CTG) and by life cycle stage (X =  tachyzoite, O =  bradyzoite). Distances calculated using the 'plotMDS' function in the 'limma' Bioconductor package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111297#pone.0111297-Smyth1" target="_blank">[30]</a>. C) Boxplot represents absolute value of expression level difference between conditions. Interquartile regions are in gold, median differences are plotted as a solid black line. The root mean square deviation for each comparison is represented as a blue cross. From left to right, the first three groups are the intrastrain comparisons, tachyzoite vs. bradyzoite. The next three groups are interstrain comparisons between tachyzoite (unstressed) groups. The final three are are interstrain comparisons between bradyzoite (alkaline-stressed) groups).</p

Differential expression of KEGG pathways between strains in the tachyzoite stage.

<p>Differential expression of KEGG pathways between strains in the tachyzoite stage.</p

Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation.

Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged dominant-negative form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). Proteomics studies revealed that GCN5b interacts with AP2-domain proteins, apicomplexan plant-like transcription factors, as well as a "core complex" that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle