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Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage.

Croken MM, Ma Y, Markillie LM, Taylor RC, Orr G, Weiss LM, Kim K - PLoS ONE (2014)

Bottom Line: These functions are likely associated with cell-cycle arrest.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro 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 in vitro bradyzoites. With an FDR of 10%, we identified 241 genes differentially expressed between CTG tachyzoites and in vitro 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 in vitro 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.

No MeSH data available.


Related in: MedlinePlus

Patterns of gene expression vary more by strain than by developmental stage.A) A representative strain from T. gondii 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 [30]. 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).
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pone-0111297-g001: Patterns of gene expression vary more by strain than by developmental stage.A) A representative strain from T. gondii 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 [30]. 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).

Mentions: We analyzed steady state mRNA levels from three different strains of T. gondii, RH (Type I, the most common laboratory strain), PLK (Type II, a clone of ME49 the genome reference strain), and CTG (Type III). We grew each strain under both “normal” tachyzoite tissue culture (pH 7, 5% CO2) conditions as well as bradyzoite-cyst inducing stress conditions (pH 8, low CO2), resulting in six total groups. Each condition was sampled in three biological replicates at a single time point. We had expected that this simple experimental design would yield groups of genes linked to the stress response, but the most striking observation is that the genes' steady state RNA levels vary much more between strains than between different stages of their life cycles. Figure 1B illustrates multi-dimensional scaling (MDS) of samples by condition, based on the top five hundred most divergent genes between each condition. The conditions cluster first by strain and then by life cycle stage. While tachyzoites and bradyzoites from different strains have many functional similarities, these results reinforce the divergence of gene expression of these parasites despite relatively modest differences in genome sequence.


Distinct strains of Toxoplasma gondii feature divergent transcriptomes regardless of developmental stage.

Croken MM, Ma Y, Markillie LM, Taylor RC, Orr G, Weiss LM, Kim K - PLoS ONE (2014)

Patterns of gene expression vary more by strain than by developmental stage.A) A representative strain from T. gondii 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 [30]. 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230917&req=5

pone-0111297-g001: Patterns of gene expression vary more by strain than by developmental stage.A) A representative strain from T. gondii 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 [30]. 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).
Mentions: We analyzed steady state mRNA levels from three different strains of T. gondii, RH (Type I, the most common laboratory strain), PLK (Type II, a clone of ME49 the genome reference strain), and CTG (Type III). We grew each strain under both “normal” tachyzoite tissue culture (pH 7, 5% CO2) conditions as well as bradyzoite-cyst inducing stress conditions (pH 8, low CO2), resulting in six total groups. Each condition was sampled in three biological replicates at a single time point. We had expected that this simple experimental design would yield groups of genes linked to the stress response, but the most striking observation is that the genes' steady state RNA levels vary much more between strains than between different stages of their life cycles. Figure 1B illustrates multi-dimensional scaling (MDS) of samples by condition, based on the top five hundred most divergent genes between each condition. The conditions cluster first by strain and then by life cycle stage. While tachyzoites and bradyzoites from different strains have many functional similarities, these results reinforce the divergence of gene expression of these parasites despite relatively modest differences in genome sequence.

Bottom Line: These functions are likely associated with cell-cycle arrest.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro 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 in vitro bradyzoites. With an FDR of 10%, we identified 241 genes differentially expressed between CTG tachyzoites and in vitro 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 in vitro 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.

No MeSH data available.


Related in: MedlinePlus