Genomic Data Reveal Toxoplasma gondii Differentiation Mutants Are Also Impaired with Respect to Switching into a Novel Extracellular Tachyzoite State

Abstract

Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states – genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.

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

“…Taken together, these GSEA results support the hypothesis that extracellular tachyzoites represent a distinct state of parasite asexual development [11]. The GSEA results further suggest a continuum of stress response or stress preparedness between the three developmental stages examined in these experiments.…”

“…Plotted normalized enrichment scores (NES) for oocyst development gene sets for the datasets reported by Fritz [9]. A positive NES indicates that the gene set is associated with unstressed, tachyzoite parasites, while a negative NES indicates that the gene set is associated with alkaline stressed, in vitro bradyzoites as reported by Lescault [11]. Stars indicate significant enrichment (FWER-adjusted p < 0.05).…”

The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions

“…Extracellular tachyzoites are arrested in G1 (Croken et al, 2014) and differ in acetylation (Jeffers and Sullivan, 2012) and metabolism (MacRae et al, 2012), suggesting that extracellular forms represent a unique tachyzoite state (Lescault et al, 2010). We examined the ubiquitinome of extracellular tachyzoites that had naturally egressed from host cells.…”

A GCN2-Like Eukaryotic Initiation Factor 2 Kinase Increases the Viability of Extracellular Toxoplasma gondii Parasites

“…The mechanisms employed by tachyzoites to overcome the dramatic changes in their extracellular environment are poorly understood but have recently emerged as a new area of intensive research. Microarray analyses have revealed significant changes in the transcriptome between intra-and extracellular tachyzoites (12,21). Generally, intracellular parasites favor expression of genes involved in metabolism and DNA replication, while Toxoplasma cells in the extracellular environment activate genes focused on invasion, motility, and signal transduction.…”