Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development

Abstract

BackgroundConsiderable work has been carried out to understand the biology of tachyzoites and bradyzoites of Toxoplasma gondii in large part due to in vitro culture methods for these stages. However, culturing methods for stages that normally develop in the gut of the definitive felid host, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. Here, we begin to unravel the molecular aspects of enteric stages by providing new data on merozoite stage gene expression.ResultsTo profile gene expression differences in enteric stages we harvested merozoites from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by comparing it to previously published data for the oocyst, tachyzoite, and bradyzoite stages. Principal component analysis highlighted the unique profile of merozoites, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen one (SAG1) and many dense granule proteins do not label merozoites: our microarray data confirms that these genes were not expressed at this stage. Also, the expression for many rhoptry and microneme proteins was drastically reduced while the expression for many surface antigens was increased at the merozoite stage. Gene Ontology and KEGG analysis revealed that genes involved in transcription/translation and many metabolic pathways were upregulated at the merozoite stage, highlighting unique growth requirements of this stage. To functionally test these predictions, we demonstrated that an upstream promoter region of a merozoite specific gene was sufficient to control expression in merozoites in vivo.ConclusionsMerozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-350) contains supplementary material, which is available to authorized users.

Transcriptomic insights into the early host-pathogen interaction of cat intestine with Toxoplasma gondii

“…GO enrichment and KEGG pathway analyses showed that DEGs involved in the metabolic process and catalytic activity were the most enriched at all time points post-infection. A previous study has reported the upregulation of cell growth/maintenance (Translation and Transcription GO categories) and metabolism (Glycolysis and TCA Cycle GO categories)-related genes in the T. gondii merozoite stage during intestinal infection of mice [26]. This intraepithelial intestinal stage of T. gondii proliferates asexually using endopolygeny, whereby multiple daughters are generated from a single parental organism [27].…”

Secreted protein kinases regulate cyst burden during chronic toxoplasmosis

“…Unfortunately, the Pru∆ ku80 strain is not capable of completing the entire life cycle (Wang, Harmon, O'Malley, & Sibley, ; Sibley laboratory, unpublished data), despite the ease of generating gene knockouts in this background. This possibility is especially pertinent as most canonical ROP proteins are downregulated during the life stages in, or derived from, the cat (Behnke Zhang, Dubey, & Sibley ; Fritz et al, ; Hehl et al, ). Several studies have detected putative ROPK transcripts that display upregulation in merozoites and oocysts.…”

Proteomic Differences between Developmental Stages of Toxoplasma gondii Revealed by iTRAQ-Based Quantitative Proteomics

“…The sexual cycle of T. gondii occurs exclusively in the enteroepithelial cells of the intestine of the felid's host (Behnke et al, 2014) and culminates in the production of oocysts, which are adapted to survive under adverse climatic conditions (Dubey, 2010). Toxoplasma gondii oocysts are shed non-sporulated (non-infective) in cat feces, and in a few days they undergo a sporulation process in the environment and with appropriate climatic conditions they mature to sporulated oocysts.…”