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.
Highlights
Considerable 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
To determine that the purified parasites were T. gondii, a portion of the sample was fixed and stained with sera from mice immunized with type II Me49 parasites (Figure 1B)
In order to place the newly acquired merozoite gene expression in context of the life cycle, the data were analyzed in combination with a recently published dataset by Fritz HM et al 2012 [27] covering day 0, 4, 10 oocyst, day 2 tachyzoite (TD2), and day 4, 8, 21 bradyzoite (BD4, BD8, BD21) development (Figure 1C)
Summary
Considerable 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. Intracellular parasites represent a significant portion of human disease burden throughout the world. The apicomplexan parasite Toxoplasma gondii is one of the most successful intracellular parasites and it is estimated up to a third of the human population has been infected [1]. This high infection rate results in approximately 1.5 million new infections in the U.S per year. The ability of Toxoplasma to infect such a large number of individuals, approximately 30 million in the U.S, results in meaningful disease burden in those individuals where the parasite circumvents normal modes of control [6]
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