Toxoplasma gondiiAsexual Development: Identification of Developmentally Regulated Genes and Distinct Patterns of Gene Expression

Abstract

Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of ϳ4,400 Toxoplasma cDNAs, representing a minimum of ϳ600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.Toxoplasma gondii is a ubiquitous protozoan parasite able to infect a broad range of warm-blooded animals, including humans (12,21). This parasite is a member of the Apicomplexa family, which includes the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). Toxoplasma is distributed worldwide, with human infection rates ranging from 15 to 85% depending on the country. The prevalence of this parasite is due to its effective means of dissemination and ability to resist immune system clearance. Both properties depend on the complex developmental biology of this single-celled eukaryote. For example, one form of dissemination is through the sexual cycle, which occurs only in felines.

Stage-Specific Expression of Surface Antigens by Toxoplasma gondii as a Mechanism to Facilitate Parasite Persistence

“…Toxoplasma stage conversion involves a differential expression of numerous genes in a stage-specific manner (4,15), among which are the members of the surface Ag (SAG)1-related sequence (SRS) superfamily that encode GPI-anchored surface proteins (Ͼ160 putative genes) (16). The prototypic SAG1 is the most abundant TZ SRS Ag (17).…”

“…In this study (26), BAG1 and MAG1 were thought to be the only BZ-specific molecules immunogenic in infection. Recent data, however, demonstrated that MAG1 mRNA (15) and protein (27) are expressed in both the TZ and BZ stages, making it likely that a MAG1-specific response may have been elicited during acute infection with TZs. Because BAG1 shares a significant homology with other small heat shock proteins across species (28), crossreactivity of an immune response to other microorganisms remains a distinct possibility.…”

Genome-wide identification of genes upregulated at the onset of gametocytogenesis in Plasmodium falciparum

“…This approach had to face two technical difficulties. Unlike described developmental studies in protozoa or fungi [13][14][15][16], induction of P. falciparum sexual differentiation in vitro is poorly controlled, and it routinely achieves gametocyte conversion rates of 10-20% of the parasite culture. In addition sexually committed schizonts cannot be physically purified, and enrichment of stage I gametocytes requires exposure to drug treatment that we preferred to avoid in our experimental design.…”

The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites

“…The stage conversion is of biological and clinical significance because the bradyzoite cysts are poorly susceptible to chemotherapy, and considered the source of reactivation causing fatal toxoplasmosis in immunocompromised patients. A more global view of the stage-specificity of gene expression is progressively accessible through the generation of several stage-specific expressed sequence tag (EST) databases [2] and the recent use of microarrays [4] and proteomics technologies [5]. Despite large gaps, the knowledge of the mechanisms and machinery implicated in gene regulation and life-stage differentiation in T. gondii and P. falciparum clearly emerges and contributes to the development of novel tools necessary to regulate gene expression and study their function.…”