Regulation of Developmental Differentiation in the Protozoan Parasite Toxoplasma gondii

Abstract

Toxoplasma gondii is an important pathogen of newborn infants and immunocompromised patients, such as those receiving organ transplants or suffering from the acquired immunodeficiency syndrome (AIDS). Whereas disease in newborns depends on congenital transmission of the parasite during primary infection of the mother, toxoplasmic encephalitis (TE) in AIDS patients seems to reflect reactivation of a latent infection [12]. It is still unclear whether conversion between bradqzoites and tachyzoites or formation and rupture of the cyst wall is important for pathogenesis of acute or reactivated toxoplasmosis [8].Recent progress in the generation of stage-specific monoclonal antibodies and genetic manipulation has made it possible to further dissect the mechanisms that are involved during tachyzoitebradyzoite interconversion [3, 151, In-vitro studies performed by independent investigators demonstrated that differentiation from the tachyzoite stage to the bradyzoite stage is inducable by external stress factors, such as pH modification of the cell culture medium or immunomodulation [4, 141. This study tnes to give new insights into the kinetics of tachyzoite to bradyzoite differentiation and what is known about the regulation of developmental differentiation of T. gondii.Mice were intraperitoneally infected with tachyzoites of T. gondii strain NTE. One day later, tachyzoites were harvested from the peritoneal cavity and were used to infect human foreslun fibroblasts. Differentiation into the bradyzoite stage was induced by shifting the pH of the cell culture medium above pH 8.0 [13]. The kinetics of the expression of stagespecific antigens, such as SAGl (tachyzoite) or BAGl (bradyzoite) and P115 (cyst wall) was determined by an immunofluorescence assay and by immunoelectron microscopy utilizing corresponding monoclonal antibodies (MAbs). Cysts from brains of mice that were mfected three months before were used as a controls for mature bradyzoites.In order to evaluate whether chemotherapeutic agents can also induce stage differentiation, murine bone marrowderived macrophages were infected with tachyzoites of T. gondii strain NTE, and were incubated in the presence of pyimethamine at different concentrations. Since previous experiments have shown that maximal expression of bradyzoite-specific antigens occurs on day 4 after infection, all cultures were evaluated on this day by determining the relative number of parasitophorous vacuoles expressing the MAb 4FS-reactive bradyzoite-specific antigen in an immunofluorescence assay [3].To determine whether the promoter of the bradyzoite-specific gene BAG1 [l] is stage-specifically regulated, the 5' promoter region of this gene was cloned upstream of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Since the 3' flanking sequence has been shown to be not important for regulation of the B,4GI promoter, the 3' flanking region of SAGl was cloned downstream of the reporter gene. This construct was used to transiently transfect tachyzoites of the NTE strain followed b...

Toxoplasma gondii : an ultrastructural study of host-cell invasion by the bradyzoite stage

“…Most importantly, although some molecules are expressed by both stages of the parasite Cesbron-Delauw et al 1989;Achbarou et al 1991;Torpier et al 1993), there are major dierences in molecular structure. In particular, the surface of the bradyzoite bears four unique molecules (Tomavo et al 1991) and lacks the tachyzoite surface molecules SAG1 and SAG 2 (Woodison and Smith 1990;Bohne et al 1993b;Soete et al 1993;Gross et al 1996;Lane et al 1996), which have been implicated in tachyzoite invasion Smith 1992, 1996;Mineo and Kasper 1994).…”

Stage-specific expression of NcSAG4 as a marker of chronic Neospora caninum infection in a mouse model

“…It would be complicated to show this using this mouse model due to the low parasite burden found in the brain beyond day 64 p.i. Some authors have suggested that the majority of Tg SAG4 expression (and other T. gondii developmentally-regulated genes) takes place during the tachyzoite-to-bradyzoite conversion; expression then decreases once encystation and/or chronic infection have been established (Gross et al 1996; Cleary et al 2002; Contini et al 2006). Thus, Cleary et al (2002) developed a microarray analysis of T. gondii cDNA in which they confirmed bradyzoite-specific expression of Tg SAG4A .…”

The role of stage-specific oligonucleotide primers in providing effective laboratory support for the molecular diagnosis of reactivated Toxoplasma gondii encephalitis in patients with AIDS

“…Several bradyzoite genes encoding for specific antigens have been identified and cloned including SAG4, BAG1/hsp30, LDH2 and MAG1 [7,12,20,22]. The cloning and characterisation of these stagespecifically expressed genes has further contributed to the molecular analysis of this developmental differentiation [3,36,37].…”