Uwe Gross

Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants.

Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite‐specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite‐specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C‐terminal region of small heat‐shock proteins from plants. Stage‐specific expression of the naturally occurring 30kDa antigen in bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen, Immuno‐electron microscopy indicated a cytosolic location of this antigen in bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite‐specific primers amplified transcripts in bradyzoites only, not in tachyzoites.

Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain.

Abstract In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2+ neurons, GFAP+ astrocytes, F4/80+ microglia, and O1+ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection.

Stage Differentiation of the Protozoan Parasite Toxoplasma Gondii

The obligate intracellular parasite Toxoplasma gondii is able to persist lifelong in its hosts by differentiating from the replicative tachyzoite stage into cyst forming latent bradyzoites. Beside the clinical relevance of stage conversion and its importance for pathogenesis and prevention of toxoplasmic encephalitis, reversible stage differentiation in T. gondii is an interesting model system of protozoan differentiation in general. In recent years a variety of molecular techniques have been developed for T. gondii, including transfection systems and the development of many selectable markers. Together with tissue culture models in which stage differentiation from tachyzoites to bradyzoites can be induced these techniques provide the tools for a molecular dissection of the differentiation pathways. Three aspects of stage conversion are highlighted in this review, including the alteration of the parasite surface, alterations in parasite metabolism and the induction of genes associated with stress response.

Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements

BAG1 is a small heat-shock protein of Toxoplasma gondii that is specifically expressed in the cyst-forming bradyzoite stage of the parasite. Upregulation of BAG1 mRNA occurs early during the differentiation pathway from tachyzoites to bradyzoites. In order define genomic elements involved in bradyzoite-specific gene regulation, chloramphenicol acetyltransferase (CAT)-reporter gene studies were performed with 5 flanking sequences of the BAG1 gene. Tachyzoites, transiently transfected with the BAG1/cat construct, exhibited very low CAT activity (200 fold less than in parasites transfected with a tubulin promoter/cat construct). After induction of bradyzoite differentiation by alkaline pH shift, however, CAT activity increased 50 fold, demonstrating bradyzoite-specific expression of the CAT reporter gene under control of 5 flanking sequences of BAG1. Stage-specific regulation of BAG1/CAT was independent of the 3-flanking region, since constructs containing 3-flanking sequences of the tachyzoite-specific SAG1 gene showed identical regulation to those containing the BAG1 3-flanking region. The kinetics of BAG1/CAT induction in stably transfected parasites is similar to the kinetics of endogenous BAG1 expression: increased CAT activity was first detected on day 3 after alkaline pH shift (20 fold) and was dramatically upregulated 250 fold on day 4. A series of deletions in the BAG1 5-flanking sequences demonstrated that a 324 nucleotide (nt) fragment, starting 60 nt upstream of the BAG1 transcription start, is sufficient to confer stage-specific regulation on the CAT reporter. These deletion analyses demonstrate that bradyzoite-specific expression of a heterologeous reporter gene requires only minimal genomic sequences.

Toxoplasma gondii virulence markers identified by random amplified polymorphic DNA polymerase chain reaction

Abstract Genomic DNAs from 35 Toxoplasma gondii strains were amplified by random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) using 18 arbitrary 10-mer primers. At least four primers were found to generate DNA fragments that discriminate the 35 T. gondii strains into a genotype of virulent strains and a genotype of avirulent strains. Primer B12 was found to generate a virulence-specific fragment and primers B5, C8, and C20 were found to generate avirulence-specific fragments, which in all cases clearly identified either the virulence phenotype or the avirulence phenotype, respectively. In addition, the DNA polymorphic bands detected were analyzed by parsimony and distance analysis. A similar genetic relationship among the T. gondii strains was determined by the two phylogenetic methods, which use completely different assumptions. Consistent with the division of the 35 strains into a genotype of virulent strains and a genotype of avirulent strains, both analyses revealed 2 clonal lineages directly correlated with murine virulence. These results strongly support the hypothesis that the genus Toxoplasma may actually contain two clonal lineages correlated with virulence, which have evolved independently following their initial separation.

Coexistence of heterogeneous populations ofToxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody

The expression of bradyzoite-specific antigens (Bsa) ofToxoplasma gondii was studied in murine bonemarrow-derived macrophages that had previously been infected with tachyzoites. Growth conditions that allowed only restricted replication ofToxoplasma gondii resulted in heterogeneous populations; (1) Bsa-positive and Bsa-negative parasites could be observed within one parasitophorous vacuole (heterogeneous vacuole community), and (2) homogeneous Bsa-positive and homogeneous Bsa-negative vacuole communities coexisted within one macrophage host cell. These observations suggest that stage conversion does not seem to be a synchromous event for a vacuole community.

Toxoplasma gondii strain-specific transcript levels of SAG1 and their association with virulence

Abstractu2002Toxoplasma gondii isolates can be classified into mouse-virulent and -avirulent strains. Since the major surface antigen of Toxoplasma gondii, SAG1, has been shown to be important for the invasion process and might thus be involved in mouse virulence as well, we analyzed the corresponding gene of mouse-virulent and -avirulent strains. In addition to eight mismatches, mouse-virulent strains harbored five copies of an upstream 27-bp repeat in the promotor region of SAG1 as compared with four copies in avirulent strains. Reverse-transcriptase polymerase chain reaction revealed that SAG1 expression levels in the mouse-virulent T. gondii strains were at least 4-fold those in the avirulent strains. Since SAG1 seems to mediate invasion, it is suggested that the possibly higher steady-state expression of SAG1 in mouse-virulent strains of T. gondii is associated with virulence and facilitates faster invasion by these strains in comparison with avirulent strains.

Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study.

Abstract Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo.

Toxoplasma gondii: Uptake of fetuin and identification of a 15-kDa fetuin-binding protein

Lectin-binding studies demonstrated the presence of a 68-kDa glycoprotein in tachyzoites ofToxoplasma gondii harvested from P388D1 macrophage cell cultures but not in tachyzoites maintained in peritoneal cavities of NMRI mice. This protein was identified as the embryonic protein fetuin that regularly is contained in fetal calf serum, a component of cell-culture media. Uptake of fetuin byT. gondii was demonstrated by intracellular localization of this protein. As shown by latex agglutination and immunofluorescence, no specific binding of fetuin to the parasites surface was detected. Using affinity chromatography of fetuin-agarose, it was demonstrated that fetuin bound specifically to a 15-kDa antigen of tachyzoites. As revealed by inhibition studies with sialic acid and the lectinSambucus nigra agglutinin, the 15-kDa protein probably recognized glycan structures of fetuin.

Toxoplasmosis in pregnancy: which is the best treatment approach?

Antiparasitic drugs have been shown to be effective in the treatment of active Toxoplasma-infections in immunosuppressed individuals, whereas understanding and treatment of gestational and congenital toxoplasmosis is still “a work in progress”. Under the prevailing view that the available[for full text, please go to the a.m. URL]