Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue.

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, can cause severe disease in humans with an immature or suppressed immune system. The outcome of Toxoplasma infection is highly dependent on the strain type, as are many of its in vitro growth properties. Here we use genetic crosses between type II and III lines to show that strain-specific differences in the modulation of host cell transcription are mediated by a putative protein kinase, ROP16. Upon invasion by the parasite, this polymorphic protein is released from the apical organelles known as rhoptries and injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways and consequent downstream effects on a key host cytokine, interleukin (IL)-12. Our findings provide a new mechanism for how an intracellular eukaryotic pathogen can interact with its host and reveal important differences in how different Toxoplasma lineages have evolved to exploit this interaction.

Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii.

Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion of DNA replication. Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were approximately 60 and approximately 30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains examined. Parasites containing a 1-1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a near-diploid ( approximately 1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.

Toxoplasma gondii bradyzoites form spontaneously during sporozoite-initiated development.

Tachyzoites (VEG strain) that emerge from host cells infected with Toxoplasma gondii sporozoites proliferate relatively fast and double their number every 6 h. This rate of growth is intrinsic, as neither the number of host cells invaded nor host cell type appears to influence emergent tachyzoite replication. Fast tachyzoite growth was not persistent, and following approximately 20 divisions, the population uniformly shifted to slower growth. Parasites 10 days post-sporozoite infection doubled only once every 15 h and, unlike emergent tachyzoites, they grew at this slower rate over several months of continuous cell culture. The spontaneous change in tachyzoite growth rate preceded the expression of the bradyzoite-specific marker, BAG1. Within 24 h of the growth shift, 2% of the population expressed BAG1, and by 15 days post-sporozoite infection, 50% of the parasites were positive for this marker. Spontaneous BAG1 expression was not observed in sporozoites or in tachyzoites during fast growth (through day 6 post-sporozoite inoculation), although these tachyzoites could be induced to express BAG1 earlier by culturing sporozoite-infected cells at pH 8.3. However, alkaline treatment also reduced the replication of emergent tachyzoites to the rate of growth-shifted parasites, supporting a link between reduced parasite growth and bradyzoite differentiation. The shift to slower growth was closely correlated with virulence in mice, as the initially fast-growing emergent tachyzoites were avirulent (100% lethal dose, >10(4) parasites), while a mutant VEG strain (MS-J) that is unable to growth shift caused 100% mortality in mice inoculated with 10 parasites. Parasites recovered from gamma interferon knockout mice inoculated with emergent tachyzoites grew at a slow rate and expressed BAG1, confirming that the replication switch occurs in animals and in the absence of a protective immune response.

Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins.

Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invasion and the second (PV2) 18 to 24 h postinoculation. PV1 is structurally distinctive due to its large size, yet it lacks a tubulovesicular network (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, and M. W. White, Mol. Biochem. Parasitol. 75:75-86, 1995). Confirming the finding that sporozoites have a different electron-dense-granule composition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5' nucleoside triphosphate hydrolases (NTPase). NTPase first appears 12 h postinfection. Other tachyzoite dense-granule proteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst extracts, and antibodies against these proteins stained granules in the sporozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA1, GRA2, or GRA4 during PV1 formation. Even after NTPase induction, these proteins were retained within cytoplasmic granules rather than being secreted into PV1. Only GRA5 was secreted by the sporozoite during host cell invasion, becoming associated with the membrane surrounding PV1. Microinjection of sporozoite-infected cells with fluorescent dyes showed that PV1 is impermeable to fluorescent dyes with molecular masses as small as 330 Da, indicating that PV1 lacks channels through which molecules can pass from the host cytoplasm into the vacuole. By contrast, lucifer yellow rapidly diffused into PV2, demonstrating the presence of molecular channels. These studies indicate that PV1 and PV2 are morphologically, immunologically, and functionally distinct, and that PV2 appears to be identical to the tachyzoite vacuole. The inaccessibility of PV1 to host cell nutrients may explain why parasite replication does not occur in this vacuole.