Identification of the microsporidian Encephalitozoon cuniculi as a new target of the IFNγ-inducible IRG resistance system.

The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation.

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3% of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3 percent of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Primary skeletal muscle cells trigger spontaneous Toxoplasma gondii tachyzoite-to-bradyzoite conversion at higher rates than fibroblasts.

Toxoplasma gondii is one of the most common eukaryotic parasites and an important opportunistic pathogen of humans. The interconversion from proliferative tachyzoites into quiescent encysted bradyzoites plays pivotal roles in the lifelong persistence of T. gondii in its host and the pathogenesis of toxoplasmosis. Stage conversion and persistence in skeletal muscle tissue may be particularly important for the food-borne transmission of T. gondii to humans via raw or undercooked meat products. Here, we have followed the transition of tachyzoites to bradyzoites after infection of skeletal muscle cells (SkMC). Primary murine myoblasts were differentiated to multinucleated syncytial myotubes that displayed regular contractions in vitro and expressed myogenic markers Myf5 and MyoD. Tachyzoites of T. gondii invaded SkMC within 4h of infection and started to replicate within 24h of infection. Remarkably, intracellular tachyzoites readily differentiated to bradyzoites in SkMC without the need of exogenous stress factors. Double immunofluorescence labelling revealed significantly higher percentages of bradyzoite-containing vacuoles in SkMC than in murine fibroblasts at 24h until 6 days after infection. Furthermore, transcript levels of bradyzoite-specific ENO1 but not tachyzoite-specific ENO2 strongly increased in T. gondii-infected SkMC until 6 days of infection. These findings indicate that the commitment of T. gondii to differentiate to bradyzoites in SkMC does not require exogenous stress factors but could be rather regulated by cell-type specific factors.

Stress-related and spontaneous stage differentiation of Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasitic protozoan that infects a variety of avian and mammalian hosts including up to one third of the human population worldwide. Developmental differentiation between distinct stages, i.e. sporozoites, tachyzoites and bradyzoites is fundamental for the parasite life cycle and for transmission between hosts. It is also interconnected with the pathogenesis of overt toxoplasmosis and makes T. gondii an important opportunistic pathogen of humans. In order to delineate the underlying mechanisms, several cell culture differentiation systems have been developed which mimic the transition from fast-replicating tachyzoites to slowly proliferating bradyzoites in vitro. Since exogenous stress factors, i.e. alkaline pH, IFN-gamma and other proinflammatory cytokines, chemicals or drugs, heat shock, and deprivation of nutrients have been shown to increase the efficacy of bradyzoite development in vitro, Toxoplasma stage differentiation is largely viewed as a stress-related response to hostile environmental conditions. However, tachyzoite to bradyzoite differentiation also occurs spontaneously in vitro and this raises questions about the importance of stress conditions for triggering stage conversion. High frequencies of spontaneous bradyzoite development in primary and permanent skeletal muscle cells, i.e. cells that preferentially harbour bradyzoite-containing tissue cysts in vivo suggest that the host cell type may be critical. Furthermore, the host cell transcriptome, including the expression of distinct host cell genes, has recently been shown to trigger bradyzoite development and cyst formation. Together, these results strongly indicate that the complex cellular environment, besides exogenous stress factors, may govern the developmental differentiation of T. gondii.