An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

Cellulose fibrils formation and organisation of cytoskeleton during encystment are essential for Acanthamoeba cyst wall architecture.

Acanthamoebae success as human pathogens is largely due to the highly resistant cysts which represent a crucial problem in treatment of Acanthamoeba infections. Hence, the study of cyst wall composition and encystment play an important role in finding new therapeutic strategies. For the first time, we detected high activity of cytoskeletal elements - microtubular networks and filamentous actin, in late phases of encystment. Cellulose fibrils - the main components of endocyst were demonstrated in inter-cystic space, and finally in the ectocyst, hereby proving the presence of cellulose in both layers of the cyst wall. We detected clustering of intramembranous particles (IMPs) and their density alterations in cytoplasmic membrane during encystment. We propose a hypothesis that in the phase of endocyst formation, the IMP clusters represent cellulose microfibril terminal complexes involved in cellulose synthesis that after cyst wall completion are reduced. Cyst wall impermeability, due largely to a complex polysaccharide (glycans, mainly cellulose) has been shown to be responsible for Acanthamoeba biocide resistance and cellulose biosynthesis pathway is suggested to be a potential target in treatment of Acanthamoeba infections. Disruption of this pathway would affect the synthesis of cyst wall and reduce considerably the resistance to chemotherapeutic agents.

Response of cell lines to actual and simulated inoculation with Cryptosporidium proliferans.

The need for an effective treatment against cryptosporidiosis has triggered studies in the search for a working in vitro model. The peculiar niche of cryptosporidia at the brush border of host epithelial cells has been the subject of extensive debates. Despite extensive research on the invasion process, it remains enigmatic whether cryptosporidian host-parasite interactions result from an active invasion process or through encapsulation. We used HCT-8 and HT-29 cell lines for in vitro cultivation of the gastric parasite Cryptosporidium proliferans strain TS03. Using electron and confocal laser scanning microscopy, observations were carried out 24, 48 and 72 h after inoculation with a mixture of C. proliferans oocysts and sporozoites. Free sporozoites and putative merozoites were observed apparently searching for an appropriate infection site. Advanced stages, corresponding to trophozoites and meronts/gamonts enveloped by parasitophorous sac, and emptied sacs were detected. As our observations showed that even unexcysted oocysts became enveloped by cultured cell projections, using polystyrene microspheres, we evaluated the response of cell lines to simulated inoculation with cryptosporidian oocysts to verify innate and parasite-induced behaviour. We found that cultured cell encapsulation of oocysts is induced by parasite antigens, independent of any active invasion/motility.

In vitro excystation of Cryptosporidium muris oocysts and viability of released sporozoites in different incubation media.

This study aimed to evaluate and document the excystation process of Cryptosporidium muris oocysts in various incubation media, and to monitor the behaviour of excysting and freshly excysted sporozoites. A test of oocyst viability, using fluorescent double staining with fluorescein diacetate and propidium iodide, was performed prior to each experimental assay. Light microscope observations confirmed that relatively often only three sporozoites were released; the fourth one either left the oocyst later together with a residual body or remained trapped within the oocyst wall. These results suggest that successful oocyst excystation is not limited by the viability of all four sporozoites. Darkening of oocysts to opaque and their specific movement (the so-called "oocyst dancing") preceded the final excystation and liberation of sporozoites, while the dormant oocysts appeared refractive. The process of excystation in C. muris is not gradual as generally described in cryptosporidia but very rapid in an eruptive manner. Experiments were performed using oocysts stored at 4 °C for various time periods, as well as oocysts freshly shed from host rodents (Mastomys coucha) of different ages. The most suitable medium supporting high excystation rate (76 %) and prolonged motility of sporozoites was RPMI 1640, enriched with 5 % bovine serum albumin (BSA). Our results emphasize that to reliably evaluate the success of in vitro excystation of cryptosporidia, not only the number of released sporozoites in a set time period should be taken into consideration but also their subsequent activity (motility), as it is expected to be essential for the invasion of host cells.

Life cycle of Cryptosporidium muris in two rodents with different responses to parasitization.

This study focuses on mapping the life cycle of Cryptosporidium muris in two laboratory rodents; BALB/c mice and the southern multimammate rat Mastomys coucha, differing in their prepatent and patent periods. Both rodents were simultaneously experimentally inoculated with viable oocysts of C. muris (strain TS03). Animals were dissected and screened for the presence of the parasite using a combined morphological approach and nested PCR (SSU rRNA) at different times after inoculation. The occurrence of first developmental stages of C. muris in stomach was detected at 2.5 days post-infection (dpi). The presence of Type II merogony, appearing 36 h later than Type I merogony, was confirmed in both rodents. Oocysts exhibiting different size and thickness of their wall were observed from 5 dpi onwards in stomachs of both host models. The early phase of parasitization in BALB/c mice progressed rapidly, with a prepatent period of 7.5-10 days; whereas in M. coucha, the developmental stages of C. muris were first observed 12 h later in comparison with BALB/c mice and prepatent period was longer (18-21 days). Similarly, the patent periods of BALB/c mice and M. coucha differed considerably, i.e. 10-15 days vs chronic infection throughout the life of the host, respectively.