ABSTRACT
Examining host-pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host-parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host-parasite interaction.
ABSTRACT
Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.
ABSTRACT
Cytohormonal profile of unstained vaginal smears were studied under phase contrast microscope to define various cell morphologies in detail and build up of smear pattern serially in various stages of menstrual cycle and first trimester of pregnancy. The findings were correlated clinically and found comparable to Papanicolaou stained smears. The maturation index was calculated more objectively by phase contract microscopy because of the refractile appearance of the pyknotic nuclei of superficial cells. The prognostication of abortion was done by combining abnormal maturation index and the presence of syncytiotrophoblasts in vaginal smears. Under phase contrast microscope, the sensitivity of abnormal smear was 87.5%, the specificity was 95%, the positive predictive value was 96.55% and the overall accuracy was 90% as compared to positive Papanicolaou smear whose sensitivity though 93%, had low specificity of 50% only. Phase contrast study appears to offer certain distinct advantage over the conventional light microscopy for quick, comprehensive and quantitative assessment of the study material. It allows the physician to detect changes in the material obtained from the patients without detour of laboratory fixation and staining. It is technically easy as errors in interpretation due to unsatisfactory fixation, staining and artefacts are obviated. Thus phase contrast microscopy offers an alternative and easy method of cytohormonal evaluation of wet and unstained smears.
