Single-cell analysis of Toxoplasma cat stages identifies multiple developmental trajectories.

Toxoplasma gondii"s sexual development is limited to the epithelial cells of the small intestine of felids. Sexual development is a key to genetic diversity and this process is crucial to dissemination, as a single felid can shed millions of environmentally stable oocysts. Due to challenges associated with cultivating presexual and ultimately sexual stages, there is still much to learn about how this developmental program is triggered and executed by the parasite in response to infection of the feline host. Prior studies (Hehl et al., 2015 and Behnke et al,. 2014) demonstrated clear differences in the transcriptomes of asexual and sexual stages using bulk transcriptomics, and metabolic differences in the feline host play a key role in triggering the transition into sexual stages (Martorelli di Genova et al., 2019). Important recent work (Artunas et al., 2024, among others) has identified multiple parasite transcription factors as critical suppressors of presexual development, permitting the study of presexual stages and opening ways to using genetics to drive the full sexual cycle in vitro. To further investigate the sexual developmental phase of Toxoplasma and identify previously unknown regulators of the distinct phases of sexual development, we have conducted multiple single-cell transcriptomics analyses of Toxoplasma parasites isolated from the small intestines of infected cats. To do this we used flow cytometry to sort fluorescent reporter strains of T. gondii VEG from scraped small intestine samples and obtained single-cell transcriptomic data from 16,966 cells across two experiments. The data made it possible to classify, characterize, and parse each cell at the transcriptome level, which led to the identification of rare cell populations, including some that we hypothesize are macrogametes and microgametes. Candidate genes emerging from this study have now been targeted in a CRISPR-CAS9 screen and we are using Perturb-seq and loss of function assays on feline enteroepithelial stages to determine which factors are required for sexual development and/or the regulation of sexual stage gene networks. We aim to identify previously unknown drivers of differentiation, especially those that mediate gametogenesis. Our single-cell data also extends what is known about gene expression changes throughout sexual development and should be useful to those in the field working towards inducing gametogenesis, mating, and oocyst formation in feline-free conditions.