Asexual Development and Virulence Are Not Affected by Knockout of Five Sexual-Stage Candidate Genes in Toxoplasma gondii

Abstract

Simple Summary Toxoplasma gondii is a common parasite that infects humans and nearly all birds and mammals, posing a serious threat to immunocompromised individuals and pregnant women, and causing significant economic losses to the livestock industry. Its sexual reproduction occurs exclusively in cat intestines, a poorly understood stage critical for transmission. Based on our single-cell transcriptomic data from infected cat intestines, we selected five previously uncharacterized genes that are highly expressed in sexual stages, including three zinc finger protein genes and two subtilisin-like protease genes. Using CRISPR-Cas9 gene-editing technology, we successfully constructed knockout mutants and systematically evaluated their tachyzoite phenotypes to confirm phenotypic neutrality before future feline infection experiments. The results show that knockout of these five genes did not significantly affect in vitro parasite growth, virulence in mice, or brain cyst formation, corroborating the transcriptional predictions. Analysis of publicly available ToxoDB transcriptomic data confirmed the expression patterns of four of these genes observed in our previous analysis. These findings indicate that the five genes are dispensable for the asexual reproduction and virulence of T. gondii and suggest that these genes may be involved in sexual reproduction within the cat intestine, a possibility that requires direct experimental validation. This study provides well-characterized knockout strains and candidate genes, establishing a foundation for investigating sexual-stage biology and developing transmission-blocking strategies against T. gondii infection.