Many eukaryotic microbes have complex life cycles that include both sexual and asexual phases with strict species specificity. Whereas the asexual cycle of the protistan parasite Toxoplasma gondii can occur in any warm-blooded mammal, the sexual cycle is restricted to the feline intestine. The molecular determinants that identify cats as the definitive host for T. gondii are unknown. Here, we defined the mechanism of species specificity for T. gondii sexual development and break the species barrier to allow the sexual cycle to occur in mice. We determined that T. gondii sexual development occurs when cultured feline intestinal epithelial cells are supplemented with linoleic acid. Felines are the only mammals that lack delta-6-desaturase activity in their intestines, which is required for linoleic acid metabolism, resulting in systemic excess of linoleic acid. We found that inhibition of murine delta-6-desaturase and supplementation of their diet with linoleic acid allowed T. gondii sexual development in mice. This mechanism of species specificity is the first defined for a parasite sexual cycle. This work highlights how host diet and metabolism shape coevolution with microbes. The key to unlocking the species boundaries for other eukaryotic microbes may also rely on the lipid composition of their environments as we see increasing evidence for the importance of host lipid metabolism during parasitic lifecycles. Pregnant women are advised against handling cat litter, as maternal infection with T. gondii can be transmitted to the fetus with potentially lethal outcomes. Knowing the molecular components that create a conducive environment for T. gondii sexual reproduction will allow for development of therapeutics that prevent shedding of T. gondii parasites. Finally, given the current reliance on companion animals to study T. gondii sexual development, this work will allow the T. gondii field to use of alternative models in future studies.

The obligate intracellular parasite Toxoplasma gondii is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how T. gondii manipulates host metabolism to support its overall growth rate and non-essential metabolites. To investigate this question, we measured changes in the joint host-parasite metabolome over a time course of infection. Host and parasite transcriptomes were simultaneously generated to determine potential changes in expression of metabolic enzymes. T. gondii infection changed metabolite abundance in multiple metabolic pathways, including the tricarboxylic acid cycle, the pentose phosphate pathway, glycolysis, amino acid synthesis, and nucleotide metabolism. Our analysis indicated that changes in some pathways, such as the tricarboxylic acid cycle, were mirrored by changes in parasite transcription, while changes in others, like the pentose phosphate pathway, were paired with changes in both the host and parasite transcriptomes. Further experiments led to the discovery of a T. gondii enzyme, sedoheptulose bisphosphatase, which funnels carbon from glycolysis into the pentose phosphate pathway through an energetically driven dephosphorylation reaction. This additional route for ribose synthesis appears to resolve the conflict between the T. gondii tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in T. gondii central carbon metabolism that allows T. gondii to energetically-drive ribose synthesis without using NADP+.

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The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle.

Protozoan parasites that infect humans are widespread and lead to varied clinical manifestations, including life-threatening illnesses in immunocompromised individuals. Animal models have provided insight into innate immunity against parasitic infections; however, species-specific differences and complexity of innate immune responses make translation to humans challenging. Thus, there is a need for in vitro systems that can elucidate mechanisms of immune control and parasite dissemination. We have developed a human microphysiological system of intestinal tissue to evaluate parasite-immune–specific interactions during infection, which integrates primary intestinal epithelial cells and immune cells to investigate the role of innate immune cells during epithelial infection by the protozoan parasite, Toxoplasma gondii , which affects billions of people worldwide. Our data indicate that epithelial infection by parasites stimulates a broad range of effector functions in neutrophils and natural killer cell–mediated cytokine production that play immunomodulatory roles, demonstrating the potential of our system for advancing the study of human-parasite interactions.