Toxoplasma gondii is an exceptionally tractable apicomplexan
Toxoplasma gondii is an exceptionally tractable apicomplexan parasite that expresses a limited yet representative subset of apicomplexan cyclase orthologs, making it an ideal model for investigating cyclic nucleotide signaling in Apicomplexa. Of the five putative nucleotide cyclases, only TgACα1 and TgACβ have been functionally characterized, both being classified as non-essential based on genetic disruption (Jia et al., 2017). The sole predicted guanylate cyclase (TgGC) awaits functional assignment but possesses an interesting domain structure that resembles a fusion of two seemingly unrelated eukaryotic genes: a P-type ATPase and a guanylate cyclase. P-type ATPases are a class of integral membrane proteins that utilize Benzoylmesaconitine australia from ATP to transport ions, or flip lipids, across biological membranes (Palmgren and Nissen, 2011). The hybrid domain structure of TgGC is restricted to Apicomplexa and related protists, but its functional significance has not been resolved in any organism (Gould and de Koning, 2011). Knowing which enzymes initiate microneme secretion for motility and how they operate is critical for understanding how these parasites sense and respond to their environment to transmit from one host cell to the next.
Here we performed a CRISPR knockout screen of all T. gondii cyclases, demonstrating that only TgGC was refractory to deletion. Conditional knockdown using an auxin-inducible degron (AID) system (Brown et al., 2017) revealed that TgGC is an initiator of motility in T. gondii by controlling microneme secretion. Transgenic complementation with mutant versions of TgGC also defines an unexpected and important role for the P-type ATPase domain. Finally, we adapted the AID system for regulating parasite protein expression in mice, demonstrating a critical role for TgGC in vivo. Altogether, this work identifies a multi-domain GC that initiates essential biological adaptations in T. gondii, a role that may extend to other members of this deadly phylum of parasites.
Discussion Apicomplexan parasites have adapted cyclic nucleotide signaling to precisely regulate the timing and amplitude of essential motile processes (Baker et al., 2017), yet it is unclear how these signals are actually initiated in these organisms. Here we performed a reverse genetic screen of enzymes predicted to synthesize cyclic nucleotides in the model apicomplexan T. gondii. From this screen, we found that a guanylate cyclase (TgGC) is essential for the lytic life cycle of T. gondii, controlling motile processes including gliding motility, invasion, and egress. The defect in motility produced by loss of TgGC was explained by a profound defect in cGMP-dependent protein secretion that was reversed by supplementing cell-permeable cGMP. A systematic analysis of the multi-domain architecture of TgGC through genetic complementation with different domain and point mutants revealed a critical requirement for both P-type ATPase and guanylate cyclase activities. To determine if TgGC contributes to T. gondii infection of a mammalian host, we developed a system for investigating the function of essential T. gondii proteins in an animal model of toxoplasmosis. We found that auxin-induced depletion of TgGC in mice infected with T. gondii protected mice from lethal toxoplasmosis, indicating that TgGC is required for T. gondii fitness both in vitro and in vivo. Conditional knockdown studies have demonstrated that T. gondii expresses two essential cyclic nucleotide-dependent kinases, TgPKAr-TgPKAc1 (Jia et al., 2017) and TgPKG (Brown et al., 2017), implying that both cAMP and cGMP are also essential in T. gondii. However, the enzymes responsible for producing cAMP and cGMP (i.e., cyclases) have yet to be identified and functionally described. Our ability to knock out each of the adenylate cyclases individually was likely the result of functional redundancy. Since loss of TgACα1, TgACα2, and TgACβ each had significant costs to parasite fitness, it is of interest to know whether loss of two or more simultaneously would produce synthetic lethality. As our goal for this study was to identify essential cyclases, we focused our attention to the guanylate cyclase (TgGC) that was refractory to genetic deletion.