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    • Since TgGC resisted several knockout attempts

    2021-10-14

    Since TgGC resisted several knockout attempts with CRISPR-Cas9, we utilized traditional epitope tagging and an auxin-inducible degron (AID) system (Brown et al., 2017, Brown et al., 2018, Long et al., 2017) to detect and regulate TgGC expression. TgGC localizes to the apical cap region of the plasma membrane in both intracellular and extracellular parasites. The apical end of apicomplexans is a defining feature of the phylum, demarcated by an apical cytoskeletal complex that focuses [Ala92]-p16 (84-103) of proteins from specialized organelles (e.g., micronemes and rhoptries) necessary for motile processes including extracellular migration, invasion, and egress (Carruthers and Tomley, 2008). Loss of TgGC effectively blocked motile processes in T. gondii, which was explained by a concomitant block in apical microneme secretion. We noted that loss of TgGC exactly phenocopies loss of TgPKG with regard to replication, microneme secretion, invasion, and egress (Brown et al., 2017), suggesting that they are functionally linked by cGMP signaling. In strong support of this possibility, cell-permeable cGMP could stimulate microneme secretion in parasites lacking TgGC but not in parasites lacking TgPKG. From this we conclude that the major role of TgGC is to produce cGMP for PKG-dependent microneme secretion, which cannot be compensated for by other cyclases or signaling pathways. Perhaps the most prominent yet puzzling feature of TgGC is that it appears to be a fusion of two seemingly unrelated eukaryotic genes: a P-type ATPase and a guanylate cyclase. Once thought to have evolved in Alveolates (Biswas et al., 2009), our phylogenetic analysis suggests that hybrid P-type ATPase-guanylate cyclase genes may have evolved earlier in a common ancestor of the Stramenopile, Alveolate, Rhizaria (SAR) supergroup of protists or arisen separately within each superphyla. Initial clues for the importance of this gene fusion have come from Plasmodium, which expresses two stage-specific guanylate cyclases (Carucci et al., 2000), where GCα is likely essential for asexual blood stages (Kenthirapalan et al., 2016) and GCβ regulates ookinete motility and invasion in the mosquito midgut (Hirai et al., 2006, Moon et al., 2009). An important distinction between Plasmodium GCα and GCβ lies in the P-type ATPase domain, which has degenerated in GCβ, suggesting that the two GCs may have different modes of regulation and/or perform separate functions. Interestingly, purified recombinant guanylate cyclase domains of P. falciparum GCα and GCβ displayed disparate GC activities, where only the GC domain of PfGCβ was active (Carucci et al., 2000). Recombinant PfGCα could be intrinsically defective due to the heterologous expression system or require the P-type ATPase domain for GC activity. Also, the sufficiency of the GC domain of PfGCβ to produce cGMP does not exclude the possibility that the P-type ATPase domain acts as a regulatory module for GC activity. Previous studies on heterologous expression of a P-type ATPase-GC gene from Paramecium resulted in processing into separate domains (Linder et al., 1999). A recent study examining TgGC expression by immunoblotting in parasite lysate also failed to detect full-length TgGC (Jia et al., 2017), which could represent an artifact of post-lysis proteolysis or imply that the P-type ATPase domain and GC domain are naturally processed to serve independent functions. Here we presented five lines of evidence that TgGC functions as a full-length, multi-domain protein: (1) full-length (477 kDa + tags) TgGC was detected by immunoblotting for epitope tags at either terminus, though breakdown products were also evident; (2) IF microscopy of 6Ty-GC-mAID-3HA protein in fixed parasites displayed overlapping staining patterns when probing for both termini, as expected for an intact protein; (3) when 6Ty-GC-mAID-3HA was depleted using the C-terminal degron, both N- and C-terminal staining was lost based on IF microscopy and immunoblotting which requires linked termini; (4) ectopic expression of full-length TgGC cDNA, but not truncated or split forms, could rescue loss of endogenous TgGC; and (5) point mutations to conserved catalytic residues in either the P-type ATPase domain or the GC domain rendered TgGC non-functional based on genetic complementation. Taken together, these findings support a model where full-length TgGC requires dual catalytic activity with both domains linked in a single protein for proper function. We suspect the requirements for the co-expression of both domains in a single protein will also apply to orthologs among apicomplexans as supported by a recent study of GCβ in P. yoelli, which was published during review of the present manuscript (Gao et al., 2018).