The cGMP signaling pathway is known to be one of the major regulators of diverse functions in eukaryotes; however, its function in protozoa is poorly understood. In this work, a guanylate cyclase coupled to an N-terminal P4-ATPase was reported in the intracellular parasite Toxoplasma gondii. An in silico analysis indicated activation of guanylate cyclase through heterodimerization of its cyclase domains and provided valuable insight into possible functions of its ATPase domain. This protein (477-kDa), termed TgATPaseP-GC in this study, localizes in the plasma membrane at the apical pole of the parasite. TgATPaseP-GC is refractory to genetic deletion and its CRISPR/Cas9-assisted cleavage prematurely terminates the lytic cycle of T. gondii. Furthermore, a Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP in tachyzoites and inhibited parasite growth due to impairments of motility-dependent processes of exit and entry. Despite its time-limited function, TgATPaseP-GC is constitutively expressed throughout the lytic cycle, implying post-translational regulation of cGMP signaling. Last but not least, the presence of TgATPaseP GC orthologs in other alveolate implies a divergent repurposing of cGMP signaling pathways in protozoa. Furthermore, an optogenetic approach was used to express the cGMP pathway by a photo-activated rhodopsin guanylate cyclase (RhoGC) in T. gondii. This system allowed controlled elevation of cGMP by light in a rapid and reversible manner. RhoGC excitation significantly stimulated parasite motility, the impact of which was also monitored with increased intrusion and egress; in contrast to the genetic knockdown of TgATPaseP-GC. The system enables the mediators of the cGMP signaling pathway to be identified by phosphoproteomics.
cGMP signaling is known as one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. In the scope of this thesis, an exclusive guanylate cyclase coupled with N-terminal P4-ATPase was reported in an obligate intracellular parasite Toxoplasma gondii. In silico analysis indicated an activation of the guanylate cyclase by heterodimerization of its two cyclase domains and offered valuable insights into possible functions of its ATPase domain. This bulky protein (477-kDa), termed in this study as TgATPaseP-GC to reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9-assisted disruption aborts the lytic cycle of T. gondii. besides, Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP in tachyzoites and inhibited the parasite growth due to impairments of motility-dependent egress and invasion events. Notably, despite its temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans. Furthermore, an optogenetic approach was utilized to induce cGMP pathway by a photo-activated rhodopsin-guanylate cyclase (RhoGC) in T. gondii. The system enabled a light control of cGMP elevation on crucial steps of lytic cycle in a fast, spatial and reversible manner. Excitation of RhoGC significantly stimulated the parasite motility of which impact was also monitored with an increased host-cell invasion and egress; as opposed to the genetic knockdown of TgATPaseP-GC. Having an established optogenetic system in the parasite allows to identify downstream targets of cGMP signaling via phosphoproteomic analysis.