Abstract
cGMP signaling is one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. Herein, we report an exclusive guanylate cyclase coupled with N-terminal P4-ATPase in a common parasitic protist, Toxoplasma gondii This bulky protein (477-kD), termed TgATPaseP-GC to fairly reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite, whereas the corresponding cGMP-dependent protein kinase (TgPKG) is distributed in the cytomembranes. 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 and inhibited the parasite growth due to impairments in the motility-dependent egress and invasion events. Equally, repression of TgPKG by a similar strategy recapitulated phenotypes of the TgATPaseP-GC-depleted mutant. Notably, despite a 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, and offers an excellent drug target against the parasitic protists.
Highlights
CGMP is regarded as a common intracellular second messenger, which relays endogenous and exogenous cues to the downstream mediators, and thereby regulates a range of cellular processes in prokaryotic and eukaryotic organisms (Lucas et al, 2000; Hall & Lee, 2018)
Our genome searches identified a single putative guanylate cyclase (GC) in the parasite database (ToxoDB) (Gajria et al, 2008), comprising multiple P-type ATPase motifs at its N terminus and two nucleotide cyclase domains at the C terminus
This study characterized an alveolate-specific protein, termed TgATPaseP-GC which imparts a central piece of cGMP signaling conundrum in T. gondii
Summary
CGMP is regarded as a common intracellular second messenger, which relays endogenous and exogenous cues to the downstream mediators (kinases, ion channels, etc.), and thereby regulates a range of cellular processes in prokaryotic and eukaryotic organisms (Lucas et al, 2000; Hall & Lee, 2018). PKG (or cGMP-dependent protein kinase) on the other hand is a major mediator of cGMP signaling in most eukaryotic cells; it phosphorylates a repertoire of effector proteins to exert a consequent subcellular response. Much of our understanding of cGMP-induced transduction is derived from higher organisms, namely, mammalian cells, which harbor four soluble GC subunits (α1, α2, β1, and β2) functioning as heterodimers, and seven membrane-bound GCs (GC-A to GC-G), occurring mostly as homodimers (Lucas et al, 2000; Potter, 2011). The type I PKGs have two soluble alternatively spliced isoforms (α and β) functioning as homodimers, whereas the type II PKGs are membrane-bound proteins (MacFarland, 1995; Pilz & Casteel, 2003), which form apparent monomers (De Jonge, 1981) as well as dimers (Vaandrager et al, 1997)
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