Crosstalk between PKA and PKG controls pH ‐dependent host cell egress of Toxoplasma gondii

Abstract

Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.

Essential cGMP Signaling in Toxoplasma Is Initiated by a Hybrid P-Type ATPase-Guanylate Cyclase

“…Curiously, it has yet to be demonstrated that any P-type ATPase/GC gene fusion can produce a full-length protein (Linder et al, 1999). A recent study examining TgGC expression by immunoblotting 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.…”

“…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.…”

“…Importantly, PKG also controls a final step in microneme secretion as loss of PKG expression or activity cannot be overcome by systemically elevated Ca 2+ , PA, or cGMP (Brown et al, 2017; Brown et al, 2016; Bullen et al, 2016). For balance, cAMP- dependent protein kinase (PKA) has been proposed as a negative regulator of microneme secretion to prevent premature secretion and egress during parasite replication (Jia et al, 2017). Cyclic nucleotides are synthesized from nucleoside triphosphates by nucleotide cyclases.…”

“…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.…”

Calcium negatively regulates secretion from dense granules inToxoplasma gondii

“…At these concentrations only minor reduction in GRA1 secretion was seen, however, as BIPPO concentrations were further increased through 1 -5 µM a clear decrease in secreted GRA1 was observed ( Figure 1B). To validate that BIPPO was acting through PKG to inhibit dense granule secretion, the PKG inhibitor 'compound 2' was tested to see if it could block the secretion responses of BIPPO (Brochet et al, 2014;Donald et al, 2002;Jia et al, 2017;Wiersma et al, 2004). When tachyzoites were pre-treated with compound 2, all constitutive and BIPPOinducible MIC2 secretion was lost, as well as the BIPPO-induced inhibition of dense granule secretion ( Figure 1C).…”