Signaling differentiation in Toxoplasma gondii through the cAMP dependent protein kinase pathway

Abstract

Toxoplasma gondii is an obligate intracellular parasite. The asexual cycle is composed of two stages. The ability of T.gondii to switch between these stages allows the parasite to evade the immune system and cause disease. The conversion from tachyzoites to bradyzoites is believed to be a stress induced response. Models of stress induced differentiation have been developed and in model systems the cAMP pathway plays a vital role in differentiation. We hypothesized that the cAMP signaling pathway would be important in signaling differentiation in T.gondii.;To test whether the cyclic nucleotide pathways played a role in differentiation we created a novel reporter parasite. Enzymatic assays were used to quantitatively measure replication rates and bradyzoite gene expression after treatment with different agents. Agents that stimulate the cAMP pathway did not cause bradyzoite differentiation while inhibition of the cAMP dependent kinase did cause differentiation. Inhibition of the cGMP dependent kinase caused differentiation.;To further explore the role of cAMP in differentiation, we cloned two isoforms of the catalytic subunit of the cAMP dependent protein kinase (PKAc) from T.gondii. PKA1 and PKA2 are 50% identical and are single copy genes. Both genes are expressed with PKA2 transcript being more abundant. Both isoforms had similar activity and were inhibited by known PKA inhibitors. Both isoforms fractionated to the particulate fraction but localize differently by immunofluorescence. Overexpression of PKA1 but not PKA2 inhibits tachyzoite replication. The different phenotypes observed with overexpression along with their different location suggest that these isoforms are non redundant.;We have begun to explore ways PKA could mediate differentiation. T.gondii's Cdc2 orthologue, TPK2, is a critical regulator of cell cycle and has a consensus PKA phosphorylation site. TPK2 is phosphorylated by bovine PKA. We have developed tachyzoite cDNA arrays to examine changes in gene expression. Microarray analysis using H89 and Compound1 show limited transcriptional changes at 24 hours while treatment with sodium nitroprusside resulted in more changes.;In summary, we have implicated the cAMP pathway in controlling differentiation and components of the cAMP pathway have been cloned. This work provides evidence that the cAMP pathway is involved in differentiation.