Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni

Abstract

cAMP-dependent protein kinases (PKAs) are the main transducers of cAMP signalling in eukaryotic cells. Recently we reported the identification and characterisation of a PKA catalytic subunit (SmPKA-C) in Schistosoma mansoni that is required for adult schistosome viability in vitro. To gain further insights into the role of SmPKA-C in biological processes during the schistosome life cycle, we undertook a quantitative analysis of SmPKA-C mRNA expression in different life cycle stages. Our data shows that SmPKA-C mRNA expression is developmentally regulated, with the highest levels of expression in cercariae and adult female worms. To evaluate the biological role of SmPKA-C in these developmental stages, cercariae and adult worms were treated with various concentrations of PKA inhibitors. Treatment of cercariae with H-89 or PKI 14-22 amide resulted in loss of viability suggesting that, as in adults, PKA is an essential enzyme activity in this infectious larval stage. In adult worms, in vitro exposure to sub-lethal concentrations of H-89 or PKI 14-22 amide resulted in inhibition of egg production in a dose-dependent manner. Furthermore, using a murine model of schistosome infection where S. mansoni fecundity is impaired, we show that reduced rates of egg production in vivo correlate with significant reductions in SmPKA-C mRNA expression and PKA activity. Finally, restoration of parasite egg production in vivo also resulted in normalisation of SmPKA-C mRNA expression and PKA activity. Taken together, our data suggest that PKA signalling is required for cercarial viability and may play a specific role in the reproductive activity of adult worms.

Fig. 1

Schistosoma mansoni protein kinase A catalytic subunit (SmPKA-C) mRNA expression and PKA activity in the larval stages of S. mansoni. A) Relative SmPKA-C mRNA levels in S. mansoni larval stages as determined by quantitative PCR. Data were normalized to the levels detected in a standard preparation of adult male S. mansoni RNA. B) Detection of PKA activity in lysate of S. mansoni cercariae using a fluorescence-based kinase assay. ◆, Cercarial lysate, ●, recombinant human PKA-Cα. C and D) Survival of S. mansoni cercariae in the presence of PKA-C inhibitors. Cercariae in groups of 20 were maintained in water containing varying concentrations of H-89 (C) or PKI 14-22 amide (D). Survival in the presence of each inhibitor was plotted against time. Inhibitor concentrations are as follows: 0 and 1 μM (●); 10 μM (◆); 50 μM (▲); 100 μM (■). Data are representative of two independent experiments. RFU, relative fluorescence units.

Fig. 2

Schistosoma mansoni protein kinase A catalytic subunit (SmPKA-C) mRNA expression and PKA activity in the intra-mammalian stages of S. mansoni. A) Relative SmPKA-C mRNA levels in adult male and female S. mansoni worms and schistosomula as determined by quantitative PCR. Data were normalized to the levels detected in a standard preparation of adult male S. mansoni RNA. B) Detection of PKA activity in lysates of adult male and female S. mansoni worms using a fluorescence-based kinase assay. ●, recombinant human PKA-Cα, ▲, male lysate, △, female lysate. Data are representative of two independent experiments. RFU, relative fluorescence units.

Fig. 3

Effects of sub-lethal PKA inhibition on egg production by Schistosoma mansoni worm pairs in vitro. Adult S. mansoni worm pairs were maintained in medium containing varying concentrations of H-89 (A) or PKI 14-22 amide (B). Eggs produced per pair in the presence of each inhibitor were plotted against time. Treatment and control groups containing six worm pairs each were used for each inhibitor concentration. *, P < 0.05 relative to control. Data are representative of two independent experiments.

Fig. 4

Schistosoma mansoni protein kinase A catalytic subunit (SmPKA-C) mRNA expression and PKA activity in S. mansoni from RAG-1^-/- mice. A) Relative SmPKA-C mRNA levels in adult S. mansoni worms from wild type (WT) and RAG-1^-/- mice, as determined by quantitative PCR. B) PKA activity in adult S. mansoni from wild type (WT) and RAG-1^-/- mice. ▲, Wild type mice; ●, RAG-1^-/- mice. C) PKA activity in adult S. mansoni from RAG^-/- mice after treatment with forskolin for 2 h. ▲, forskolin-treated worms; ●, DMSO control-treated worms. Data are representative of three independent experiments. RFU, relative fluorescence units.

Fig. 5

Restoration of Schistosoma mansoni protein kinase A catalytic subunit (SmPKA-C) mRNA expression, PKA activity and egg production in RAG-1^-/- mice. To restore parasite egg production in vivo, RAG-1^-/- mice were infected with S. mansoni and injected i.p. twice each week with 20 μg of lipopolysaccharide (LPS) for 6 weeks, while infected control RAG-1^-/- mice received injections of PBS. Parasites were then isolated for analysis at 6 weeks p.i.. A) Relative SmPKA-C mRNA levels in adult S. mansoni worms from wild type (WT), control RAG-1^-/- and LPS-treated RAG-1^-/- mice as determined by quantitative PCR. Data were normalized to the levels of SmPKA-C mRNA detected in adult S. mansoni from WT mice. B) PKA activity in S. mansoni from WT, control RAG-1^-/- and LPS-treated RAG-1^-/- mice. ▲, WT mice; ●, control RAG-1^-/- mice; Δ, LPS-treated RAG-1^-/- mice. C) Number of eggs per schistosome pair deposited in the livers of WT, PBS-treated control RAG-1^-/- and LPS-treated RAG-1^-/- mice. Data are representative of two independent experiments. RFU, relative fluorescence units.