PKA activity is essential for adult <i>S. mansoni</i> viability <i>in vitro</i>.

<p>Adult <i>S. mansoni</i> worm pairs were maintained in medium containing varying concentrations of H-89 (A and B) or PKI 14–22 amide (C and D). Survival in the presence of each inhibitor was plotted against time (A and C). For H-89, concentrations of inhibitor are as follows: 0 and 1 µM (•); 10 µM (♦); 25 µM (▪); 50, 100, 250, and 500 µM (▾). For PKI 14–22 amide concentrations of inhibitor are as follows: 0, 1, 10, 25, and 50 µM (•); 100 µM (♦); 250 and 500 µM (▪). Treatment groups containing 6 worm pairs each (12 worms total) were used for each concentration. B and D, micrographs of representative worm pairs incubated in the presence of 100 µM H-89 (B) or100 µM PKI 14–22 amide (D). Scale bars = 1 mm. e, parasite eggs. Data are representative of three independent experiments.</p

Schistosome PKA activity is sensitive to adenylyl cyclase modulation.

<p>Kinase activity was measured in kinase reactions containing Sm lysate from adult worm pairs (A and C) that were previously treated with SQ22536 (A) or forskolin (C). Treatment groups contained 10 worm pairs (20 worms total) each and were performed in triplicate. Kinase reactions containing recombinant human PKA-Cα (B and D) were treated directly with SQ22536 (B) or forskolin (D). ▴, inhibitor-treated; •, no inhibitor. Data are representative of two independent experiments.</p

Identification of SmPKA-C as a PKA-C subunit from <i>S. mansoni</i>.

<p>A, Amino acid alignment of both SmPKA-C and putative splice variant peptide sequences with PKA-C sequences from <i>Caenorhabditis elegans</i> (Cekin-1; NP_493605), <i>Drosophila melanogaster</i> (DmPKA; NP_476977), <i>Mus musculus</i> (MmPKAa; P05132) and <i>Homo sapiens</i> (HsPKAa; P17612). B, Phylogenetic analysis comparing both SmPKA-C amino acid sequences to the following PKA-C sequences from other organisms: <i>Homo sapiens</i> PKA-Cα (HsPKAa; P17612), <i>H. sapiens</i> PKA-Cβ (HsPKAb; P22694), <i>H. sapiens</i> PKA-Cγ (HsPKAg; P22612), <i>Mus musculus</i> PKA-Cα (MmPKAa; P05132), <i>M. musculus</i> PKA-Cβ (MmPKAb; P68181), <i>Rattus norvegicus</i> PKA-Cα (RnPKAa; P27791), <i>R. norvegicus</i> PKA-Cβ (RnPKAb; P68182), <i>D. melanogaster</i> (DmPKA; NP_476977), <i>Aplysia californica</i> (AcPKA; CAA45014), and <i>Acyrthosiphon pisum</i> (ApPKA; XP_001946114). C, SmPKA-C transcript is expressed in cDNA of all the life cycle stages of <i>S. mansoni</i>. 1, egg, 2, miracidium, 3, sporocyst, 4, cercaria, 5, schistosomulum, 6, adult female, 7, adult male.</p

SmPKA-C encodes for an essential kinase activity.

<p>A, Electroporation of adult worms with 30 µg of SmPKA-C dsRNA (▪) resulted in 75% mortality while worms treated with control dsRNA (•) exhibited 100% survival. B, Three days after electroporation, SmPKA-C transcript levels were decreased in worms electroporated with 10 µg of SmPKA-C dsRNA, while transcript levels were unaffected in control dsRNA-treated worms, as determined by real-time PCR. C, 7 days after electroporation, SmPKA-C dsRNA-treated (•) and control dsRNA-treated worms (▴) were incubated for 2 h in 100 µM forskolin. PKA activity was significantly decreased after electroporation with 10 µg SmPKA-C dsRNA compared to control dsRNA-treated worms.</p

Detection of a putative schistosome PKA in parasite lysates.

<p>A, Western blotting of lysates from adult <i>S. mansoni</i> (Sm lysate) and two human cell lines (293FT, HT1080) using a polyclonal antibody specific for the human PKA-Cα subunit revealed the presence of a putative PKA-C subunit of the expected molecular weight of 40 kDa in the schistosome lysate. B, Putative PKA activity is detectable in lysate of adult <i>S. mansoni</i> worms using a fluorescence-based kinase assay. ▴, Sm lysate, • recombinant human PKA-Cα. C–F, Kinase activity was measured in kinase reactions containing Sm lysate (C and E) and recombinant human PKA-Cα (D and F) and the PKA inhibitors H-89 (C and D) and PKI 14–22 (E and F). ▴, inhibitor-treated; •, no inhibitor. B–F, each time point represents the mean of three biological replicates.</p