Journal: PLoS Biology
Article Title: Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion
Figure Lengend Snippet: Deletion of PfPDEβ bypasses the need for PKG activity upstream of MyoA S 19 phosphorylation in mature schizonts and results in premature proteolytic shedding of AMA1. (A) PKG-independent phosphorylation of MyoA in PfPDEβ-null parasites. Western blot of PKG inhibitor–treated (+C2) and E64-treated PfPDEβ ΔcatHA schizont proteins from DMSO- and RAP-treated PfPDEβ ΔcatHA cultures, probed with a phospho-S 19 MyoA antibody. The blot was reprobed with a polyclonal anti-MyoA antibody to determine the relative amounts of MyoA present in each sample. The lower panel shows total protein levels in the gel prior to blotting. The schematic shows the effect of various compounds on egress: Compound 2 and BAPTA-AM block egress upstream of PVM rupture, BIPPO induces egress, and E64 prevents erythrocyte plasma membrane rupture. (B) The PKA inhibitor H89 inhibits MyoA S 19 phosphorylation. Western blot analysis of E64-arrested wild-type schizonts (mock-treated PfPDEβ ΔcatHA ) treated with increasing concentrations of the PKA inhibitor H89. A sample was taken before inhibitor addition (start) and a Compound 2–blocked sample served as negative control. Blots were probed with antibodies against phospho-S 19 MyoA, total MyoA, and GAPDH as a loading control. (C) The PDE inhibitor BIPPO induces MyoA phosphorylation. Western blot analysis of Compound 2–arrested mock- or RAP-treated PfPDEβ ΔcatHA schizonts, following incubation with increasing concentrations of the PDE inhibitor BIPPO. A sample was taken before inhibitor addition to show the absence of MyoA S 19 phosphorylation (start). Blots were probed with the same antibodies as in (B). (D) BAPTA-AM inhibits MyoA phosphorylation. Effects of BAPTA-AM on MyoA S 19 phosphorylation in Compound 2–arrested (top panel) or E64-arrested (bottom panel) PfPDEβ ΔcatHA schizonts following mock or RAP treatment. Western blots were additionally probed with the same antibodies as in B and C. (E) Dual-stained IFAs showing the cellular distribution of AMA1 in RAP- and mock-treated (DMSO) PfPDEβ ΔcatHA schizonts. Schizonts were co-stained with an AMA1 antibody (red) and a HA antibody (green) to detect the presence or absence of PfPDEβ. DAPI (blue) was used to visualise nuclear material. Scale bar, 5 μm. The bar chart (right) shows quantitation of proportions of schizonts displaying apical or peripheral AMA1 staining in E64-arrested PfPDEβ ΔcatHA schizonts. Data presented are from a representative experiment in which > 50 schizonts per condition were counted by two researchers. Error bars, 1 SD. (F) Western blot analysis of culture supernatants from RAP- and mock-treated PfPDEβ ΔcatHA purified schizonts sampled over 90 minutes of culture. The blot was probed with antibodies specific for the micronemal proteins AMA1 and EBA175, as well as SERA5, to measure schizont rupture. Positions of molecular weight markers are indicated (left). The blot is representative of at least three independent experiments. (G) Quantitation of AMA1 shedding by IFA in merozoites released from RAP- and mock-treated PfPDEβ ΔcatHA schizonts. Purified merozoites were stained with an antibody against the AMA1 ectodomain. Thumbnails show examples for translocated (surface), shed (−), and micronemal (apical) AMA1 in green. Nuclei were stained with DAPI (blue). The bar charts show the mean proportions of merozoites exhibiting surface (left) and apical (right) AMA1 staining for each condition. More than 100 merozoites were counted by two researchers. Error bars, 1 SD. *, significant by unpaired t test ( p -value = 0.005); n.s., not significant ( p -value = 0.306). AMA1, apical membrane antigen-1; BAPTA-AM, 1,2-bis( o -aminophenoxy)ethane- N , N , N′ , N′ -tetraacetic acid-acetoxymethyl ester; BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; EBA175, erythrocyte-binding antigen 175; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, haemagglutinin; IFA, immunofluorescence assay; MyoA, myosin A; n.s., not significant; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PVM, parasitophorous vacuole membrane; RAP, rapamycin; SERA5, serine repeat antigen 5.
Article Snippet: Small molecules and antibodies WR99210 was a kind gift from Jacobus Pharmaceuticals (New Jersey), RAP and the cysteine protease inhibitor E64 were purchased from Sigma, the PKG inhibitor Compound 2 was synthesised by MRC Technology (London, United Kingdom), the PKA inhibitor H89 was obtained from TOCRIS Biosciences, the PDE inhibitor BIPPO [ ] was a kind gift from Philip E. Thompson (Monash University, Australia).
Techniques: Activity Assay, Western Blot, Blocking Assay, Negative Control, Incubation, Staining, Quantitation Assay, Purification, Molecular Weight, Immunofluorescence, Binding Assay