Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase

Abstract

Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP) exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG) has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG) is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG) has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA), maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER) was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony.

Figure 1. PfPKG expression peaks in late blood stages and is carbonate-soluble.

(A) Western blots of synchronised cultures of the PfPKG-HA-3A clone and WT parasites (3D7 clone), 24 hours (mostly mid trophozoites), 30 hours (mostly late trophozoites), 41 hours (mostly early schizonts) and 46 hours (mostly late schizonts) post invasion were detected with anti-HA and anti-humanPKG, respectively. Blots were re-probed with an antibody against Pfαtubulin to estimate the relative total protein loading between lanes. (B) Sequential solubilisation of parasite proteins from saponin-released late trophozoites and schizonts. S1: soluble protein fraction (5 mM Tris-HCl, freeze thaw); S2: peripheral membrane fraction (extraction with 100 mM Na₂CO₃); S3: integral membrane fraction (extraction with 4% SDS/0.5% TX-114/0.5×PBS). Equal volumes of the three supernatants were analysed by SDS-PAGE and Western blots were probed for the integral membrane protein PfPMV , stripped and re-probed simultaneously for PfGAPDH and PfPKG-HA. Densitometric analysis of the scan of the blot presented revealed that 89.3% of PfPKG-HA is present in fraction S1, while fractions S2 and S3 contain 9.5% and 1.2%, respectively. (C) Immunofluorescent anti-HA detection in fixed smears of erythrocytic stages of the PfPKG-HA-3A clone. Representative images of (i) a ring stage parasite, (ii) three early trophozoites, (iii) an early schizont, (iv, v) late schizonts (approximate hours post invasion: (i) 4–10, (ii) 20–26, (iii) 33–39, (iv, v) 45–48) and (vi) a stage III gametocyte are shown together with bright field images (first column) and parasite nuclei stained with DAPI (second column). Bars ∼5 µM.

Figure 2. Subcellular location of PfPKG in mature schizonts.

Dual immunofluorescent detection of PfPKG-HA in fixed smears of early and late schizonts of the PfPKG-HA-3A clone together with (A) PfGAPDH , (B) PfBiP , (C) PfPMV , (D) PfRab11A and (E) PfGAP45 . Representative images are shown for each antibody, together with bright field images (first column) and parasite nuclei stained with DAPI (in the merged image). Bars ∼5 µM. To quantify co-localisation, Pearson coefficients of the individual stains were calculated using Imaris image analysis software (Bitplane).

Figure 3. Parasite morphology and global protein phosphorylation pattern of PKG inhibitor-treated P. falciparum schizonts.

(A) Immunofluorescent staining of DMSO/compound 1-treated WT schizonts using antibodies detecting (i) PfGAP45 , (ii) PfSUB1 and (iii) PfAMA1 . Representative images are shown for each staining together with parasite nuclei stained with DAPI. Bars ∼5 µM. (B) Metabolic labelling of phosphoproteins in P. falciparum schizonts. Autoradiographs of (i) 3D7 WT and (ii) gatekeeper mutant 3D7 PfPKG_T618Q schizonts, treated with ³²P-orthophosphate and DMSO (−) or compound 2 (+) prior to lysis, ÄKTA anion exchange chromatography (fractions 10–14 are shown) and separation by SDS-PAGE. Rectangular boxes highlight bands that show a differential signal following inhibitor-treatment in WT, but not PfPKG_T618Q schizonts.