Abstract
During infection of their human definitive host, schistosomes transform rapidly from free-swimming infective cercariae in freshwater to endoparasitic schistosomules. The ‘somules’ next migrate within the skin to access the vasculature and are surrounded by host molecules that might activate intracellular pathways that influence somule survival, development and/or behaviour. However, such ‘transactivation’ by host factors in schistosomes is not well defined. In the present study, we have characterized and functionally localized the dynamics of protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) activation during early somule development in vitro and demonstrate activation of these protein kinases by human epidermal growth factor, insulin, and insulin-like growth factor I, particularly at the parasite surface. Further, we provide evidence that support the existence of specialized signalling domains called lipid rafts in schistosomes and propose that correct signalling to ERK requires proper raft organization. Finally, we show that modulation of PKC and ERK activities in somules affects motility and reduces somule survival. Thus, PKC and ERK are important mediators of host-ligand regulated transactivation events in schistosomes, and represent potential targets for anti-schistosome therapy aimed at reducing parasite survival in the human host.
Highlights
Using anti-phospho protein kinase C (PKC) (Ser660) and anti-phospho PKC (Thr410) antibodies that recognize activation motifs in S. mansoni PKCs that are conserved with human PKCs15, three phosphorylated PKCs were consistently detected in somules with apparent molecular weights of ~78, ~81, and ~116 kDa (Fig. 1a)
Digital analysis of western blot bands from four separate experiments revealed that activation of the ~78, ~116, and ~132 kDa PKCs did not change significantly over four days when compared to 3 h somules; ~81 kDa PKC activation was consistently upregulated at 72 h and was sustained at 96 h when compared to 3 h somules (Fig. 1a) (~2.4 fold increase; p ≤ 0.001)
Localization of activated PKCs within intact somules during early development with anti-phospho PKC (Ser660) antibodies using confocal laser scanning microscopy revealed that activated PKC was differentially distributed over time, with activation at the tegument at 3 h and 16 h, which declined thereafter, with greater activation seen within the somule body at 96 h (Fig. 1b)
Summary
Immunomodulatory molecules are released by cercariae during invasion[4] and each cercaria undergoes complex transformation into a biochemically distinct skin somule[5] which develops a specialised syncytial tegument that remains into adulthood This unique host-interactive layer has been the focus of much research, especially because it expresses potential drug and vaccine targets[6,7,8,9,10]. Signalling across cellular plasma membranes is considered to occur through dynamic membrane/lipid rafts, which are nanoscale microdomains present in the lipid bilayer that assemble cholesterol and sphingolipids and subsets of transmembrane or glycosylphosphatidylinisotol (GPI)-anchored proteins[20] These structures are presumed to selectively concentrate intracellular signalling molecules providing platforms for where protein kinases, scaffolding molecules, and substrates are brought into close proximity enabling rapid signal transduction[21]. In schistosomes putative caveolae-like structures, possibly formed by caveolin-like molecules, have been described in the surface (tegumental) membrane, and membrane fractions characteristic of detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) or detergent-resistant membranes (DRMs) have been prepared[25], indicating lipid raft presence in vivo[23]
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