Ressurreicao, Margarida de Azevedo Gomes de Carvalho (2013) Protein kinase signalling in 'Schistosoma mansoni'. (PhD thesis), Kingston University, .
Abstract
The present study focused on protein kinase C (pKC), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) signalling in the human parasite Schistosoma mansoni, with a focus on life-stages which are human infective and dependent (cercariae, schistosomules and adult worms). Western blotting with anti-phospho antibodies, detected two phosphorylated PKC 081 kDa and ~ 116 kDa) and two phosphorylated ERK (~43 kDa and ~48 kDa) isotypes predicted in the S. mansoni genome in addition to the previously identified ~78 kDa PKC and ~42 kDa p38 MAPK (Ludtmann et al., 2009; Ressurreíçâo et al., 20lla,b). Additionally, an unusually large ~132 kDa PKC-like protein was detected that is not predicted in the genome. These proteins possessed enzymatic activities, responded to conventional activators and inhtbitors, and their activation profile was dissimilar between life-stages suggesting isotype distinct roles in each developmental stage. In vitro challenge with praziquantel stimulated activity of specific PKC and ERK isotypes, showing a putative involvement in the mode of action of this anthelmintic drug. In situ localization revealed the activated kinases associated with several regions including tegument, sensory, neuromuscular and reproductive structures; additionally, phosphorylated ERK was associated with the excretory system. PKC, ERK and p38 MAPK function was assessed through pharmacological and environmental assays. PKC and ERK were found to playa role in pairing, motility, ventral sucker attachment and egg output of adult worms and motility of schistosomules. Maintenance of unpaired adult worms in different sex ratio environments resulted in changes in PKC, ERK and p38 MAPK activity (both in male and females) showing importance in transduction of chemotatic and/or thigmotatic stimuli .. Light and temperature changes affected kinase activity in cercariae mainly at the cercariae sensory papillae and parasite surface. Moreover, combined linoleic acid (LA) and CFDA-SE based assays developed for induction and monitoring of cercarial gland release showed that PKC, ERK and p38 MAPK are involved in mechanisms that underpin cercariae host detection/penetration and that pharmacological inhibition of these enzymes partially blocked LA induced release, while the PKC activator accelerated it. In schistosomules, epidermal growth factor and insulin stimulated ERK and PKC activity whereas insulin-like growth factor I and mouse red blood cells up¬regulated PKC activity only suggesting association with parasite nutrition and host-parasite communication. Internalization of fluorescently labelled transferrin via the schistosomule tegument was delayed with PKC inhibitors suggesting a role in transferrin uptake. Taken together, these data contribute significantly to our understanding of cell signalling in schistosomes and how such signalling regulates parasite function, and should open up new avenues of investigation for development of anti-schistosome drugs.
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