Exploring the function of protein kinases in schistosomes: perspectives from the laboratory and from comparative genomics

Eukaryotic protein kinases are well conserved through evolution. The genome of Schistosoma mansoni, which causes intestinal schistosomiasis, encodes over 250 putative protein kinases with all of the main eukaryotic groups represented. However, unraveling functional roles for these kinases is a considerable endeavor, particularly as protein kinases regulate multiple and sometimes overlapping cell and tissue functions in organisms. In this article, elucidating protein kinase signal transduction and function in schistosomes is considered from the perspective of the state-of-the-art methodologies used and comparative organismal biology, with a focus on current advances and future directions. Using the free-living nematode Caenorhabditis elegans as a comparator we predict roles for various schistosome protein kinases in processes vital for host invasion and successful parasitism such as sensory behavior, growth and development. It is anticipated that the characterization of schistosome protein kinases in the context of parasite function will catalyze cutting edge research into host-parasite interactions and will reveal new targets for developing drug interventions against human schistosomiasis

Molecular characterization of host-parasite cell signalling in 'Schistosoma mansoni' during early development

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

Protein Kinase C and extracellular signal-regulated Kinase regulate movement, attachment, pairing and egg release in 'Schistosoma mansoni'

Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using 'smart' antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance

A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

BACKGROUND: Motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni. RESULTS: Fluorescence confocal microscopy revealed that non-motile miracidia trapped within eggs prior to hatching displayed phosphorylated (activated) p38 MAPK associated with their ciliated surface. In contrast, freshly-hatched, rapidly swimming, miracidia lacked phosphorylated p38 MAPK. Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument. Freshly-hatched miracidia possessed swim velocities between 2.17 - 2.38 mm/s. Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min. In contrast, SB 203580, a p38 MAPK inhibitor, increased swim velocity by up to 15% over this duration. Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage. CONCLUSIONS: This study supports a role for p38 MAPK in the regulation of ciliary-beat. Given the evolutionary conservation of signalling processes and cilia structure, we hypothesize that p38 MAPK may regulate ciliary beat and beat-frequency in a variety of eukaryotes

Cell signalling in 'Schistosoma mansoni': a role for p38 MAPK

“Schistosoma mansoni” is a parasitic trematode that can cause intestinal schistosomiasis in humans. This study focused on mitogen-activated protein kinases (MAPKs), particularly p38 MAPK signalling in different “S. mansoni” life stages, and the functional role of p38 MAPK in miracidia, cercariae and during transformation of miracidia into mother sporocysts. Using anti-phospho-p38 MAPK monoclonal antibodies and western blotting, a phosphorylated (activated) p38 MAPK-like protein was detected at approximately 43 kDa in miracidia, transforming miracidia, and adult worms. Immunoprecipitation and kinase assays incorporating ATF-2 substrate, a downstream target of p38 MAPK, revealed that the identified kinase possessed p38 MAPK activity which was inhibited by the p38 MAPK inhibitor, SB203580. Phosphorylation of p38 MAPK was low in intact miracidia; however, phosphorylation increased significantly when miracidia were exposed to the p38 MAPK activator, anisomycin. During in vitro transformation of miracidia into mother sporocysts, p38 MAPK phosphorylation levels increased to 3.7 times that of 4 h larvae after 28 h transformation. Transformation experiments carried out with anisomycin revealed that shedding of ciliated plates from miracidia occurred significantly faster than in control samples. Fluorescence confocal microscopy of miracidia challenged with this compound for 30 min showed that phosphorylated p38 MAPK was predominately associated with the cilliary plates. Moreover, video analysis of freshly-hatched miracidia incubated in anisomycin showed that this compound reduced swimming speed significantly, with total stoppage after 45 min exposure; in the presence of SB203580 the swimming speed of miracidia increased slightly. In addition, the phosphorylation status of p38 MAPK was observed in freshly-emerged cercariae by confocal microscopy; high levels of phosphorylation were seen associated with the head region and anisomycin did not seem to increase p38 MAPK phosphorylation. Video analysis of cercariae swimming in anisomycin revealed that this compound reduced swimming speed, with little swimming evident after 15 min. This study significantly enhances knowledge of p38 MAPK signalling in “S. mansoni” and supports a functional role of this kinase in motility and transformation of the parasite

Protein kinase signalling in 'Schistosoma mansoni'

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

A role for p38 mitogen-activated protein kinase in early post-embryonic development of 'Schistosoma mansoni'

The importance of p38 mitogen-activated protein kinase (p38 MAPK) to Schistosoma mansoni miracidium to mother-sporocyst development was investigated. Western blotting revealed that phosphorylation (activation) of p38 MAPK was low in larvae after 4h development in vitro but increased markedly during transformation, with ∼2.7- and ∼3.7-fold increases after 19h and 28h culture, respectively. Immunohistochemistry of larvae undergoing transformation revealed activated p38 MAPK associated with regions including the tegument, neural mass and germinal cells. Inhibition of larval p38 MAPK with SB203580 reduced significantly the rate of development of miracidia to mother sporocysts, whereas activation of p38 MAPK with anisomycin had the opposite effect. These results provide insight into p38 MAPK signalling in schistosomes and support a role for p38 MAPK in the early post-embryonic development of S. mansoni

Functional mapping of protein kinase A reveals its importance in adult schistosoma mansoni motor activity

Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and 'smart' anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology