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A role for p38 mitogen-activated protein kinase in early post-embryonic development of Schistosoma mansoni
Molecular & Biochemical Parasitology 180 (2011) 51–55
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Short communication
A role for p38 mitogen-activated protein kinase in early post-embryonic development of Schistosoma mansoni Margarida Ressurreic¸ão a,b , David Rollinson b , Aidan M. Emery b , Anthony J. Walker a,∗ a b
School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, United Kingdom Wolfson Wellcome Biomedical Laboratories, Zoology Department, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 4 July 2011 Accepted 8 July 2011 Available online 19 July 2011 Keywords: Schistosomes Miracidia In vitro transformation p38 MAPK phosphorylation Post-embryonic development Mother sporocysts
a b s t r a c t 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 4 h development in vitro but increased markedly during transformation, with ∼2.7- and ∼3.7-fold increases after 19 h and 28 h 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. © 2011 Elsevier B.V. All rights reserved.
During the life-cycle of Schistosoma mansoni, the non-feeding, free-living miracidium moves from a freshwater environment to the endoparasitic environment of a snail host where it undergoes striking morphological, physiological and metabolic alteration. As the parasite moves through the different milieu it is thought to respond to environmental stimuli using cellular pathways that communicate signals received at the parasite surface to various cells inducing changes in gene expression [1]. It is expected therefore, that such pathways play a role in schistosome development, reproduction and immune evasion by the parasite among other biological activities (reviewed in [2]). Among the family of mitogen-activated protein kinases (MAPKs), p38 MAPK is commonly referred to as a stress-response cell signalling protein that is activated by environmental and mechanical stimuli such as UV light, hyperosmolarity and redox changes; however, it is also affected by other stimuli, including growth, migratory and death signals [3]. These modulatory signals induce phosphorylation of p38 MAPK which triggers both its translocation to the nucleus and its catalytic activity (reviewed in [4]]. P38 MAPK is highly conserved across organisms in diverse phyla [4] including several eukaryotic pathogens such as Plasmodium falciparum [5], Toxoplasma gondii [6], Echinococcus multilocularis [7], and Brugia malayi [8]. Inhibition of p38 MAPK with
∗ Corresponding author. Tel.: +44 020 8417 2466; fax: +44 020 8417 2497. E-mail address: [email protected] (A.J. Walker). 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.07.002
pyridinylimidazole agents, specific p38 MAPK inhibitors [9], has been shown to alter the transformation, replication and survival of some pathogens highlighting the potential for the development of chemotherapies against this kinase for the treatment of a variety of human diseases (e.g., [5–7]). Although procedures for in vitro transformation of S. mansoni miracidia to mother sporocysts exist, few studies have focused on the signal-based molecular control of development of these early post-embryonic life-stages. Exceptions include studies investigating the role of protein kinase C (PKC) [10], tyrosine phosphorylation [11], and protein kinase A (PKA) in transformation of miracidia to mother sporocysts, with PKA being identified via a mediumthroughput small-molecule screen of chemical compounds that inhibit or delay sporocyst development [12]. Recently, we characterized S. mansoni p38 MAPK and detected phosphorylated (activated) p38 MAPK in protein extracts of miracidia and adult worms using anti-phospho p38 MAPK monoclonal antibodies [13]. The S. mansoni p38 MAPK was found to display significant homology to both vertebrate and invertebrate p38 MAPKs and we further demonstrated that S. mansoni p38 MAPK regulates cilia beat frequency in miracidia [13]. In the present study, we report the nature of p38 MAPK signalling during the miracidium to mother-sporocyst transition and the importance of p38 MAPK to this process, providing novel insight into the role of p38 MAPK during S. mansoni development. To determine S. mansoni p38 MAPK phosphorylation (activation) during the miracidium to mother-sporocyst transition, an
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M. Ressurreic¸ão et al. / Molecular & Biochemical Parasitology 180 (2011) 51–55
Fig. 1. Immunodetection of phosphorylated (activated) p38 mitogen-activated protein kinase (p38 MAPK) in S. mansoni miracidia, and larvae undergoing in vitro transformation to mother sporocysts. (A) p38 MAPK phosphorylation increases and is sustained during development. Larvae (1500 per sample) were recovered at various time points over 44 h, proteins extracted and processed for western blotting (upper panel) using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies as described in materials and methods in Supplementary Material; blots were subsequently stripped and re-probed with anti-actin antibodies to confirm equal protein loading between lanes. Blots from four independent experiments were analyzed and mean relative intensities (±SEM) calculated (lower panel, graph). Values are represented with
M. Ressurreic¸ão et al. / Molecular & Biochemical Parasitology 180 (2011) 51–55
in vitro transformation assay [10] was performed during which transforming larvae were sampled at various time points and processed for western blotting using anti-phospho p38 MAPK antibodies that we validated previously for use with S. mansoni [13] (for details regarding the experimental aspects of in vitro transformation and immunological techniques see materials and methods in Supplementary Material). As larval development progressed there was a significant increase in p38 MAPK phosphorylation (p ≤ 0.01; Fig. 1A). After 4 h culture, p38 MAPK phosphorylation levels remained low and were similar to those observed in our previous study with freshly hatched swimming miracidia (0 h) in which phosphorylation was virtually undetectable by western blotting [13]. However, p38 MAPK phosphorylation increased significantly, to ∼2.7 times that of 4 h larvae after 19 h (p ≤ 0.05) and ∼3.7 times (p ≤ 0.01) that of 4 h larvae after 28 h culture (Fig. 1A). Such phosphorylation was sustained through to the mother sporocyst stage (44 h) (Fig. 1A). In an attempt to localize activated p38 MAPK within developing larvae immunohistochemistry and laser scanning confocal microscopy were employed. Examination of freshly hatched miracidia, miracidia recently placed in culture, post-miracidia undergoing development to mother sporocysts, and mother sporocysts, revealed active p38 MAPK associated with various anatomical regions (Fig. 1B and C). Consistent with our previously published observations [13], p38 MAPK activity in freshly hatched swimming miracidia was relatively low and where evident was localized to the region occupied by the cilia/ciliated plates (Fig. 1B). All larvae incubated with secondary antibody alone (negative control) displayed negligible fluorescence (results for miracidia are shown in Fig. 1B). After 4 h culture, and consistent with the western blotting data (Fig. 1A), larvae displayed minimal levels of p38 MAPK phosphorylation which, when present, was mainly associated with the surface of the parasite (Fig. 1C); this pattern of staining at the parasite surface was however distributed in a random fashion between individual larvae, a situation similar to that seen in freshly hatched swimming miracidia (Fig. 1B and [13]). After 4 h culture, low levels of staining were also observed in the germinal cells (Fig. 1C). In contrast, transforming larvae (19–28 h) displayed high levels of phosphorylated p38 MAPK particularly associated with the new tegument, remains of the neural mass and germinal cells (Fig. 1C). At these time points, the majority of larvae were undergoing transformation (data not shown). Finally, after 44 h culture, when the majority of larvae were mother sporocysts, phosphorylated p38 MAPK was found associated with various regions of the parasite including the germinal cells and their associated nuclei, but was also particularly evident in stained structures resembling the excretory vesicles (flame cells) observed by Collins et al. [14] (Fig. 1C). These results are generally consistent with the western blotting data (Fig. 1A), that reveal low levels of p38 MAPK activity during early stages of post-embryonic development with increased activity as transformation progresses. Recently, using immunoprecipitation, p38 MAPK activity assays and western blotting we reported that the p38 MAPK inhibitor SB203580 directly attenuated S. mansoni p38 MAPK activity whereas the activator anisomycin stimulated it [13]. The key
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residues of mammalian p38 MAPK that interact with SB203580 were also found to be conserved in S. mansoni p38 MAPK [13]. These pharmacological agents were therefore employed in the present study to investigate the potential role of p38 MAPK in early post-embryonic development of S. mansoni. Larvae were cultured in vitro for up to 50 h with SB203580 or anisomycin and the speed of larval development assessed. Initial experiments demonstrated that 0.02% (v/v) DMSO (vehicle) did not affect the in vitro transformation efficiency of S. mansoni when compared to control (CBSS) cultures, and that overall transformation rates were between 75% and 85% (data not shown). In addition, enumeration of live parasites at each time point revealed that neither SB203580 nor anisomycin affected parasite survival throughout the duration of the assay. SB203580 (1 M), significantly reduced the efficiency of transformation of miracidia to mother sporocysts (Fig. 2A) and after 21 h, 19% more miracidia remained compared with CBSS controls (p ≤ 0.01; Fig. 2B). Moreover, after 25 h culture there was a 46% reduction in transformation efficiency (to fully transformed mother sporocysts) (p ≤ 0.01; Fig. 2A); coincident with this, significantly more miracidia were observed in the SB203580-treated group. This suppressive effect of SB203580 on development into mother sporocysts persisted until end of the transformation assay; after 50 h, the transformation efficiency of larvae in SB203580 to fully mature mother sporocysts was 43% that of the CBSS culture (p ≤ 0.01; Fig. 2A). However, almost all larvae exposed to SB203580 had progressed from the miracidial stage after 29 h culture (Fig. 2B) but had not transformed into fully mature mother sporocysts after 50 h (Fig. 2A). Thus SB203580 appears to not only retard development of the miracidium to post-miracidium stage but also suppresses development of the post-miracidium to the mother sporocyst stage. In contrast, when S. mansoni were exposed to anisomycin (20 M), development of miracidia into post-miracidia occurred significantly faster than in CBSS alone (Fig. 2B) with fewer miracidia being present until 17 h into the assay (p ≤ 0.01). After only 2 h, 35% more larvae in the anisomycin-treated culture had progressed from the miracidium stage compared to the CBSS controls (Fig. 2B), and there were 37% more post-miracidia present (data not shown) (p ≤ 0.01). The positive effect of anisomycin on the speed of transformation was striking. After 5 h, 21% of the parasites cultured with anisomycin were mother sporocysts compared to none in the control group (p ≤ 0.001; Fig. 2A) and there were 5 times more mother sporocysts present in the anisomycin-treated cultures at 17 h compared to the controls (p ≤ 0.001; Fig. 2A). However, between 17 h and 50 h, the transformation efficiency of larvae exposed to anisomycin appeared similar to the CBSS controls (Fig. 2A). Post-embryonic development of S. mansoni from a miracidium to mother sporocyst within its compatible molluscan host is crucial to the subsequent asexual replication of daughter sporocysts and cercariae, providing the opportunity for vertebrate–host infection. Currently, the kinase-mediated cell signalling pathways regulating this transformation process have been barely explored, notable exceptions being studies demonstrating a restrictive role for PKC [10] and PKA [12]. Recently we studied p38 MAPK in S. mansoni miracidia and found that this enzyme was active in stationary miracidia within eggs but was inactive in swimming miracidia [13].
reference to p38 MAPK phosphorylation levels in larvae cultured for 4 h that have been assigned a value of 1 (shown as dotted line). *p ≤ 0.05 and **p ≤ 0.01 (ANOVA, with Fishers post hoc test) when compared to phosphorylation levels at 4 h. (B and C) In situ distribution of phosphorylated p38 MAPK in intact acetone-fixed S. mansoni larvae undergoing development visualized using laser scanning confocal microscopy. Larvae were stained as described in Supplementary Material with anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies. (B, left panel) freshly hatched miracidium displaying low levels of p38 MAPK phosphorylation associated mainly with the region corresponding to ciliated surface (CS) of the parasite; the terebratorium (TB) is indicated; (right panel) freshly hatched miracidium incubated without primary antibodies but with secondary antibodies (negative control); (Bar = 15 m). (C) Columns (from left to right): transmitted light, z-axis projections shown in maximum pixel brightness mode, individual z-sections through the parasite at different stages of development. Images show: post-miracidium after 4 h culture displaying low levels of fluorescence; larva undergoing transformation at 19 h with staining associated with (T) tegument, (GC) germinal cells and (NM) neural mass; larva after 28 h with immunoreactivity associated with germinal cells and tegument; mother sporocyst after 44 h culture with phosphorylated p38 MAPK associated with germinal cells, tegument and structures that resemble excretory vesicles (indicated by arrows). Images shown represent the most common expression profiles for activated p38 MAPK from all captured images of larvae across two independent experiments (Bar = 30 m).
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Fig. 2. Inhibition of p38 MAPK during in vitro transformation of S. mansoni miracidia to mother sporocysts restricts larval development whereas activation promotes it. Larvae were either treated with 20 M anisomycin (p38 MAPK activator), 1 M SB203580 (p38 MAPK inhibitor) in Chernin’s balanced salt solution (CBSS) or remained untreated. Parasites were cultured and staged as described in materials and methods in Supplementary Material. (A) Efficiency of larval transformation, and (B) percentage of miracidia remaining in the assay at various time points, determined by visual scoring of 90 randomly selected parasites at each time point across three experiments. Values shown are means (±S.E.M). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 (ANOVA, with Fishers post hoc test) when compared to the CBSS control groups at the equivalent time point.
Moreover, we found that exposure of miracidia to the p38 MAPK activator anisomycin for 30 min induced a marked increase of p38 MAPK activity in cilia resulting in attenuated swim speed and subsequent accelerated release of ciliated plates, providing insights into a possible functional role for p38 MAPK in S. mansoni development. In light of these findings, the present study employed the same anti-phospho p38 MAPK monoclonal antibodies to detect phosphorylated p38 MAPK in S. mansoni larvae and further explore the role of this kinase in the in vitro development of miracidia into mother sporocysts. The increase in p38 MAPK activity between either miracidia/4 h larvae and larvae cultured for 19 h could be due in part to an increase in the environmental osmolarity, as p38 MAPK is known in other organisms to be activated by osmotic change [15]. In this context, it is well established that a trigger for initialization of S. mansoni miracidial transformation is increased osmolarity, such as that experienced by miracidia upon movement from spring water (∼10 mOsm/L) to a suitable transformation media such as the Chernin’s balanced salt solution (CBSS; ∼140 mOs/L) used here which resembles the osmolarity levels of Biomphalaria glabrata
haemolymph (see Supplementary Material). This change causes the cessation of miracidial swimming, release of ciliated plates and creation of a new tegumental syncytium for the maturing mother sporocysts [16]. However, if changes in osmolarity are responsible for increased p38 MAPK activity after 19 h then this represents a delayed response. The current study focused on p38 MAPK activation during larval transformation in vitro, nevertheless, it would be interesting to study earlier effects (e.g., 15 min) of exposure to CBSS or host-snail haemolymph on S. mansoni p38 MAPK activation to ascertain whether or not p38 MAPK activity is induced significantly by osmotic shock. Differential patterns of phosphorylated p38 MAPK were observed in situ between different larval stages. Expression of phosphorylated p38 MAPK in the neural mass, a structure surrounded by the neural ring and comprising the nervous system was found in transforming larvae and in some transformed mother sporocysts (44 h). This highlights a possible involvement of p38 MAPK in signalling the degeneration of the neural mass as p38 MAPK is known to be involved in neuronal apoptotic processes (e.g., [17]) and in B. glabrata the S. mansoni larval neural mass degenerates
M. Ressurreic¸ão et al. / Molecular & Biochemical Parasitology 180 (2011) 51–55
within 24 h [18]. The abnormal persistence of the neural mass in some larvae beyond 24 h transformation in vitro is probably due to degeneration occurring more rapidly in vivo. The germinal cells of transforming larvae and mother sporocysts that give rise to daughter sporocysts also displayed immunoreactivity towards phosphorylated p38 MAPK suggesting that p38 MAPK is involved in regulation of germinal development. Studies with the intracellular parasite T. gondii show that when p38 MAPK is inhibited in infected fibroblasts, intracellular T. gondii tachyzoite replication is suppressed in a dose-dependent fashion [6]. In addition, when phosphorylated, one of the Caenorhabditis elegans orthologs of human p38 MAPK (PMK-1) activates SKN-1, a transcription factor with a role in embryonic mesodermal development [19]. Hence, phosphorylation of p38 MAPK in the germinal cells of S. mansoni larvae may be crucial to asexual replication. In larvae undergoing transformation and in fully transformed mother sporocysts, phosphorylated p38 MAPK was also found associated with the new tegumental syncytium and in mother sporocysts p38 MAPK activation appeared associated with structures resembling the flame cells. The increased phosphorylation of p38 MAPK in the tegument during development is interesting as this structure would protect the parasite from the host immune system and facilitate molecular communication between parasite and host. PKC was not active in the tegument of developing larvae [10], thus activation of specific signalling cascades seem likely to influence development of the new tegument. Inhibition of p38 MAPK with SB203580 significantly delayed S. mansoni larval development after 21 h but did not affect larval survival. On the other hand, activation of p38 MAPK with anisomycin accelerated the transformation process during the first 17 h of the assay. Although SB203580 was used at a low concentration (1 M) to ensure its specificity, this inhibitor could potentially affect the activity of other signalling molecules; however, taken together, the contrasting effects of SB203580 and anisomycin on larval transformation provide strong support for p38 MAPK playing a major role in this process. Western blotting and confocal laser scanning microscopy revealed that under non-stimulated conditions p38 MAPK activity is low during the initial stages of transformation but increases after 17 h culture. This finding coupled with effects of SB203580 and anisomycin support the notion that activation of p38 MAPK serves as a trigger for larval transformation driving development forward. Whether this effect on development is general, for example p38 MAPK playing a role in remodelling of various tissues or is more specific, for example signalling through the neural mass to enable transformation, requires further investigation. Knockdown of S. mansoni p38 MAPK expression by RNA interference (RNAi) might help further in this endeavour and although we did not attempt RNAi in this study, the ability to suppress p38 MAPK expression by RNAi in schistosomes is worthy of investigation. The deleterious effect of SB203580 on developmental progression of S. mansoni is somewhat similar to that observed in other organisms from diverse taxa during post-embryonic development and morphogenesis. For example, pharmacological inhibition of p38 MAPK blocks completion of metamorphosis in the marine polychaete Hydroides elegans [20] and suppresses wing pattern development in Drosophila melanogaster [21]. It has also been suggested that due to their p38 MAPK inhibitory properties pyridinylimidazoles such as the SmithKline Beecham compound SB203580 may represent a novel, potentially broadly acting class of anti-parasitic agents [6]. We are therefore now focusing research effort into characterizing p38 MAPK signalling in the definitive-host stages of S. mansoni with
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a view to studying the effects of these drugs on the development of schistosomula. Acknowledgements We are indebted to Mike Anderson and Jayne King of the Natural History Museum (London) for the maintenance and passage of parasites. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2011.07.002. References [1] Loverde PT, Osman A, Hinck A. Schistosoma mansoni: TGF-beta signaling pathways. Exp Parasitol 2007;117:304–17. [2] Bahia D, Andrade LF, Ludolf F, Mortara RA, Oliveira G. Protein tyrosine kinases in Schistosoma mansoni. Mem Inst Oswaldo Cruz 2006;101(Suppl. 1):137–43. [3] Shi Y, Gaestel M. In the cellular garden of forking paths: how p38 MAPKs signal for downstream assistance. Biol Chem 2002;383:1519–36. [4] Martin-Blanco E. p38 MAPK signalling cascades: ancient roles and new functions. BioEssays 2002;22:637–45. [5] Brumlik MJ, Nkhoma S, Kious MJ, et al. Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication. Exp Parasitol 2011;128:170–5. [6] Wei S, Marches F, Daniel B, Sonda S, Heidenreich K, Curiel T. Pyridinylimidazole p38 mitogen-activated protein kinase inhibitors block intercellular Toxoplasma gondii replication. Int J Parasitol 2002;32:969–77. [7] Gelmedin V, Caballero-Gamiz R, Brehm K. Characterization and inhibition of a p38-like mitogen-activated protein kinase (MAPK) from Echinococcus multilocularis: antiparasitic activities of p38 MAPK inhibitors. Biochem Pharmacol 2008;76:1068–81. [8] Patel A, Chojnowski AN, Gaskill K, De Martini W, Goldberg RL, Siekierka JJ. The role of a Brugia malayi p38 MAPK kinase ortholog (Bm-MPK1) in parasite anti-oxidative stress responses. Mol Biochem Parasitol 2011;2:90–7. [9] Wilson KP, MsCaffrey PG, Hsieao K, et al. The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAPK kinase. Chem Biol 1997;4:223–31. [10] Ludtmann MHR, Rollinson D, Emery AM, Walker AJ. Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni. Int J Parasitol 2009;39:1223–33. [11] Walker AJ, Rollinson D. Specific tyrosine phosphorylation induced in Schistosoma mansoni miracidia by haemolymph from schistosome susceptible, but not resistant, Biomphalaria glabrata. Parasitology 2008;135:337–45. [12] Taft AS, Norante FA, Yoshino TP. The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen. Exp Parasitol 2010;125:84–94. [13] Ressurreic¸ão M, Rollinson D, Emery AM, Walker AJ. p38 MAPK regulates cilliary motion in a eukaryote. BMC Cell Biol 2011;12:6. [14] Collins III JJ, King RS, Cogswell A, Williams DL, Newmark PA. An atlas for Schistosoma mansoni organs and life-cycle stages using cell type-specific markers and confocal microscopy. PLoS Negl Trop Dis 2011;5:e1009. [15] Whitmarsh AJ. A central role for p38 MAPK in the early transcriptional response to stress. BMC Biol 2010;8:47. [16] Samuelson JC, Quinn JJ, Caulfield JP. Hatching, chemokinesis, and transformation of miracidia of Schistosoma mansoni. J Parasitol 1984;70:321–31. [17] Cardaci S, Filomeni G, Rotilio G. Ciriolo MR. p38MAPK/p53 signalling axis mediates neuronal apoptosis in response to tetrahydrobiopterin-induced oxidative stress and glucose uptake inhibition: implication for neurodegeneration. Biochem J 2010;430:439–51. [18] Pan SC. Schistosoma mansoni: the ultrastructure of larval morphogenesis in Biomphalaria glabrata and of associated host–parasite interactions. Jpn J Med Sci Biol 1996;49:129–49. [19] An JH, Blackwell TK. SKN-1 C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev 2003;17:1882–93. [20] Wang H, Qian P. Involvement of a novel p38 mitogen-activated protein kinase in larval metamorphosis of the polychaete Hydroides elegans (Haswell). J Exp Zool (Mol Dev Evol) 2010;B314:390–402. [21] Adachi-Yamada T, Nakamura M, Irie K, et al. Matsumoto K. p38 mitogenactivated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis. Mol Cell Biol 1999;19:23220–9.
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
Short communication
A role for p38 mitogen-activated protein kinase in early post-embryonic development of Schistosoma mansoni Margarida Ressurreic¸ão a,b , David Rollinson b , Aidan M. Emery b , Anthony J. Walker a,∗ a b
School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, United Kingdom Wolfson Wellcome Biomedical Laboratories, Zoology Department, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 4 July 2011 Accepted 8 July 2011 Available online 19 July 2011 Keywords: Schistosomes Miracidia In vitro transformation p38 MAPK phosphorylation Post-embryonic development Mother sporocysts
a b s t r a c t 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 4 h development in vitro but increased markedly during transformation, with ∼2.7- and ∼3.7-fold increases after 19 h and 28 h 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. © 2011 Elsevier B.V. All rights reserved.
During the life-cycle of Schistosoma mansoni, the non-feeding, free-living miracidium moves from a freshwater environment to the endoparasitic environment of a snail host where it undergoes striking morphological, physiological and metabolic alteration. As the parasite moves through the different milieu it is thought to respond to environmental stimuli using cellular pathways that communicate signals received at the parasite surface to various cells inducing changes in gene expression [1]. It is expected therefore, that such pathways play a role in schistosome development, reproduction and immune evasion by the parasite among other biological activities (reviewed in [2]). Among the family of mitogen-activated protein kinases (MAPKs), p38 MAPK is commonly referred to as a stress-response cell signalling protein that is activated by environmental and mechanical stimuli such as UV light, hyperosmolarity and redox changes; however, it is also affected by other stimuli, including growth, migratory and death signals [3]. These modulatory signals induce phosphorylation of p38 MAPK which triggers both its translocation to the nucleus and its catalytic activity (reviewed in [4]]. P38 MAPK is highly conserved across organisms in diverse phyla [4] including several eukaryotic pathogens such as Plasmodium falciparum [5], Toxoplasma gondii [6], Echinococcus multilocularis [7], and Brugia malayi [8]. Inhibition of p38 MAPK with
∗ Corresponding author. Tel.: +44 020 8417 2466; fax: +44 020 8417 2497. E-mail address: [email protected] (A.J. Walker). 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.07.002
pyridinylimidazole agents, specific p38 MAPK inhibitors [9], has been shown to alter the transformation, replication and survival of some pathogens highlighting the potential for the development of chemotherapies against this kinase for the treatment of a variety of human diseases (e.g., [5–7]). Although procedures for in vitro transformation of S. mansoni miracidia to mother sporocysts exist, few studies have focused on the signal-based molecular control of development of these early post-embryonic life-stages. Exceptions include studies investigating the role of protein kinase C (PKC) [10], tyrosine phosphorylation [11], and protein kinase A (PKA) in transformation of miracidia to mother sporocysts, with PKA being identified via a mediumthroughput small-molecule screen of chemical compounds that inhibit or delay sporocyst development [12]. Recently, we characterized S. mansoni p38 MAPK and detected phosphorylated (activated) p38 MAPK in protein extracts of miracidia and adult worms using anti-phospho p38 MAPK monoclonal antibodies [13]. The S. mansoni p38 MAPK was found to display significant homology to both vertebrate and invertebrate p38 MAPKs and we further demonstrated that S. mansoni p38 MAPK regulates cilia beat frequency in miracidia [13]. In the present study, we report the nature of p38 MAPK signalling during the miracidium to mother-sporocyst transition and the importance of p38 MAPK to this process, providing novel insight into the role of p38 MAPK during S. mansoni development. To determine S. mansoni p38 MAPK phosphorylation (activation) during the miracidium to mother-sporocyst transition, an
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M. Ressurreic¸ão et al. / Molecular & Biochemical Parasitology 180 (2011) 51–55
Fig. 1. Immunodetection of phosphorylated (activated) p38 mitogen-activated protein kinase (p38 MAPK) in S. mansoni miracidia, and larvae undergoing in vitro transformation to mother sporocysts. (A) p38 MAPK phosphorylation increases and is sustained during development. Larvae (1500 per sample) were recovered at various time points over 44 h, proteins extracted and processed for western blotting (upper panel) using anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies as described in materials and methods in Supplementary Material; blots were subsequently stripped and re-probed with anti-actin antibodies to confirm equal protein loading between lanes. Blots from four independent experiments were analyzed and mean relative intensities (±SEM) calculated (lower panel, graph). Values are represented with
M. Ressurreic¸ão et al. / Molecular & Biochemical Parasitology 180 (2011) 51–55
in vitro transformation assay [10] was performed during which transforming larvae were sampled at various time points and processed for western blotting using anti-phospho p38 MAPK antibodies that we validated previously for use with S. mansoni [13] (for details regarding the experimental aspects of in vitro transformation and immunological techniques see materials and methods in Supplementary Material). As larval development progressed there was a significant increase in p38 MAPK phosphorylation (p ≤ 0.01; Fig. 1A). After 4 h culture, p38 MAPK phosphorylation levels remained low and were similar to those observed in our previous study with freshly hatched swimming miracidia (0 h) in which phosphorylation was virtually undetectable by western blotting [13]. However, p38 MAPK phosphorylation increased significantly, to ∼2.7 times that of 4 h larvae after 19 h (p ≤ 0.05) and ∼3.7 times (p ≤ 0.01) that of 4 h larvae after 28 h culture (Fig. 1A). Such phosphorylation was sustained through to the mother sporocyst stage (44 h) (Fig. 1A). In an attempt to localize activated p38 MAPK within developing larvae immunohistochemistry and laser scanning confocal microscopy were employed. Examination of freshly hatched miracidia, miracidia recently placed in culture, post-miracidia undergoing development to mother sporocysts, and mother sporocysts, revealed active p38 MAPK associated with various anatomical regions (Fig. 1B and C). Consistent with our previously published observations [13], p38 MAPK activity in freshly hatched swimming miracidia was relatively low and where evident was localized to the region occupied by the cilia/ciliated plates (Fig. 1B). All larvae incubated with secondary antibody alone (negative control) displayed negligible fluorescence (results for miracidia are shown in Fig. 1B). After 4 h culture, and consistent with the western blotting data (Fig. 1A), larvae displayed minimal levels of p38 MAPK phosphorylation which, when present, was mainly associated with the surface of the parasite (Fig. 1C); this pattern of staining at the parasite surface was however distributed in a random fashion between individual larvae, a situation similar to that seen in freshly hatched swimming miracidia (Fig. 1B and [13]). After 4 h culture, low levels of staining were also observed in the germinal cells (Fig. 1C). In contrast, transforming larvae (19–28 h) displayed high levels of phosphorylated p38 MAPK particularly associated with the new tegument, remains of the neural mass and germinal cells (Fig. 1C). At these time points, the majority of larvae were undergoing transformation (data not shown). Finally, after 44 h culture, when the majority of larvae were mother sporocysts, phosphorylated p38 MAPK was found associated with various regions of the parasite including the germinal cells and their associated nuclei, but was also particularly evident in stained structures resembling the excretory vesicles (flame cells) observed by Collins et al. [14] (Fig. 1C). These results are generally consistent with the western blotting data (Fig. 1A), that reveal low levels of p38 MAPK activity during early stages of post-embryonic development with increased activity as transformation progresses. Recently, using immunoprecipitation, p38 MAPK activity assays and western blotting we reported that the p38 MAPK inhibitor SB203580 directly attenuated S. mansoni p38 MAPK activity whereas the activator anisomycin stimulated it [13]. The key
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residues of mammalian p38 MAPK that interact with SB203580 were also found to be conserved in S. mansoni p38 MAPK [13]. These pharmacological agents were therefore employed in the present study to investigate the potential role of p38 MAPK in early post-embryonic development of S. mansoni. Larvae were cultured in vitro for up to 50 h with SB203580 or anisomycin and the speed of larval development assessed. Initial experiments demonstrated that 0.02% (v/v) DMSO (vehicle) did not affect the in vitro transformation efficiency of S. mansoni when compared to control (CBSS) cultures, and that overall transformation rates were between 75% and 85% (data not shown). In addition, enumeration of live parasites at each time point revealed that neither SB203580 nor anisomycin affected parasite survival throughout the duration of the assay. SB203580 (1 M), significantly reduced the efficiency of transformation of miracidia to mother sporocysts (Fig. 2A) and after 21 h, 19% more miracidia remained compared with CBSS controls (p ≤ 0.01; Fig. 2B). Moreover, after 25 h culture there was a 46% reduction in transformation efficiency (to fully transformed mother sporocysts) (p ≤ 0.01; Fig. 2A); coincident with this, significantly more miracidia were observed in the SB203580-treated group. This suppressive effect of SB203580 on development into mother sporocysts persisted until end of the transformation assay; after 50 h, the transformation efficiency of larvae in SB203580 to fully mature mother sporocysts was 43% that of the CBSS culture (p ≤ 0.01; Fig. 2A). However, almost all larvae exposed to SB203580 had progressed from the miracidial stage after 29 h culture (Fig. 2B) but had not transformed into fully mature mother sporocysts after 50 h (Fig. 2A). Thus SB203580 appears to not only retard development of the miracidium to post-miracidium stage but also suppresses development of the post-miracidium to the mother sporocyst stage. In contrast, when S. mansoni were exposed to anisomycin (20 M), development of miracidia into post-miracidia occurred significantly faster than in CBSS alone (Fig. 2B) with fewer miracidia being present until 17 h into the assay (p ≤ 0.01). After only 2 h, 35% more larvae in the anisomycin-treated culture had progressed from the miracidium stage compared to the CBSS controls (Fig. 2B), and there were 37% more post-miracidia present (data not shown) (p ≤ 0.01). The positive effect of anisomycin on the speed of transformation was striking. After 5 h, 21% of the parasites cultured with anisomycin were mother sporocysts compared to none in the control group (p ≤ 0.001; Fig. 2A) and there were 5 times more mother sporocysts present in the anisomycin-treated cultures at 17 h compared to the controls (p ≤ 0.001; Fig. 2A). However, between 17 h and 50 h, the transformation efficiency of larvae exposed to anisomycin appeared similar to the CBSS controls (Fig. 2A). Post-embryonic development of S. mansoni from a miracidium to mother sporocyst within its compatible molluscan host is crucial to the subsequent asexual replication of daughter sporocysts and cercariae, providing the opportunity for vertebrate–host infection. Currently, the kinase-mediated cell signalling pathways regulating this transformation process have been barely explored, notable exceptions being studies demonstrating a restrictive role for PKC [10] and PKA [12]. Recently we studied p38 MAPK in S. mansoni miracidia and found that this enzyme was active in stationary miracidia within eggs but was inactive in swimming miracidia [13].
reference to p38 MAPK phosphorylation levels in larvae cultured for 4 h that have been assigned a value of 1 (shown as dotted line). *p ≤ 0.05 and **p ≤ 0.01 (ANOVA, with Fishers post hoc test) when compared to phosphorylation levels at 4 h. (B and C) In situ distribution of phosphorylated p38 MAPK in intact acetone-fixed S. mansoni larvae undergoing development visualized using laser scanning confocal microscopy. Larvae were stained as described in Supplementary Material with anti-phospho p38 MAPK (Thr180/Tyr182) monoclonal antibodies. (B, left panel) freshly hatched miracidium displaying low levels of p38 MAPK phosphorylation associated mainly with the region corresponding to ciliated surface (CS) of the parasite; the terebratorium (TB) is indicated; (right panel) freshly hatched miracidium incubated without primary antibodies but with secondary antibodies (negative control); (Bar = 15 m). (C) Columns (from left to right): transmitted light, z-axis projections shown in maximum pixel brightness mode, individual z-sections through the parasite at different stages of development. Images show: post-miracidium after 4 h culture displaying low levels of fluorescence; larva undergoing transformation at 19 h with staining associated with (T) tegument, (GC) germinal cells and (NM) neural mass; larva after 28 h with immunoreactivity associated with germinal cells and tegument; mother sporocyst after 44 h culture with phosphorylated p38 MAPK associated with germinal cells, tegument and structures that resemble excretory vesicles (indicated by arrows). Images shown represent the most common expression profiles for activated p38 MAPK from all captured images of larvae across two independent experiments (Bar = 30 m).
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Fig. 2. Inhibition of p38 MAPK during in vitro transformation of S. mansoni miracidia to mother sporocysts restricts larval development whereas activation promotes it. Larvae were either treated with 20 M anisomycin (p38 MAPK activator), 1 M SB203580 (p38 MAPK inhibitor) in Chernin’s balanced salt solution (CBSS) or remained untreated. Parasites were cultured and staged as described in materials and methods in Supplementary Material. (A) Efficiency of larval transformation, and (B) percentage of miracidia remaining in the assay at various time points, determined by visual scoring of 90 randomly selected parasites at each time point across three experiments. Values shown are means (±S.E.M). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 (ANOVA, with Fishers post hoc test) when compared to the CBSS control groups at the equivalent time point.
Moreover, we found that exposure of miracidia to the p38 MAPK activator anisomycin for 30 min induced a marked increase of p38 MAPK activity in cilia resulting in attenuated swim speed and subsequent accelerated release of ciliated plates, providing insights into a possible functional role for p38 MAPK in S. mansoni development. In light of these findings, the present study employed the same anti-phospho p38 MAPK monoclonal antibodies to detect phosphorylated p38 MAPK in S. mansoni larvae and further explore the role of this kinase in the in vitro development of miracidia into mother sporocysts. The increase in p38 MAPK activity between either miracidia/4 h larvae and larvae cultured for 19 h could be due in part to an increase in the environmental osmolarity, as p38 MAPK is known in other organisms to be activated by osmotic change [15]. In this context, it is well established that a trigger for initialization of S. mansoni miracidial transformation is increased osmolarity, such as that experienced by miracidia upon movement from spring water (∼10 mOsm/L) to a suitable transformation media such as the Chernin’s balanced salt solution (CBSS; ∼140 mOs/L) used here which resembles the osmolarity levels of Biomphalaria glabrata
haemolymph (see Supplementary Material). This change causes the cessation of miracidial swimming, release of ciliated plates and creation of a new tegumental syncytium for the maturing mother sporocysts [16]. However, if changes in osmolarity are responsible for increased p38 MAPK activity after 19 h then this represents a delayed response. The current study focused on p38 MAPK activation during larval transformation in vitro, nevertheless, it would be interesting to study earlier effects (e.g., 15 min) of exposure to CBSS or host-snail haemolymph on S. mansoni p38 MAPK activation to ascertain whether or not p38 MAPK activity is induced significantly by osmotic shock. Differential patterns of phosphorylated p38 MAPK were observed in situ between different larval stages. Expression of phosphorylated p38 MAPK in the neural mass, a structure surrounded by the neural ring and comprising the nervous system was found in transforming larvae and in some transformed mother sporocysts (44 h). This highlights a possible involvement of p38 MAPK in signalling the degeneration of the neural mass as p38 MAPK is known to be involved in neuronal apoptotic processes (e.g., [17]) and in B. glabrata the S. mansoni larval neural mass degenerates
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within 24 h [18]. The abnormal persistence of the neural mass in some larvae beyond 24 h transformation in vitro is probably due to degeneration occurring more rapidly in vivo. The germinal cells of transforming larvae and mother sporocysts that give rise to daughter sporocysts also displayed immunoreactivity towards phosphorylated p38 MAPK suggesting that p38 MAPK is involved in regulation of germinal development. Studies with the intracellular parasite T. gondii show that when p38 MAPK is inhibited in infected fibroblasts, intracellular T. gondii tachyzoite replication is suppressed in a dose-dependent fashion [6]. In addition, when phosphorylated, one of the Caenorhabditis elegans orthologs of human p38 MAPK (PMK-1) activates SKN-1, a transcription factor with a role in embryonic mesodermal development [19]. Hence, phosphorylation of p38 MAPK in the germinal cells of S. mansoni larvae may be crucial to asexual replication. In larvae undergoing transformation and in fully transformed mother sporocysts, phosphorylated p38 MAPK was also found associated with the new tegumental syncytium and in mother sporocysts p38 MAPK activation appeared associated with structures resembling the flame cells. The increased phosphorylation of p38 MAPK in the tegument during development is interesting as this structure would protect the parasite from the host immune system and facilitate molecular communication between parasite and host. PKC was not active in the tegument of developing larvae [10], thus activation of specific signalling cascades seem likely to influence development of the new tegument. Inhibition of p38 MAPK with SB203580 significantly delayed S. mansoni larval development after 21 h but did not affect larval survival. On the other hand, activation of p38 MAPK with anisomycin accelerated the transformation process during the first 17 h of the assay. Although SB203580 was used at a low concentration (1 M) to ensure its specificity, this inhibitor could potentially affect the activity of other signalling molecules; however, taken together, the contrasting effects of SB203580 and anisomycin on larval transformation provide strong support for p38 MAPK playing a major role in this process. Western blotting and confocal laser scanning microscopy revealed that under non-stimulated conditions p38 MAPK activity is low during the initial stages of transformation but increases after 17 h culture. This finding coupled with effects of SB203580 and anisomycin support the notion that activation of p38 MAPK serves as a trigger for larval transformation driving development forward. Whether this effect on development is general, for example p38 MAPK playing a role in remodelling of various tissues or is more specific, for example signalling through the neural mass to enable transformation, requires further investigation. Knockdown of S. mansoni p38 MAPK expression by RNA interference (RNAi) might help further in this endeavour and although we did not attempt RNAi in this study, the ability to suppress p38 MAPK expression by RNAi in schistosomes is worthy of investigation. The deleterious effect of SB203580 on developmental progression of S. mansoni is somewhat similar to that observed in other organisms from diverse taxa during post-embryonic development and morphogenesis. For example, pharmacological inhibition of p38 MAPK blocks completion of metamorphosis in the marine polychaete Hydroides elegans [20] and suppresses wing pattern development in Drosophila melanogaster [21]. It has also been suggested that due to their p38 MAPK inhibitory properties pyridinylimidazoles such as the SmithKline Beecham compound SB203580 may represent a novel, potentially broadly acting class of anti-parasitic agents [6]. We are therefore now focusing research effort into characterizing p38 MAPK signalling in the definitive-host stages of S. mansoni with
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a view to studying the effects of these drugs on the development of schistosomula. Acknowledgements We are indebted to Mike Anderson and Jayne King of the Natural History Museum (London) for the maintenance and passage of parasites. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2011.07.002. References [1] Loverde PT, Osman A, Hinck A. Schistosoma mansoni: TGF-beta signaling pathways. Exp Parasitol 2007;117:304–17. [2] Bahia D, Andrade LF, Ludolf F, Mortara RA, Oliveira G. Protein tyrosine kinases in Schistosoma mansoni. Mem Inst Oswaldo Cruz 2006;101(Suppl. 1):137–43. [3] Shi Y, Gaestel M. In the cellular garden of forking paths: how p38 MAPKs signal for downstream assistance. Biol Chem 2002;383:1519–36. [4] Martin-Blanco E. p38 MAPK signalling cascades: ancient roles and new functions. BioEssays 2002;22:637–45. [5] Brumlik MJ, Nkhoma S, Kious MJ, et al. Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication. Exp Parasitol 2011;128:170–5. [6] Wei S, Marches F, Daniel B, Sonda S, Heidenreich K, Curiel T. Pyridinylimidazole p38 mitogen-activated protein kinase inhibitors block intercellular Toxoplasma gondii replication. Int J Parasitol 2002;32:969–77. [7] Gelmedin V, Caballero-Gamiz R, Brehm K. Characterization and inhibition of a p38-like mitogen-activated protein kinase (MAPK) from Echinococcus multilocularis: antiparasitic activities of p38 MAPK inhibitors. Biochem Pharmacol 2008;76:1068–81. [8] Patel A, Chojnowski AN, Gaskill K, De Martini W, Goldberg RL, Siekierka JJ. The role of a Brugia malayi p38 MAPK kinase ortholog (Bm-MPK1) in parasite anti-oxidative stress responses. Mol Biochem Parasitol 2011;2:90–7. [9] Wilson KP, MsCaffrey PG, Hsieao K, et al. The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAPK kinase. Chem Biol 1997;4:223–31. [10] Ludtmann MHR, Rollinson D, Emery AM, Walker AJ. Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni. Int J Parasitol 2009;39:1223–33. [11] Walker AJ, Rollinson D. Specific tyrosine phosphorylation induced in Schistosoma mansoni miracidia by haemolymph from schistosome susceptible, but not resistant, Biomphalaria glabrata. Parasitology 2008;135:337–45. [12] Taft AS, Norante FA, Yoshino TP. The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen. Exp Parasitol 2010;125:84–94. [13] Ressurreic¸ão M, Rollinson D, Emery AM, Walker AJ. p38 MAPK regulates cilliary motion in a eukaryote. BMC Cell Biol 2011;12:6. [14] Collins III JJ, King RS, Cogswell A, Williams DL, Newmark PA. An atlas for Schistosoma mansoni organs and life-cycle stages using cell type-specific markers and confocal microscopy. PLoS Negl Trop Dis 2011;5:e1009. [15] Whitmarsh AJ. A central role for p38 MAPK in the early transcriptional response to stress. BMC Biol 2010;8:47. [16] Samuelson JC, Quinn JJ, Caulfield JP. Hatching, chemokinesis, and transformation of miracidia of Schistosoma mansoni. J Parasitol 1984;70:321–31. [17] Cardaci S, Filomeni G, Rotilio G. Ciriolo MR. p38MAPK/p53 signalling axis mediates neuronal apoptosis in response to tetrahydrobiopterin-induced oxidative stress and glucose uptake inhibition: implication for neurodegeneration. Biochem J 2010;430:439–51. [18] Pan SC. Schistosoma mansoni: the ultrastructure of larval morphogenesis in Biomphalaria glabrata and of associated host–parasite interactions. Jpn J Med Sci Biol 1996;49:129–49. [19] An JH, Blackwell TK. SKN-1 C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev 2003;17:1882–93. [20] Wang H, Qian P. Involvement of a novel p38 mitogen-activated protein kinase in larval metamorphosis of the polychaete Hydroides elegans (Haswell). J Exp Zool (Mol Dev Evol) 2010;B314:390–402. [21] Adachi-Yamada T, Nakamura M, Irie K, et al. Matsumoto K. p38 mitogenactivated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis. Mol Cell Biol 1999;19:23220–9.