Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway

BACKGROUND: Schistosoma mansoni uses Biomphalaria glabrata as an intermediate host during its complex life cycle. In the snail, the parasite initially transforms from a miracidium into a mother sporocyst and during this process excretory-secretory products (ESPs) are released. Nitric oxide (NO) and its reactive intermediates play an important role in host defence responses against pathogens. This study therefore aimed to determine the effects of S. mansoni ESPs on NO production in defence cells (haemocytes) from schistosome-susceptible and schistosome-resistant B. glabrata strains. As S. mansoni ESPs have previously been shown to inhibit extracellular signal-regulated kinase (ERK) phosphorylation (activation) in haemocytes from susceptible, but not resistant, B. glabrata the regulation of NO output by ERK in these cells was also investigated. RESULTS: Haemocytes from resistant snails challenged with S. mansoni ESPs (20 mug/ml) over 5 h displayed an increase in NO production that was 3.3 times greater than that observed for unchallenged haemocytes; lower concentrations of ESPs (0.1-10 mug/ml) did not significantly increase NO output. In contrast, haemocytes from susceptible snails showed no significant change in NO output following challenge with ESPs at any concentration used (0.1-20 mug/ml). Western blotting revealed that U0126 (1 muM or 10 muM) blocked the phosphorylation (activation) status of ERK in haemocytes from both snail strains. Inhibition of ERK signalling by U0126 attenuated considerably intracellular NO production in haemocytes from both susceptible and resistant B. glabrata strains, identifying ERK as a key regulator of NO output in these cells. CONCLUSION: S. mansoni ESPs differentially influence intracellular NO levels in susceptible and resistant B. glabrata haemocytes, possibly through modulation of the ERK signalling pathway. Such effects might facilitate survival of S. mansoni in its intermediate host

Human growth factor-mediated signalling through lipid rafts regulates stem cell proliferation, development and survival of Schistosoma mansoni

Although the mechanisms by which schistosomes grow and develop in humans are poorly defined, their unique outer tegument layer, which interfaces with host blood, is considered vital to homeostasis of the parasite. Here, we investigated the importance of tegument lipid rafts to the biology of Schistosoma mansoni in the context of host–parasite interactions. We demonstrate the temporal clustering of lipid rafts in response to human epidermal growth factor (EGF) during early somule development, concomitant with the localization of anteriorly orientated EGF receptors (EGFRs) and insulin receptors, mapped using fluorescent EGF/insulin ligand. Methyl-β-cyclodextrin (MβCD)-mediated depletion of cholesterol from lipid rafts abrogated the EGFR/IR binding at the parasite surface and led to modulation of protein kinase C, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and Akt signalling pathways within the parasite. Furthermore, MβCD-mediated lipid raft disruption, and blockade of EGFRs using canertinib, profoundly reduced somule motility and survival, and attenuated stem cell proliferation and somule growth and development particularly to the fast-growing liver stage. These findings provide a novel paradigm for schistosome development and vitality in the host, driven through host–parasite interactions at the tegument, that might be exploitable for developing innovative therapeutic approaches to combat human schistosomiasis

Identification of a major causative agent of human cercarial dermatitis, Trichobilharzia franki (Muller and Kimmig 1994), in southern England and its evolutionary relationships with other European populations.

Background Trichobilharzia is the most species rich and widely distributed genus of schistosomes and is known throughout Europe and North America as an agent of human cercarial dermatitis. The disease is caused by an acute allergic reaction in the skin that develops as a consequence of repeated contact with water containing schistosomatid cercariae. However, despite historical outbreaks of the disease, there are no published records of accurately identified Trichobilharzia species from the UK. Methods Two hundred Radix auricularia (L.) were sampled from a recreational fishing lake in Hampshire and emerging schistosomatid cercariae were collected for microscopy and DNA extraction. General morphological description of the cercariae was performed, alongside sequencing and phylogenetic analysis of the 28S ribosomal DNA for accurate species identification as well as comparisons of ITS1 in order to identify evolutionary affinities with other European populations. All molecular comparisons were performed using published sequences. Results The phylogenetic analysis of 28S sequences identified the cercariae as Trichobilharzia franki. Two unique British ITS1 haplotypes were identified which were most closely related to haplotypes of T. franki populations from France. Haplotype network analysis indicated the mixing of T. franki populations throughout Europe. It is suggested that parasite distribution is the probable result of the movement of migratory waterfowl. Conclusions This is the first accurate record of T. franki in the UK. The movement of T. franki with waterfowl could pose a considerable human health risk, as in mainland Europe, and signifies T. franki-associated human cercarial dermatitis as a re-emerging disease in the UK

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

Sensory protein kinase signalling in ' Schistosoma mansoni ' Cercariae : host location and invasion

Schistosoma mansoni cercariae display specific behavioural responses to abiotic/biotic stimuli enabling them to locate and infect the definitive human host. Here we report the effect of such stimulants on signalling pathways of cercariae in relation to host finding and invasion. Cercariae exposed to various light/temperature regimes displayed modulated protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) activities, with distinct responses at 37°C and intense light/dark, when compared to 24°C under normal light. Kinase activities were localized to regions including the oral sensory papillae, acetabular ducts, tegument, acetabular glands, and nervous system. Furthermore, linoleic acid (LA) modulated PKC and ERK activities concurrent with the temporal release of acetabular gland components. Attenuation of PKC, ERK and p38 MAPK activities significantly reduced gland component release, particularly in response to LA, demonstrating the importance of these signalling pathways to host penetration mechanisms

'Schistosoma mansoni' excretory-secretory products induce protein kinase signalling, hyperkinesia, and stem cell proliferation in the opposite sex

Adult male and female schistosomes in copula dwell within human blood vessels and lay eggs that cause the major Neglected Tropical Disease human schistosomiasis. How males and females communicate to each other is poorly understood; however, male-female physical interaction is known to be important. Here, we investigate whether excretory-secretory products (ESPs), released into the external milieu by mature Schistosoma mansoni, might induce responses in the opposite sex. We demonstrate that ESPs adhere to the surface of opposite sex worms inducing the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways, particularly in the parasite tegument. Furthermore, we show that mature worms stimulated signalling in juvenile worms. Strikingly, we demonstrate that ESPs from the opposite sex promote stem cell proliferation, in an ERK- and p38 MAPK-dependent manner, in the tegument and within the testes of males, and the ovaries and vitellaria of females. Hyperkinesia also occurs following opposite sex ESP exposure. Our findings support the hypothesis that male and female schistosomes may communicate over distance to modulate key processes underlying worm development and disease progression, opening unique avenues for schistosomiasis control

Molecular epidemiological analyses reveal extensive connectivity between 'Echinostoma revolutum (sensu stricto)' populations across Eurasia and species richness of zoonotic echinostomatids in England

Echinostoma revolutum (sensu stricto) is a widely distributed member of the Echinostomatidae, a cosmopolitan family of digenetic trematodes with complex life cycles involving a wide range of definitive hosts, particularly aquatic birds. Integrative taxonomic studies, notably those utilising nad1 barcoding, have been essential in discrimination of E. revolutum (s.s.) within the ‘Echinostoma revolutum’ species complex and investigation of its molecular diversity. No studies, however, have focussed on factors affecting population genetic structure and connectivity of E. revolutum (s.s.) in Eurasia. Here, we used morphology combined with nad1 and cox1 barcoding to determine the occurrence of E. revolutum (s.s.) and its lymnaeid hosts in England for the first time, in addition to other echinostomatid species Echinoparyphium aconiatum, Echinoparyphium recurvatum and Hypoderaeum conoideum. Analysis of genetic diversity in E. revolutum (s.s.) populations across Eurasia demonstrated haplotype sharing and gene flow, probably facilitated by migratory bird hosts. Neutrality and mismatch distribution analyses support possible recent demographic expansion of the Asian population of E. revolutum (s.s.) (nad1 sequences from Bangladesh and Thailand) and stability in European (nad1 sequences from this study, Iceland and continental Europe) and Eurasian (combined data sets from Europe and Asia) populations with evidence of sub-population structure and selection processes. This study provides new molecular evidence for a panmictic population of E. revolutum (s.s.) in Eurasia and phylogeographically expands the nad1 database for identification of echinostomatids