Effect of amphotericin B on the infection success of Schistosoma mansoni in Biomphalaria glabrata

In the present study, we examined the effect of amphotericin B on larval stages (miracidia and primary sporocyst) of the helminth Schistosoma mansoni, the causative agent of human schistosomiasis. Amphotericin B (AmB) is a polyene macrolide that disturbs the function of the cell membrane; it is widely used as prophylactic antimycotic agent in in vitro culture. We show for the first time that S. mansoni miracidia infectivity is considerably reduced after AmB treatment. Moreover we demonstrate that AmB does not affect the development, growth, viability, and behavior of miracidia and primary sporocysts. Our data indicate that AmB effects on S. mansoni sporocyst prevalence are linked to the oxidative properties of AmB. These may alter the capacity of sporocysts to respond to the oxidative stress generated by the snail immune defence system.     

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.     

Female biased sex-ratio in Schistosoma mansoni after exposure to an allopatric intermediate host strain of Biomphalaria glabrata

For parasites that require multiple hosts to complete their development, the interaction with the intermediate host may have an impact on parasite transmission and development in the definitive host. The human parasite Schistosoma mansoni needs two different hosts to complete its life cycle: the freshwater snail Biomphalaria glabrata (in South America) as intermediate host and a human or rodents as final host. To investigate the influence of the host environment on life history traits in the absence of selection, we performed experimental infections of two B. glabrata strains of different geographic origin with the same clonal population of S. mansoni. One B. glabrata strain is the sympatric host and the other one the allopatric host. We measured prevalence in the snail, the cercarial infectivity, sex-ratio, immunopathology in the final host and microsatellite frequencies of individual larvae in three successive generations.     

Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.     

N-Glycosylation patterns of hemolymph glycoproteins from Biomphalaria glabrata strains expressing different susceptibility to Schistosoma mansoni infection

Comparative analyses of the N-glycosylation pattern of hemolymph glycoproteins from Biomphalaria glabrata strains Puerto Rico (BgPR) and Salvador (BgBS-90), differing in their susceptibility towards Schistosoma mansoni infection, were performed by Western blotting, enzyme-linked immunosorbent assays, two-dimensional high-performance liquid chromatography and mass spectrometry. Obtained data demonstrated an enhanced expression of serologically cross-reacting, fucosylated carbohydrate epitopes by the highly susceptible BgPR-strain in comparison to the resistant BgBS-90-strain. In particular, glycoproteins of BgPR snails exhibited larger amounts of glycans with (β1-2)-linked xylose or terminal Fuc(α1-3)GalNAc(β1-4)[±Fuc(α1-3)]GlcNAc(β1-)-units which are known to mediate cross-reactivity with schistosomal glycoconjugates. This finding could be corroborated by immunohistochemical studies showing again an enhanced expression of such carbohydrate epitopes in BgPR tissue. Hence, our results provide evidence for a correlation of B. glabrata susceptibility towards S. mansoni infection and the expression of carbohydrate determinants shared by the parasite and its intermediate host.     

Non-random organization of the Biomphalaria glabrata genome in interphase Bge cells and the spatial repositioning of activated genes in cells co-cultured with Schistosomamansoni

Biomphalaria glabrata is a major intermediate host for the parasitic trematode Schistosoma mansoni, a causative agent of human schistosomiasis. To decipher the molecular basis of this host-parasite interaction, the Bge embryonic cell line provides a unique in vitro model system to assess whether interactions between the snail and parasite affect the cell and genome biology in either organism. The organization of the B. glabrata genome in Bge cells was studied using image analysis through positioning territories of differently sized chromosomes within cell nuclei. The snail chromosome territories are similar in morphology as well as in non-random radial positioning as those found in other derived protostome and deuterostome organisms. Specific monitoring of four gene loci, piwi, BgPrx, actin and ferritin, revealed non-random radial positioning of the genome. This indicates that specific parts of the snail genome reside in reproducible nuclear addresses. To determine whether exposure to parasite is reflected in genome organization, the interphase spatial positioning of genes was assessed after co-culturing Bge cells with either normal or irradiation attenuated miracidia for 30 min to 24 h. The loci of actin and ferritin, genes that are up-regulated in the snail when subjected to infection, were visualized by fluorescence in situ hybridisation (FISH) and their radial nuclear positions i.e. their position in the interphase nucleus with respect to the nuclear edge/envelope, mapped. Interestingly, large scale gene repositioning correlated to temporal kinetics of gene expression levels in Bge cells co-cultured with normal miracidia while irradiated parasites failed to elicit similar gene expression or gene loci repositioning as demonstrated using the ferritin gene. This indicates that normal but not attenuated schistosomes provide stimuli that evoke host responses that are reflected in the host’s nuclear architecture. We believe that this is not only the first time that gene-repositioning studies have been attempted in a mollusc but also demonstrates a parasite influencing the interphase genome organization of its host.     

Bursts of transposition from non-long terminal repeat retrotransposon families of the RTE clade in Schistosoma mansoni

The genus Schistosoma is composed of blood flukes that infect vertebrates, from which three species are major causative agents of human schistosomiasis, a tropical disease that affects more than 200 million people. Current models of the recent evolution of Schistosoma indicate multiple events of migration and speciation from an Asian ancestral species. Transposable elements are important drivers of genome evolution and have been hypothesised to have an important role in speciation. In this work, we describe a comprehensive inventory of Schistosoma mansoni and Schistosoma japonicum retrotransposons, based on their recently published genomic data. We find a considerable difference in retrotransposon representation between the two species (22% and 13%, respectively). A large part of this difference can be attributed to higher representation of two previously described families of S. mansoni retrotransposons (SR2 and Perere-3/SR3), compared with the representation of their closest relative families in S. japonicum. A more detailed analysis suggests that these two S. mansoni families were the subject of recent bursts of transposition that were not paralleled by their S. japonicum counterparts. We hypothesise that these bursts could be a consequence of the evolutionary pressure resulting from migration of Schistosoma from Asia to Africa and their establishment in this new environment, helping both speciation and adaptation.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.     

Schistosoma mansoni Annexin 2: Molecular characterization and immunolocalization

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.     

Development of Schistosoma mansoni in the laboratory rat analyzed by light and confocal laser scanning microscopy

The kinetic of maturation (schistogram) of Schistosoma mansoni worms grown in laboratory rats was studied by light and confocal laser scanning microscopy. Infected rats with the BH strain were weekly euthanized 3-9 weeks pi. Recovered flukes stained with hydrochloric carmine were preserved as whole-mounts and analyzed by confocal and brightfield microscopy. Worms displayed varying degrees of maturation of the reproductive system at weeks 3-6. Male worms showed complete maturation of the reproductive system at week 6, while female worms completed their maturation at week 7. Males presented few tubercles in tegument in all weeks. Despite the presence of a developing embryo within the ootype, no uterine egg was found. The schistogram in rats follows a pattern similar to that observed in mice hosts.     

Schistosoma mansoni: Effect of dietary zinc supplement on egg granuloma in Swiss mice treated with praziqantel

Schistosomiasis is one of the most important parasitic diseases in Egypt and chemotherapy is considered the most effective method of control. This study was conducted to assess the effectiveness of zinc administration against Schistosoma mansoni infection by evaluating the activities of arylesterase and paraoxonase (PON1) enzymes, and the degree of liver damage.One hundred and twenty albino mice were divided into two groups; one was an infected control and the other a treated group which was further subdivided into three according to the praziquantel and zinc supplementation given. Blood and liver samples, collected 10 weeks post-infection, were subjected to parasitological, histopathological, and enzyme assays, and immunological studies. The results showed that dietary zinc supplementation led to marked reduction in worm load, and egg deposition in the liver and intestine.Histopathological examination showed marked reduction in the number and diameter of hepatic granulomas in the treated groups. The activity of arylesterase and PON1 enzymes were partially restored in infected animals receiving zinc. IL-10 mRNA expression was higher in the treated groups than in the infection control group. In conclusion, zinc administration could be a promising adjuvant therapy for S. mansoni infection.     

The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1ΔC) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1ΔC were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.     

Schistosoma mansoni: Developmental arrest of miracidia treated with histone deacetylase inhibitors

In the present study, we examined the effect of the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VA) and sodium-butyrate on the metamorphosis of larvae of the human blood-fluke Schistosoma mansoni from the free-swimming miracidia into the intramolluskal sporocyst. We show that HDAC inhibitors block transformation in concentration dependant manner. TSA reversibly blocks this developmental process: only 13 ± 11% of TSA treated miracidia transform into sporocysts in-vitro, compared to 92 ± 3% in the mock-treated control. Other enzyme inhibitors such as cycloheximide or hydroxyurea had no effect on metamorphosis. For treatment of up to 4 h, the effect of TSA was completely reversible. Our data indicates that HDAC activity is necessary for the transformation of S. mansoni miracidia during infection of the snail host.     

Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni

cAMP-dependent protein kinases (PKAs) are the main transducers of cAMP signalling in eukaryotic cells. Recently we reported the identification and characterisation of a PKA catalytic subunit (SmPKA-C) in Schistosoma mansoni that is required for adult schistosome viability in vitro. To gain further insights into the role of SmPKA-C in biological processes during the schistosome life cycle, we undertook a quantitative analysis of SmPKA-C mRNA expression in different life cycle stages. Our data shows that SmPKA-C mRNA expression is developmentally regulated, with the highest levels of expression in cercariae and adult female worms. To evaluate the biological role of SmPKA-C in these developmental stages, cercariae and adult worms were treated with various concentrations of PKA inhibitors. Treatment of cercariae with H-89 or PKI 14-22 amide resulted in loss of viability suggesting that, as in adults, PKA is an essential enzyme activity in this infectious larval stage. In adult worms, in vitro exposure to sub-lethal concentrations of H-89 or PKI 14-22 amide resulted in inhibition of egg production in a dose-dependent manner. Furthermore, using a murine model of schistosome infection where S. mansoni fecundity is impaired, we show that reduced rates of egg production in vivo correlate with significant reductions in SmPKA-C mRNA expression and PKA activity. Finally, restoration of parasite egg production in vivo also resulted in normalisation of SmPKA-C mRNA expression and PKA activity. Taken together, our data suggest that PKA signalling is required for cercarial viability and may play a specific role in the reproductive activity of adult worms.     

Binding of von Willebrand factor and plasma proteins to the eggshell of Schistosoma mansoni

Schistosoma mansoni eggs have to cross the endothelium and intestinal wall to leave the host and continue the life cycle. Mechanisms involved in this essential step are largely unknown. Here we describe direct binding to the S. mansoni eggshell of von Willebrand factor and other plasma proteins involved in haemostasis. Using deletion-mutants, we demonstrated that it is the A1 domain of von Willebrand factor that binds to the eggshell. Our results suggest that binding of plasma proteins to the eggshell promotes binding to the endothelium, initiating the passage of the egg through the blood-vessel wall to be excreted in the end.