Schistosoma mansoni: Development of acetabular glands of cercaria at ultrastructural level

Cercariae of Schistosoma mansoni in daughter sporocysts in Biomphalaria glabrata were studied with the electron microscope to observe the maturing process of their acetabular glands. The undifferentiated acetabular gland displays its enlarged basal area (fundus) and extended narrow process (duct) before other organ systems are recognized. Its fundus contains a prominent nucleus and subcellular organelles typical of active secretory cells.The secretory granules of the postacetabular glands are formed in a milieu of dilated rough endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) homogeneously granular ones, and (2) other granular ones with electron dense bodies in their matrices. Mostly, the homogeneously granular ones are produced first in the fundus and are forced into the ducts as the other type is formed.The secretory granules of preacetabular glands are formed from translucent vacuoles which arise from an environment of endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) one type has a homogeneous dense matrix, and (2) the other type has a less dense matrix containing electron-lucid bodies.The duct of an undifferentiated acetabular gland has either filamentous material or microtubules dispersed in its cytoplasm. Once microtubules are formed, they persist during the life of the cercaria. The microtubules are believed to have possibly two functions: (1) to support the long duct, and (2) to assist the movement of the secretory granules into the channels of the ducts where they remain until released during host penetration.Few of the subcellular organelles associated with secretory granules formation are seen in the duct except the area in close proximity to the fundus; thus, the few secretory granules produced in the duct are in this region.     

Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes

The purified proteolytic fraction of Schistosoma mansoni cercarial enzymes (PCF) was inhibited by Diisopropylphosphofluoridate (DFP). Its esterolytic activity was not affected by either of two specific active center reagents of proteolytic enzymes: (1) 1-chloro-3-tosylamido-7-ammo-2-heptanone (trypsin) and (2) l-1-tosylamido-2-phenylethyl chloromethyl ketone (chymotrypsin). Furthermore, PCF did not destroy the biological activity of bradykinin on the isolated guinea pig ileum, nor did it release bradykinin from purified dog plasma bradykininogen.     

Populational structure of Schistosoma mansoni assessed by DNA microsatellites

DNA microsatellites were used as molecular markers to analyse the population structure of the laboratory LE strain and of 10 field isolates of Schistosoma mansoni, the aetiologic agent of schistosomiasis. Out of 16,000 DNA sequences analysed in databases, 622 microsatellite loci were identified in 481 sequences (3.0%). The AT repetitions were the most frequent, followed by AAT and AC. Six loci showing perfect repetitions were selected and used in the polymerase chain reaction to evaluate polymorphisms in the number of repeats. Two groups of worms were studied. The first group consisted of 78 individuals, 39 of each sex, of the LE strain. The second group of worms consisted of 10 field isolates: seven from humans and three from snails. Four of the six loci were polymorphic, containing 11-17 alleles per locus. No linkage disequilibrium was observed among loci and none of the loci was sex linked. In both groups of worms, a significant deviation from Hardy-Weinberg equilibrium was observed. The observed heterozygosity was always lower than the expected one. The polymerase chain reaction primers were S. mansoni specific. The LE strain showed a lower total number of alleles or a lower average number of alleles/polymorphic locus than the field isolates, suggesting that 41 years of laboratory maintenance exerted selective pressure on the LE strain. The S. mansoni populations from the field were most genetically undifferentiated (R(ST)<0.027), suggesting a high gene flow among them. Our results showed the usefulness of microsatellites for population analysis of S. mansoni, offering a new alternative for a better understanding of schistosomiasis epidemiology.     

Structural bioinformatics study of PNP from Schistosoma mansoni

The parasite Schistosoma mansoni lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. Schistosomiasis is endemic in 76 countries and territories and amongst the parasitic diseases ranks second after malaria in terms of social and economic impact and public health importance. The PNP is an attractive target for drug design and it has been submitted to extensive structure-based design. The atomic coordinates of the complex of human PNP with inosine were used as template for starting the modeling of PNP from S. mansoni complexed with inosine. Here we describe the model for the complex SmPNP-inosine and correlate the structure with differences in the affinity for inosine presented by human and S. mansoni PNPs.     

Schistosoma mansoni: human skin ceramides are a chemical cue for host recognition of cercariae

Schistosoma mansoni cercariae recognize the human host with a sequence of behavioral responses particularly to chemical host cues. After attaching to the skin surface, cercariae are stimulated by so far unknown skin components to hold enduring contact with the skin and to start creeping towards entry sites. We studied the chemical stimulus of human skin for the cercarial enduring contact response by fractionation of human and pig skin surface extracts and offering the fractions to the cercariae via membrane filters. Enduring contact was stimulated exclusively by ceramides, specific lipids of the uppermost skin layers. This chemical cue differs from the 6 chemical host signals used by S. mansoni cercariae in other behavioral steps of host invasion, and thus underlines the specialization of S. mansoni cercariae particularly in chemical host signals. All together, the enduring contact response of the cercariae is, like the other phases of host invasion, well adapted to the chemical properties of human skin.     

Determination of ED50 values for praziquantel in praziquantel-resistant and -susceptible Schistosoma mansoni isolates

The dose of praziquantel required to kill 50% of adult worms in vivo (i.e. the ED50) was estimated for nine different isolates of Schistosoma mansoni in infected mice. Four of the isolates were selected because they had not knowingly been in contact with the drug (i.e. they were putatively praziquantel-susceptible). Five putatively praziquantel-resistant isolates were chosen because they had been selectively bred for drug-resistance in the laboratory and/or had previously been shown to be relatively resistant to praziquantel in the field. The work was performed in three laboratories in different countries using pre-agreed and comparable experimental protocols. All four praziquantel-susceptible isolates had ED50s estimated to be <100 mg/kg (mean=70+/-7 SD; median=68), while all five putatively praziquantel-resistant isolates had estimated ED50s >100 mg/kg (mean=209+/-48 SD; median=192). Thus, the five praziquantel-resistant isolates, including two that had been subjected to drug pressure during more than 20 passages in mice, had drug ED50s that were approximately three times as great as those of the praziquantel-susceptible isolates. Two of the five isolates in the putatively resistant group had previously been passaged 15 or more times in mice without administration of drug-pressure, but had ED50s consistent with the other three isolates in the group, indicating that the trait of praziquantel-resistance did not necessarily impair biological fitness during laboratory passage. The protocols used here to estimate the praziquantel ED50s of S. mansoni isolates should be useful for establishing and monitoring the drug susceptibility/resistance profiles of parasite isolates freshly obtained from endemic areas, particularly those in which increased usage of the drug is likely to occur.     

Schistosoma mansoni: Assessment of effects of oleic acid,cercarial age and water temperature on parasite-host attraction

Although the lifecycle of Schistosoma spp. and pathophysiology of schistosomiasis have been established, the mechanism by which cercariae find their host is not well understood. Speculatively, host infection by random and accidental host contact is not as biologically plausible as a biochemical mechanism of mammalian attraction. A few studies have indicated that biochemical cues and temperature gradients may play a role in host identification, attraction and attachment triggers. This study aimed to elucidate these mechanisms more specifically through evaluation of biochemical, age and temperature influences leading to Schistosoma mansoni cercariae attraction and attachment behaviors. Oleic acid, a common unsaturated free fatty acid in the outer layer of human skin, was tested for cercariae attraction across biologically relevant concentrations. Influence of media type (beeswax, nail varnish and agar), age-dependent behavior variability and environmentally appropriate temperatures (22 and 30 °C) were also evaluated. Results indicated that oleic acid at concentrations of 0.3, 0.9 and 1.8 g/mL in beeswax significantly increased median attachment to media (median attachment of 7.50%, 4.20% and 3.71%, respectively, P < 0.001), compared with plain beeswax, with maximal attachment of 30.30% at 0.3 g/mL of oleic acid. In media containing 0.3 g/mL of oleic acid, cercarial attachment was highest for freshly emerged cercariae to 5 h post-emergence, with a significant decrease in attachment behavior at 10 h post-emergence (P < 0.01). Aquatic temperature at which cercariae were exposed to media did not yield significant results (P value >0.05). Biochemical, age and environmental factors influencing cercarial host attraction and attachment behavior have been elucidated by this study. This information will inform further development of devices for environmental surveillance and potentially improve cercarial exposure prevention strategies.     

Characterization of Schistosoma mansoni ATPDase2 gene, a novel apyrase family member

Schistosoma mansoni is a major causative agent of schistosomiasis, which constitutes a severe health problem in developing countries. We have previously described the SmATPDase1 gene, encoding a protein from the external surface of the parasites. In this work, we describe the cloning and characterization of SmATPDase2, a novel CD39-like ATP diphosphohydrolase gene in S. mansoni. In silico analysis of the protein encoded by SmATPDase2 predicts a single N-terminal transmembrane domain similar to that described for secreted human apyrase isoforms. Immuno-colocalization experiments detected both SmATPDase proteins at the S. mansoni adult worm tegument basal and apical membranes, but only SmATPDase2 in the tegument syncytium. SmATPDase2 but not SmATPDase1 protein was detected by Western blot in culture medium supernatants following incubation of adult worms in vitro, indicating that SmATPDase2 was secreted by the parasite to the medium. Taken together these data suggest a non-redundant role for SmATPDase2 in the parasite-host interplay.     

Schistosoma mansoni: lack of correlation between praziquantel-induced intra-worm calcium influx and parasite death

The schistosomicidal activity of praziquantel (PZQ) is accompanied by a large influx of calcium into the worms, suggesting that this phenomenon could be the source of the observed muscular contraction, surface disruption and eventual death of the parasite. We have incubated live adult schistosomes in a medium containing radioactive calcium and we were able to confirm that PZQ does indeed stimulate calcium entry into the parasite. An even higher calcium uptake, however, occurred in schistosomes exposed to PZQ after pre-incubation with cytochalasin D, a condition that suppresses PZQ schistosomicidal effects and allows the complete survival of the parasites. The calcium blockers nicardipine and nifedipine also failed to prevent the calcium influx induced by PZQ. Similarly, a large calcium influx occurred in 28-day-old worms exposed to PZQ, in spite of the fact that these immature worms are largely insensitive to the schistosomicidal effects of the drug. Schistosomes incubated overnight with radioactive calcium and PZQ and then returned to normal medium, retained a calcium content higher than worms pre-incubated with cytochalasin D, but the difference could be a consequence—rather than a cause—of schistosomicidal effects. These results suggest that calcium accumulation by itself, at least as measured in whole parasites maintained in vitro, may not represent an exhaustive explanation for the schistosomicidal effects of PZQ.     

Navigation within host tissues: Schistosoma mansoni and Trichobilharzia ocellata schistosomula respond to chemical gradients

After penetration of human or duck host's skin schistosomula of Schistosoma mansoni and Trichobilharzia ocellata migrate parallel to the surface in the epidermis, then they enter the dermis and venules prior to further migration. This study focuses on potential behavioural mechanisms and host cues which may enable this navigation within host tissues. We stimulated cercariae to penetrate into agar substrates and to transform to schistosomula, and analysed their orientation behaviour within chemical concentration gradients. Both species were chemotactically attracted by low molecular weight fractions of their host's serum (human, duck) and D-glucose and L-arginine were identified as attractive components in serum. They responded to gradients, which established after addition of very low concentrations of D-glucose (1 microM in T. ocellata and 2 microM in S. mansoni) and L-arginine (0.025 microM in T. ocellata and 1.0 microM in S. mansoni). The response to D-glucose was specific as other saccharides had no stimulatory activity. L-Arginine stimulated chemotactic orientation both when free and bound in peptides. However, the two species responded differently to the position of L-arginine within the peptide (terminal or subterminal), and only S. mansoni, not T. ocellata, responded to peptides occurring in serum and endothelial cells: fibronectin (1 microM), bradykinin (25 pM) and its fragment 1-5 (2.5 microM). Both species adjusted their body axis with the ventral side towards the higher concentrations of D-glucose and of L-arginine. We argue that the chemotactic orientation and the alignment of the body axis enable the parasites (i) to orientate towards deeper skin layers and avoid accidental perforation of the covering skin surface layers, (ii) to determine their position during their surface-parallel migration within the epidermis, (iii) to locate blood vessels.     

A comparison of microsatellite polymorphism and heterozygosity among field and laboratory populations of Schistosoma mansoni

The genetic diversity of a field population (recently collected in Melquiades, Brazil) and two laboratory strains (LE and NMRI) of a human blood fluke, Schistosoma mansoni, were analysed using microsatellite markers. Data from the three groups showed an extreme and consistent discrepancy in the level of polymorphism at all microsatellite loci between the field population and laboratory populations. The numbers of alleles detected in LE and NMRI populations averaged only 14 and 10% of those found in the field population, respectively. Especially apparent was the abundance of rare alleles in the Melquiades population when compared with the laboratory strains. The reduction in allelic diversity in the laboratory strains is most likely due to the founder effect and potential bottlenecks that may have occurred during decades of laboratory maintenance. Surprisingly, a much less drastic difference was found when comparing the average heterozygosity of the field population with the laboratory strains. This apparent anomaly may be explained by observed population substructuring (and a potential resultant Wahlund effect) in the natural population. Our comparison of genetic diversity between laboratory and field populations of S. mansoni emphasizes the need for studies of representative populations in schistosome vaccine development.     

The proteome of the insoluble Schistosoma mansoni eggshell skeleton

In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold’s layer, von Lichtenberg’s envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins.     

Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic β-hematin (βH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to βH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.     

Effect of somatostatin on gastrointestinal contractility in Schistosoma mansoni infected mice

Schistosoma mansoni infection induces severe gastrointestinal motility disturbances which are characterised by hyperactivity of intestinal muscle, abdominal pain, diarrhoea, vomiting and nausea. During schistosomiasis, the neuropeptide somatostatin is generated within inflammatory granulomas. However, somatostatin is also an important inhibitory modulator of gastrointestinal motility. In the present study, we have investigated the potential of somatostatin to reduce schistosomiasis-induced hyperactivity of gastrointestinal smooth muscle. Organ bath experiments were performed to study the contractility of isolated smooth muscle strips of intestine from control mice and from mice that were infected with S. mansoni for 2, 4, 8 and 16 weeks. Electrical field stimulation (0.5-8 Hz) of enteric nerves induced frequency-dependent neurogenic contractions of cholinergic origin in all regions of the small intestine. Somatostatin (0.1-1 microM) concentration-dependently inhibited the contractions to enteric nerve stimulation in the small intestine from uninfected control mice and from acutely S. mansoni infected mice (2 and 4 weeks of infection). After 8 weeks of infection with S. mansoni, this inhibitory effect of somatostatin was less pronounced and after 16 weeks of infection it was completely abolished. Histology demonstrated that chronic infection of mice with S. mansoni was associated with significant alterations in the musculature of the small intestine. These alterations may be associated with physiological changes in the responsiveness to somatostatin and suggest that the somatostatin neuroregulatory circuit of enteric neurotransmission in the small intestine is disturbed during chronic schistosomiasis mansoni.     

Energy allocation patterns in Biomphalaria alexandrina snails in response to cadmium exposure and Schistosoma mansoni infection

This study was aimed to investigate the effects of both parasitism and environmental stress on the growth, reproduction, and survival of Biomphalaria alexandrina snails. Resource allocation strategies may be influenced by both biotic and abiotic factors. Using the planorbid snail B. alexandrina and Schistosoma mansoni, this hypothesis was examined by raising snails fed the same diet under two stressors (infection and Cd exposure). The snails divided into four groups, uninfected, infected, Cd-exposed uninfected, and Cd-exposed infected snails. Egg production, growth, and survival of the snails were monitored over a 9-week period postinfection. Inhibition of snail reproductive activity by parasitism results in increased snail growth in the first week postinfection, termed gigantism, during which the snail is hypothesized to allocate excess energy normally used for reproduction to somatic growth. Infection status and Cd exposure had significant effects on snail growth and reproduction. The infected and Cd-exposed infected snails exhibiting reduced survival relative to snails of other treatments. It was found that parasite development influenced by Cd exposure. Results of this study suggest that energy allocation patterns are context-dependent in B. alexandrina snails, influenced by infection and Cd exposure.