Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment

The efficacy of praziquantel against a Puerto Rican strain of Schistosoma mansoni was assessed using both in vivo and in vitro approach. The drug effective dose (50%) in the infected mouse model was about 30 times higher when determined against 28-day-old infections than against 7-week-old parasites. Single-sex female infections were also largely refractory to treatment and single-sex male infections moderately refractory, in comparison with bisexual infections. The in vitro approach consisted of overnight exposure of parasite cultures to various drug concentrations, followed by several days of culture in drug-free medium. In vitro results confirmed in vivo data and allowed for the observation of schistosome morphological phenomena after praziquantel exposure. Early worm contraction was observed in all cases, even after exposure to sub-lethal concentrations of praziquantel or upon exposure of the largely refractory 28-day-old schistosomes. In these instances, however, worms resumed movements and normal shape upon drug removal and were able to survive. The inference of these observations on the clinical use of praziquantel and on its mechanism of action is discussed.     

Cytochalasin D abolishes the schistosomicidal activity of praziquantel

To test the hypothesis that calcium channels of schistosomes are the targets for the action of praziquantel, we subjected schistosomes in vitro to pharmacological agents capable of interfering with the functioning of calcium channels. After 1-h exposure to these agents, praziquantel was added and incubation continued overnight. Worms were then washed, resuspended in drug-free medium and observed during the following 7-10 days. About 50% of schistosomes pre-exposed to the calcium channel blockers nicardipine and nifedipine were able to survive a praziquantel concentration (3 microM) that normally killed the majority of adult male worms. Since the organization of the actin cytoskeleton controls the activity of calcium channels in a number of different systems, we also pre-exposed schistosomes to the actin depolymerizing agent cytochalasin D. This treatment rendered the parasites completely refractory to the effects of very high praziquantel levels (up to 36 microM). These results are consistent with the hypothesis that schistosome calcium channels are involved in the mechanism of action of praziquantel.     

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.     

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: evaluation of an RNAi-based treatment targeting HGPRTase gene

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential gene of the parasite Schistosoma mansoni and it is well conserved in its hosts (mouse and human) at the protein but not at the RNA level. This feature prompted us to assess RNA interference (RNAi) to combat schistosomiasis. Small interfering RNAs (siRNAs) were produced against HGPRTase, injected in infected mice and the number of worms was counted six days after injection. The total number of parasites was reduced by approximately 27% after treatment. RT-PCR analyzes showed a significant reduction in parasite target mRNA but not in host's homologue. The use of low doses of molecules did not oversaturate si- or miRNA pathways as mice survival rates were not affected by siRNAs. This is the first successful in vivo demonstration of a RNAi-based treatment against schistosomiasis. We believe that improvements in molecule delivery and an increase on siRNA dose could rapidly eliminate parasite.     

Voltage-gated calcium channel subunits from platyhelminths: potential role in praziquantel action

Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+ -dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming alpha(1) subunit and other modulatory subunits, including the beta subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel beta subunits (Ca(v)betavar) found in these parasites. In particular, we examine the role these beta subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Ca(v)betavar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant beta subunits may thus represent a platyhelminth-specific gene family.     

Effect of praziquantel on the immature stages of Schistosoma haematobium

Schistosoma mansoni is known to be refractory to praziquantel treatment in the pre-patent period of infection. Since Schistosoma haematobium has a much longer pre-patent period (10-12 weeks vs. 5-6 for the former species), we asked the question whether a correspondingly longer period of insusceptibility exists in urinary schistosomiasis. In hamsters treated at different times after infection, S. haematobium was partially refractory to praziquantel when treatment was given at week 5, but showed practically full sensitivity at 7-8 weeks and later times. Schistosoma haematobium worms obtained at different times after infection and exposed in vitro to praziquantel were refractory to low drug concentrations between 4 and 6 weeks, but were clearly affected at higher concentrations and at later time points. We conclude that S. haematobium does not have a praziquantel-insensitive window longer than in S. manson, in spite of its much longer maturation period. In addition, refractoriness of immature stages can be overcome at higher drug concentrations.     

Potential correlation between carboxylic acid metabolites in Biomphalaria alexandrina snails after exposure to Schistosoma mansoni infection

Carboxylic acids play an important role in both aerobic and anaerobic metabolic pathways of both the snail and the parasite. Monitoring the effects of infection by schistosome on Biomphalaria alexandrina carboxylic acids metabolic profiles represents a promising additional source of information about the state of metabolic system. We separated and quantified pyruvic, fumaric, malic, oxalic, and acetic acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to detect correlations between these acids in both hemolymph and digestive gland gonad complex (DGG's) samples in a total of 300 B. alexandrina snails (150 infected and 150 controls) at different stages of infection. The results showed that the majority of metabolite pairs did not show significant correlations. However, some high correlations were found between the studied acids within the control group but not in other groups. More striking was the existence of reversed correlations between the same acids at different stages of infection. Some possible explanations of the underlying mechanisms were discussed. Ultimately, however, further data are required for resolving the responsible regulatory events. These findings highlight the potential of metabolomics as a novel approach for fundamental investigations of host-pathogen interactions as well as disease surveillance and control.     

Effects of intermediate host genetic background on parasite transmission dynamics: a case study using Schistosoma mansoni

For parasites that require multiple hosts to complete their development, genetic interplay with one host may impact parasite transmission and establishment in subsequent hosts. In this study, we used microsatellite loci to address whether the genetic background of snail intermediate hosts influences life-history traits and transmission patterns of dioecious trematode parasites in their definitive hosts. We performed experimental Schistosoma mansoni infections utilizing two allopatric populations of Biomphalaria glabrata snails and assessed intensities and sex ratios of adult parasites in mouse definitive hosts. Our results suggest that the genetic background of hosts at one point in a parasite’s life cycle can influence the intensities and sex ratios of worms in subsequent hosts.     

Schistosoma mansoni: Heterologous complementation of a yeast null mutant by SmRbx, a protein similar to a RING box protein involved in ubiquitination

The SCF (Skp1-Cul1-F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1(-) yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxDelta24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.     

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.     

Adverse effects of praziquantel treatment of Schistosoma japonicum infection: involvement of host anaphylactic reactions induced by parasite antigen release

The present study using a murine model heavily infected with Schistosoma japonicum aimed to elucidate the pathogenesis of adverse effects of praziquantel treatment of schistosome-infected subjects. Inbred BALB/c mice were infected with S. japonicum (Yamanashi strain) before being treated with a single dose of praziquantel at 4 or 8 weeks p.i. All the mice treated at 8 weeks p.i. exhibited signs typical of systemic anaphylaxis until half of them died shortly after praziquantel administration. At autopsy, these mice exhibited remarkable intestinal alterations characterised by increased mucosal permeability, mucosal oedema and petechial haemorrhage, which are changes typical of immediate intestinal anaphylaxis. In these mice treated at 8 weeks p.i., degranulation of intestinal mast cells was frequently observed, which was particularly remarkable around S. japonicum eggs hatched as an effect of praziquantel. Furthermore, the plasma histamine concentration just after praziquantel treatment was much higher in mice at 8 weeks p.i. than that in uninfected mice or in S. japonicum-infected mice without drug treatment. In contrast, none of these intestinal changes was observed in untreated or uninfected control mice, or in mice administered praziquantel at 4 weeks p.i., in which worm pairs had just reached sexual maturation and begun egg-laying. The finding by ELISA that serum IgM and IgA levels specific to S. japonicum eggs decreased immediately after praziquantel treatment, together with the results of immunohistochemistry, revealed the sudden release of parasite antigens from the eggs hatched by praziquantel treatment. The results of this study demonstrate that adverse effects of praziquantel treatment of schistosomiasis characterised by abdominal signs depend on anaphylactic reactions due to parasite antigens, especially antigens from eggs hatched as an effect of praziquantel.