Design and synthesis of molecular probes for the determination of the target of the anthelmintic drug praziquantel.

Schistosomiasis is a highly prevalent neglected tropical disease caused by blood-dwelling helminths of the genus Schistosoma. Praziquantel (PZQ) is the only drug available widely for the treatment of this disease and is administered in racemic form, even though only the (R)-isomer has significant anthelmintic activity. Progress towards the development of a second generation of anthelmintics is hampered by a lack of understanding of the mechanism of action of PZQ. In this Letter, we report an efficient protocol for the small-scale separation of enantiomers of 2 (hydrolyzed PZQ) using supercritical fluid chromatography (SFC). The enantiopure 2 was then used to develop several molecular probes, which can potentially be used to help identify the protein target of PZQ and study its mode of action.

Towards an understanding of the mechanism of action of praziquantel.

Although praziquantel (PZQ) has been used to treat schistosomiasis for over 20 years its mechanism of action remains unknown. We have developed an assay based on the transcriptional response of Schistosoma mansoni PR-1 to heat shock to confirm that while 6-week post-infection (p.i.) schistosomes are sensitive to PZQ, 4-week p.i. schistosomes are not. Further, we have used this assay to demonstrate that in mice this sensitivity develops between days 37 and 40 p.i. When PZQ is linked to the fluorophore BODIPY to aid microscopic visualization, it appears to enter the cells of intact 4 and 6-week p.i. schistosomes as well as mammalian NIH 3T3 cells with ease suggesting that the differential effects of PZQ is not based on cell exclusion. A transcriptomal analysis of gene expression between 4 and 6 weeks p.i. revealed 607 up-regulated candidate genes whose products are potential PZQ targets. A comparison of this gene list with that of genes expressed by PZQ sensitive miracidia reduced this target list to 247 genes, including a number involved in aerobic metabolism and cytosolic calcium regulation. Finally, we also report the effect of an in vitro sub-lethal exposure of PZQ on the transcriptome of S. mansoni PR-1. Annotation of genes differentially regulated by PZQ exposure suggests that schistosomes may undergo a transcriptomic response similar to that observed during oxidative stress.

Transcriptomic analysis of reduced sensitivity to praziquantel in Schistosoma mansoni.

Schistosomiasis is an intravascular parasitic infection estimated to affect over 206 million people, the majority of whom live in Africa where the trematode worms Schistosoma mansoni and Schistosoma haematobium are the major causative agents. While a number of drugs have been used to treat schistosomiasis, praziquantel (PZQ) is the only one that is widely available, relatively cheap, and easy to use. The reliance on a single drug for the treatment of such a prevalent disease is a cause for concern due to the potential for resistance to render PZQ ineffective. In this study, we examine the transcriptome of three generations of a laboratory strain of S. mansoni (PR1) whose susceptibility to PZQ has been diminished across 9 passages through exposure to increasing sub-lethal doses of the drug. Miracidial susceptibility was significantly reduced after exposure to 2 × 50 mg/Kg PZQ during the first passage. Susceptibility of worms in vivo was first assessed during passage 5 when mice infected with PZQ-selected schistosomes were dosed with a lethal dose of 3 × 300 mg/kg PZQ resulting in only a 10% reduction in worm number compared to control treatment. The emergence of reduced sensitivity was marked by a shift in sex ratio from a predominantly male to a female population, a reduction in the length of females and ultimately the loss of the PZQ-selected line after passage 9. Analysis of differentially regulated transcripts did not suggest that any particular gene product or pathway was associated with drug resistance suggesting either a loss of function mutation to a single gene or an epistatic interaction of multiple gene products as the underlying cause of reduced susceptibility.

A cDNA microarray for Crassostrea virginica and C. gigas.

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.

Effect of praziquantel on the differential expression of mouse hepatic genes and parasite ATP binding cassette transporter gene family members during Schistosoma mansoni infection.

Schistosomiasis is a chronic parasitic disease caused by sexually dimorphic blood flukes of the genus Schistosoma. Praziquantel (PZQ) is the only drug widely available to treat the disease but does not kill juvenile parasites. Here we report the use of next generation sequencing to study the transcriptional effect of PZQ on murine hepatic inflammatory, immune and fibrotic responses to Schistosoma mansoni worms and eggs. An initial T helper cell 1 (Th1) response is induced against schistosomes in mice treated with drug vehicle (Vh) around the time egg laying begins, followed by a T helper cell 2 (Th2) response and the induction of genes whose action leads to granuloma formation and fibrosis. When PZQ is administered at this time, there is a significant reduction in egg burden yet the hepatic Th1, Th2 and fibrotic responses are still observed in the absence of granuloma formation suggesting some degree of gene regulation may be induced by antigens released from the dying adult worms. Quantitative real-time PCR was used to examine the relative expression of 16 juvenile and adult S. mansoni genes during infection and their response to Vh and PZQ treatment in vivo. While the response of stress genes in adult parasites suggests the worms were alive immediately following exposure to PZQ, they were unable to induce transcription of any of the 9 genes encoding ATP-binding cassette (ABC) transporters tested. In contrast, juvenile schistosomes were able to significantly induce the activities of ABCB, C and G family members, underscoring the possibility that these efflux systems play a major role in drug resistance.

The anthelmintic praziquantel is a human serotoninergic G-protein-coupled receptor ligand.

Schistosomiasis is a debilitating tropical disease caused by infection with parasitic blood flukes. Approximately 260 million people are infected worldwide, underscoring the clinical and socioeconomic impact of this chronic infection. Schistosomiasis is treated with the drug praziquantel (PZQ), which has proved the therapeutic mainstay for over three decades of clinical use. However, the molecular target(s) of PZQ remain undefined. Here we identify a molecular target for the antischistosomal eutomer - (R)-PZQ - which functions as a partial agonist of the human serotoninergic 5HT2B receptor. (R)-PZQ modulation of serotoninergic signaling occurs over a concentration range sufficient to regulate vascular tone of the mesenteric blood vessels where the adult parasites reside within their host. These data establish (R)-PZQ as a G-protein-coupled receptor ligand and suggest that the efficacy of this clinically important anthelmintic is supported by a broad, cross species polypharmacology with PZQ modulating signaling events in both host and parasite.

Characterization of two POU transcription factor family members from the urochordate Oikopleura dioica.

Three POU domain containing transcription factors have been cloned from the urochordate Oikopleura dioica. Phylogenetic analysis showed that two of these (OctA1 and OctA2) are closely related members of the class II POU domain family, and one (OctB) is a member of the class III POU domain family. All three transcription factors contained a highly conserved bipartite DNA-binding POU domain with POU specific and POU homeodomains, separated by a linker region. All three proteins were shown to bind specifically to the canonical octamer motif, ATGCAAAT. The ability of these factors to drive transcription from an octamer-containing reporter construct was assessed in vertebrate B lymphocyte cell lines. Both OctA1 and OctA2 drove transcription in murine and catfish B cell lines, however, OctB did not increase the level of transcription above background levels. It is concluded that Oct transcription factors capable of functioning in a similar fashion to vertebrate Oct1/2 were present at the phylogenetic level of the urochordates.

New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas).

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at (www.genome.clemson.edu).

Praziquantel sensitivity of Kenyan Schistosoma mansoni isolates and the generation of a laboratory strain with reduced susceptibility to the drug.

Schistosomiasis is a neglected tropical disease caused by blood-dwelling flukes of the genus Schistosoma. While the disease may affect as many as 249 million people, treatment largely relies on a single drug, praziquantel. The near exclusive use of this drug for such a prevalent disease has led to concerns regarding the potential for drug resistance to arise and the effect this would have on affected populations. In this study, we use an in vitro assay of drug sensitivity to test the effect of praziquantel on miracidia hatched from eggs obtained from fecal samples of Kenyan adult car washers and sand harvesters as well as school children. Whereas in a previous study we found the car washers and sand harvesters to harbor Schistosoma mansoni with reduced praziquantel sensitivity, we found no evidence for the presence of such strains in any of the groups tested here. Using miracidia derived from seven car washers to infect snails, we used the shed cercariae to establish a strain of S. mansoni with significantly reduced praziquantel sensitivity in mice. This was achieved within 5 generations by administering increasing doses of praziquantel to the infected mice until the parasites could withstand a normally lethal dose. This result indicates that while the threat of praziquantel resistance may have diminished in the Kenyan populations tested here, there is a strong likelihood it could return if sufficient praziquantel pressure is applied.

Transcriptional analysis of Schistosoma mansoni treated with praziquantel in vitro.

Schistosomiasis is one of the foremost health problems in developing countries and has been estimated to account for the loss of up to 56 million annual disability-adjusted life years. Control of the disease relies almost exclusively on praziquantel (PZQ) but this drug does not kill juvenile worms during the early stages of infection or prevent post-treatment reinfection. As the use of PZQ continues to grow, there are fears that drug resistance may become problematic thus there is a need to develop a new generation of more broadly effective anti-schistosomal drugs, a task that will be made easier by having an understanding of why PZQ kills sexually mature worms but fails to kill juveniles. Here, we describe the exposure of mixed-sex juvenile and sexually mature male and female Schistosoma mansoni to 1 µg/mL PZQ in vitro and the use of microarrays to observe changes to the transcriptome associated with drug treatment. Although there was no significant difference in the total number of genes expressed by adult and juvenile schistosomes after treatment, juveniles differentially regulated a greater proportion of their genes. These included genes encoding multiple drug transporter as well as calcium regulatory, stress and apoptosis-related proteins. We propose that it is the greater transcriptomic flexibility of juvenile schistosomes that allows them to respond to and survive exposure to PZQ in vivo.

Polymorphism associated with the Schistosoma mansoni tetraspanin-2 gene.

A vaccine against schistosomiasis would contribute significantly to reducing the 3-70 million disability-adjusted life years lost annually to the disease. Towards this end, inoculation with the large extracellular loop (EC-2) of Schistosoma mansoni tetraspanin-2 protein (Sm-TSP-2) has proved effective in reducing worm and egg burdens in S. mansoni-infected mice. The EC-2 loop of Schistosoma japonicum TSP-2, however, has been found to be highly polymorphic, perhaps diminishing the likelihood that this antigen can be used for vaccination against this species. Here, we examine polymorphism of the EC-2 of Sm-TSP-2 in genetically unique worms derived from six individuals from Kisumu, Kenya.

Ikaros family members from the agnathan Myxine glutinosa and the urochordate Oikopleura dioica: emergence of an essential transcription factor for adaptive immunity.

The Ikaros multigene family encodes a number of zinc finger transcription factors that play key roles in vertebrate hemopoietic stem cell differentiation and the generation of B, T, and NK cell lineages. In this study, we describe the identification and characterization of an Ikaros family-like (IFL) protein from the agnathan hagfish Myxine glutinosa and the marine urochordate Oikopleura dioica, both of which lie on the evolutionary boundary between the vertebrates and invertebrates. The IFL molecules identified in these animals displayed high conservation in the zinc finger motifs critical for DNA binding and dimerization in comparison with those of jawed vertebrates. Expression of the IFL gene in hagfish was strongest in blood, intestine, and gills. In O. dioica, transcription from the IFL gene was initiated at or around the time of hatching and maintained throughout the life span of the animal. In situ hybridization localized O. dioica IFL expression to the Fol cells, which are responsible for generating the food filter of the house. Biochemical analysis of the DNA binding and dimerization domains from M. glutinosa and O. dioici IFLs showed that M. glutinosa behaves as a true Ikaros family member. Taken together, these results indicate that the properties associated with the Ikaros family preceded the emergence of the jawed vertebrates and thus adaptive immunity.