A single locus determines praziquantel response in Schistosoma mansoni.

We previously performed a genome-wide association study (GWAS) to identify the genetic basis of praziquantel (PZQ) response in schistosomes, identifying two quantitative trait loci situated on chromosomes 2 and 3. We reanalyzed this GWAS using the latest (version 10) genome assembly showing that a single locus on chromosome 3, rather than two independent loci, determines drug response. These results reveal that PZQ response is monogenic and demonstrates the importance of high-quality genomic information.

Genomic analysis of a parasite invasion: Colonization of the Americas by the blood fluke Schistosoma mansoni.

Schistosoma mansoni, a snail-borne, blood fluke that infects humans, was introduced into the Americas from Africa during the Trans-Atlantic slave trade. As this parasite shows strong specificity to the snail intermediate host, we expected that adaptation to South American Biomphalaria spp. snails would result in population bottlenecks and strong signatures of selection. We scored 475,081 single nucleotide variants in 143 S. mansoni from the Americas (Brazil, Guadeloupe and Puerto Rico) and Africa (Cameroon, Niger, Senegal, Tanzania, and Uganda), and used these data to ask: (i) Was there a population bottleneck during colonization? (ii) Can we identify signatures of selection associated with colonization? (iii) What were the source populations for colonizing parasites? We found a 2.4- to 2.9-fold reduction in diversity and much slower decay in linkage disequilibrium (LD) in parasites from East to West Africa. However, we observed similar nuclear diversity and LD in West Africa and Brazil, suggesting no strong bottlenecks and limited barriers to colonization. We identified five genome regions showing selection in the Americas, compared with three in West Africa and none in East Africa, which we speculate may reflect adaptation during colonization. Finally, we infer that unsampled populations from central African regions between Benin and Angola, with contributions from Niger, are probably the major source(s) for Brazilian S. mansoni. The absence of a bottleneck suggests that this is a rare case of a serendipitous invasion, where S. mansoni parasites were pre-adapted to the Americas and able to establish with relative ease.

Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment.

Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29-14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni.

Ancient Hybridization and Adaptive Introgression of an Invadolysin Gene in Schistosome Parasites.

Introgression among parasite species has the potential to transfer traits of biomedical importance across species boundaries. The parasitic blood fluke Schistosoma haematobium causes urogenital schistosomiasis in humans across sub-Saharan Africa. Hybridization with other schistosome species is assumed to occur commonly, because genetic crosses between S. haematobium and livestock schistosomes, including S. bovis, can be staged in the laboratory, and sequencing of mtDNA and rDNA amplified from microscopic miracidia larvae frequently reveals markers from different species. However, the frequency, direction, age, and genomic consequences of hybridization are unknown. We hatched miracidia from eggs and sequenced the exomes from 96 individual S. haematobium miracidia from infected patients from Niger and the Zanzibar archipelago. These data revealed no evidence for contemporary hybridization between S. bovis and S. haematobium in our samples. However, all Nigerien S. haematobium genomes sampled show hybrid ancestry, with 3.3-8.2% of their nuclear genomes derived from S. bovis, providing evidence of an ancient introgression event that occurred at least 108-613 generations ago. Some S. bovis-derived alleles have spread to high frequency or reached fixation and show strong signatures of directional selection; the strongest signal spans a single gene in the invadolysin gene family (Chr. 4). Our results suggest that S. bovis/S. haematobium hybridization occurs rarely but demonstrate profound consequences of ancient introgression from a livestock parasite into the genome of S. haematobium, the most prevalent schistosome species infecting humans.

The hemolymph of Biomphalaria snail vectors of schistosomiasis supports a diverse microbiome.

The microbiome - the microorganism community that is found on or within an organism's body - is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. Microbiomes modulate the capacity of insect disease vectors (mosquitoes, tsetse flies, sandflies) to transmit parasites and disease. We investigate the diversity and abundance of microorganisms within the hemolymph (i.e. blood) of Biomphalaria snails, the intermediate host for Schistosoma mansoni, using Illumina MiSeq sequencing of the bacterial 16S V4 rDNA. We sampled hemolymph from five snails from six different laboratory populations of B. glabrata and one population of B. alexandrina. We observed 279.84 ± 0.79 amplicon sequence variants per snail. There were significant differences in microbiome composition at the level of individual snails, snail populations and species. Snail microbiomes were dominated by Proteobacteria and Bacteroidetes while water microbiomes from snail tank were dominated by Actinobacteria. We investigated the absolute bacterial load using qPCR: hemolymph samples contained 2784 ± 339 bacteria/µl. We speculate that the microbiome may represent a critical, but unexplored intermediary in the snail-schistosome interaction as hemolymph is in very close contact with the parasite at each step of its development.

Whole genome amplification and exome sequencing of archived schistosome miracidia.

Adult schistosomes live in the blood vessels and cannot easily be sampled from humans, so archived miracidia larvae hatched from eggs expelled in feces or urine are commonly used for population genetic studies. Large collections of archived miracidia on FTA cards are now available through the Schistosomiasis Collection at the Natural History Museum (SCAN). Here we describe protocols for whole genome amplification of Schistosoma mansoni and Schistosome haematobium miracidia from these cards, as well as real time PCR quantification of amplified schistosome DNA. We used microgram quantities of DNA obtained for exome capture and sequencing of single miracidia, generating dense polymorphism data across the exome. These methods will facilitate the transition from population genetics, using limited numbers of markers to population genomics using genome-wide marker information, maximising the value of collections such as SCAN.

Efficient linkage mapping using exome capture and extreme QTL in schistosome parasites.

BACKGROUND: Identification of parasite genes that underlie traits such as drug resistance and host specificity is challenging using classical linkage mapping approaches. Extreme QTL (X-QTL) methods, originally developed by rodent malaria and yeast researchers, promise to increase the power and simplify logistics of linkage mapping in experimental crosses of schistosomes (or other helminth parasites), because many 1000s of progeny can be analysed, phenotyping is not required, and progeny pools rather than individuals are genotyped. We explored the utility of this method for mapping a drug resistance gene in the human parasitic fluke Schistosoma mansoni. RESULTS: We staged a genetic cross between oxamniquine sensitive and resistant parasites, then between two F1 progeny, to generate multiple F2 progeny. One group of F2s infecting hamsters was treated with oxamniquine, while a second group was left untreated. We used exome capture to reduce the size of the genome (from 363 Mb to 15 Mb) and exomes from pooled F2 progeny (treated males, untreated males, treated females, untreated females) and the two parent parasites were sequenced to high read depth (mean = 95-366×) and allele frequencies at 14,489 variants compared. We observed dramatic enrichment of alleles from the resistant parent in a small region of chromosome 6 in drug-treated male and female pools (combined analysis: Z = 11.07, p = 8.74 × 10(-29)). This region contains Smp_089320 a gene encoding a sulfotransferase recently implicated in oxamniquine resistance using classical linkage mapping methods. CONCLUSIONS: These results (a) demonstrate the utility of exome capture for generating reduced representation libraries in Schistosoma mansoni, and (b) provide proof-of-principle that X-QTL methods can be successfully applied to an important human helminth. The combination of these methods will simplify linkage analysis of biomedically or biologically important traits in this parasite.

How should we sample snail microbiomes?

Background: The microbiome is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. The microbiome may influence transmission of pathogens by their vectors, such as mosquitoes or aquatic snails. We previously sequenced the bacterial 16S V4 ribosomal DNA of the hemolymph (blood) of Biomphalaria spp. snails, one of the vectors of the human blood fluke schistosome. We showed that snail hemolymph harbored an abundant and diverse microbiome. This microbiome is distinct from the water environment and can discriminate snail species and populations. As hemolymph bathes snail organs, we then investigated the heterogeneity of the microbiome in these organs. Results: We dissected ten snails for each of two different species ( B. alexandrina and B. glabrata ) and collected their organs (ovotestis, hepatopancreas, gut, and stomach). We also ground in liquid nitrogen four whole snails of each species. We sampled the water in which the snails were living (environmental controls). Sequencing the 16S V4 rDNA revealed organ- specific microbiomes. These microbiomes harbored a lower diversity than the hemolymph microbiome, and the whole-snail microbiome. The organ microbiomes tend to cluster by physiological function. In addition, we showed that the whole-snail microbiome is more similar to hemolymph microbiome. Conclusions: These results are critical for future work on snail microbiomes and show the necessity of sampling individual organ microbiomes to provide a complete description of snail microbiomes.

Contribution of parasite and host genotype to immunopathology of schistosome infections.

Background: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa, and viruses, but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes. Methods: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over an infection period of 12 weeks. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology), and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics. Results: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area, but not in granuloma size. Variation in organ weight was explained by egg burden and by intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokines (IFN-γ, TNF-α), eosinophil, lymphocyte, and monocyte counts. Conclusions: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotype impact immunopathology outcomes.

Contribution of parasite and host genotype to immunopathology of schistosome infections.

Background: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa, and viruses, but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes. Methods: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over an infection period of 12 weeks. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology), and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics. Results: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area, but not in granuloma size. Variation in organ weight was explained by egg burden and by intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokines (IFN-γ, TNF-α), eosinophil, lymphocyte, and monocyte counts. Conclusions: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotype impact immunopathology outcomes.

Contribution of parasite and host genotype to immunopathology of schistosome infections.

BACKGROUND: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa and viruses but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes. METHODS: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over a 12-week infection period. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology) and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics. RESULTS: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area but not in granuloma size. Variation in organ weight was explained by egg burden and intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokine levels (IFN-γ, TNF-α), eosinophils, lymphocytes and monocyte counts. CONCLUSIONS: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotypes impact immunopathology outcomes.

No evidence for schistosome parasite fitness trade-offs in the intermediate and definitive host.

BACKGROUND: The trematode parasite Schistosoma mansoni uses an aquatic snail intermediate and a vertebrate definitive host to complete its life cycle. We previously showed that a key transmission trait-the number of cercariae larvae shed from infected Biomphalaria spp. snails-varies significantly within and between different parasite populations and is genetically controlled by five loci. We investigated the hypothesis that the success of parasite genotypes showing high propagative fitness in the intermediate snail host may be offset by lower reproductive fitness in the definitive vertebrate host. METHODS: We investigated this trade-off hypothesis by selecting parasite progeny producing high or low number of larvae in the snail and then comparing fitness parameters and virulence in the rodent host. We infected inbred BALB/c mice using two Schistosoma mansoni parasite lines [high shedder (HS) and low shedder (LS) lines] isolated from F2 progeny generated by genetic crosses between SmLE (HS parent) and SmBRE (LS parent) parasites. We used the F3 progeny to infect two populations of inbred Biomphalaria glabrata snails. We then compared life history traits and virulence of these two selected parasite lines in the rodent host to understand pleiotropic effects of genes determining cercarial shedding in parasites infecting the definitive host. RESULTS: HS parasites shed high numbers of cercariae, which had a detrimental impact on snail physiology (measured by laccase-like activity and hemoglobin rate), regardless of the snail genetic background. In contrast, selected LS parasites shed fewer cercariae and had a lower impact on snail physiology. Similarly, HS worms have a higher reproductive fitness and produced more viable F3 miracidia larvae than LS parasites. This increase in transmission is correlated with an increase in virulence toward the rodent host, characterized by stronger hepato-splenomegaly and hepatic fibrosis. CONCLUSIONS: These experiments revealed that schistosome parasite propagative and reproductive fitness was positively correlated in intermediate and definitive host (positive pleiotropy). Therefore, we rejected our trade-off hypothesis. We also showed that our selected schistosome lines exhibited low and high shedding phenotype regardless of the intermediate snail host genetic background. ​.

A single locus determines praziquantel response in Schistosoma mansoni.

We previously performed a genome-wide association study (GWAS) to identify the genetic basis of praziquantel (PZQ) response in schistosomes, identifying two quantitative trait loci (QTL) situated on chromosome 2 and chromosome 3. We reanalyzed this GWAS using the latest (v10) genome assembly showing that a single locus on chromosome 3, rather than two independent loci, determines drug response. These results reveal that praziquantel response is monogenic and demonstrates the importance of high-quality genomic information.

Snails, microbiomes, and schistosomes: a three-way interaction?

Aquatic snails, the intermediate hosts of schistosomes, harbor a diverse unexplored microbiome. We speculate that this may play a critical role in host-parasite interactions. We summarize our current knowledge of snail microbiomes and highlight future research priorities.

Schistosome Sulfotransferases: Mode of Action, Expression and Localization.

Oxamniquine (OXA) is a prodrug activated by a sulfotransferase (SULT) that was only active against Schistosoma mansoni. We have reengineered OXA to be effective against S. haematobium and S. japonicum. Three derivatives stand out, CIDD-0066790, CIDD-0072229, and CIDD-0149830 as they kill all three major human schistosome species. However, questions remain. Is the OXA mode of action conserved in derivatives? RNA-interference experiments demonstrate that knockdown of the SmSULT, ShSULT, and SjSULT results in resistance to CIDD-0066790. Confirming that the OXA-derivative mode of action is conserved. Next is the level of expression of the schistosome SULTs in each species, as well as changes in SULT expression throughout development in S. mansoni. Using multiple tools, our data show that SmSULT has higher expression compared to ShSULT and SjSULT. Third, is the localization of SULT in the adult, multicellular eucaryotic schistosome species. We utilized fluorescence in situ hybridization and uptake of radiolabeled OXA to determine that multiple cell types throughout the adult schistosome worm express SULT. Thus, we hypothesize the ability of many cells to express the sulfotransferase accounts for the ability of the OXA derivatives to kill adult worms. Our studies demonstrate that the OXA derivatives are able to kill all three human schistosome species and thus will be a useful complement to PZQ.

Genetic architecture of transmission stage production and virulence in schistosome parasites.

Both theory and experimental data from pathogens suggest that the production of transmission stages should be strongly associated with virulence, but the genetic bases of parasite transmission/virulence traits are poorly understood. The blood fluke Schistosoma mansoni shows extensive variation in numbers of cercariae larvae shed and in their virulence to infected snail hosts, consistent with expected trade-offs between parasite transmission and virulence. We crossed schistosomes from two populations that differ 8-fold in cercarial shedding and in their virulence to Biomphalaria glabrata snail hosts, and determined four-week cercarial shedding profiles in F0 parents, F1 parents and 376 F2 progeny from two independent crosses in inbred snails. Sequencing and linkage analysis revealed that cercarial production is polygenic and controlled by five QTLs (i.e. Quantitative Trait Loci). These QTLs act additively, explaining 28.56% of the phenotypic variation. These results demonstrate that the genetic architecture of key traits relevant to schistosome ecology can be dissected using classical linkage mapping approaches.

Rational approach to drug discovery for human schistosomiasis.

Human schistosomiasis is a debilitating, life-threatening disease affecting more than 229 million people in as many as 78 countries. There is only one drug of choice effective against all three major species of Schistosoma, praziquantel (PZQ). However, as with many monotherapies, evidence for resistance is emerging in the field and can be selected for in the laboratory. Previously used therapies include oxamniquine (OXA), but shortcomings such as drug resistance and affordability resulted in discontinuation. Employing a genetic, biochemical and molecular approach, a sulfotransferase (SULT-OR) was identified as responsible for OXA drug resistance. By crystallizing SmSULT- OR with OXA, the mode of action of OXA was determined. This information allowed a rational approach to novel drug design. Our team approach with schistosome biologists, medicinal chemists, structural biologists and geneticists has enabled us to develop and test novel drug derivatives of OXA to treat this disease. Using an iterative process for drug development, we have successfully identified derivatives that are effective against all three species of the parasite. One derivative CIDD-0149830 kills 100% of all three human schistosome species within 5 days. The goal is to generate a second therapeutic with a different mode of action that can be used in conjunction with praziquantel to overcome the ever-growing threat of resistance and improve efficacy. The ability and need to design, screen, and develop future, affordable therapeutics to treat human schistosomiasis is critical for successful control program outcomes.

Genetic analysis of praziquantel response in schistosome parasites implicates a transient receptor potential channel.

Mass drug administration with praziquantel (PZQ) monotherapy is considered the mainstay for control and elimination of the parasites causing schistosomiasis in humans. This drug shows imperfect cure rates in the field, and parasites showing reduced PZQ response can be selected in the laboratory, but the extent of resistance in Schistosoma mansoni populations is unknown. We examined the genetic basis of the variation in response in a PZQ-selected S. mansoni population (SmLE-PZQ-R) in which 35% of the parasitic worms survive high-dose PZQ (73 micrograms per milliliter) treatment. We used genome-wide association to map loci underlying PZQ response and identified a transient receptor potential (Sm.TRPMPZQ) channel (Smp_246790) within the major chromosome 3 peak that is activated by nanomolar concentrations of PZQ. The PZQ response showed recessive inheritance and marker-assisted selection of parasites at a single Sm.TRPMPZQ SNP that produced populations of PZQ-enriched resistant (PZQ-ER) and PZQ-enriched sensitive (PZQ-ES) parasites, exhibiting >377-fold difference in PZQ response. The PZQ-ER parasites survived treatment in rodents at higher frequencies compared with PZQ-ES, and resistant parasites exhibited 2.25-fold lower expression of Sm.TRPMPZQ relative to sensitive parasites. Specific chemical blockers of Sm.TRPMPZQ enhanced PZQ resistance, whereas Sm.TRPMPZQ activators increased sensitivity. We surveyed Sm.TRPMPZQ sequence variations in 259 parasites from different global sites and identified one nonsense mutation that resulted in a truncated protein with no PZQ binding site. Our results demonstrate that Sm.TRPMPZQ underlies variation in PZQ responses in S. mansoni and provides an approach for monitoring emerging PZQ-resistant alleles in schistosome elimination programs.

Why does oxamniquine kill Schistosoma mansoni and not S. haematobium and S. japonicum?

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (SmSULT). S. haematobium and S. japonicum possess homologs of SmSULT (ShSULT and SjSULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group.

An iterative process produces oxamniquine derivatives that kill the major species of schistosomes infecting humans.

Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Strong selective pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel. Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The oxamniquine activating enzyme was identified as a S. mansoni sulfotransferase (SmSULT-OR). Structural data have allowed for directed drug development in reengineering oxamniquine to be effective against S. haematobium and S. japonicum. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust SAR program that tested over 300 derivatives and identified several new lead compounds with effective worm killing in vitro. Previous studies resulted in the discovery of compound CIDD-0066790, which demonstrated broad-species activity in killing of schistosome species. As these compounds are racemic mixtures, we tested and demonstrate that the R enantiomer CIDD-007229 kills S. mansoni, S. haematobium and S. japonicum better than the parent drug (CIDD-0066790). The search for derivatives that kill better than CIDD-0066790 has resulted in a derivative (CIDD- 149830) that kills 100% of S. mansoni, S. haematobium and S. japonicum adult worms within 7 days. We hypothesize that the difference in activation and thus killing by the derivatives is due to the ability of the derivative to fit in the binding pocket of each sulfotransferase (SmSULT-OR, ShSULT-OR, SjSULT-OR) and to be efficiently sulfated. The purpose of this research is to develop a second drug to be used in conjunction with praziquantel to treat the major human species of Schistosoma. Collectively, our findings show that CIDD-00149830 and CIDD-0072229 are promising novel drugs for the treatment of human schistosomiasis and strongly support further development and in vivo testing.