Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms.

BACKGROUND: Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis. METHODOLOGY: We used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay. PRINCIPAL FINDINGS: We identified sets of genes that were affected by PZQ in paired and unpaired mature females, however with opposite gene expression patterns (up-regulated in paired and down-regulated in unpaired mature females), indicating that PZQ effects are heavily influenced by the mating status. We also identified genes that were similarly affected by PZQ in males and females. Functional analyses of gene interaction networks were performed with parasite genes that were differentially expressed upon PZQ treatment, searching for proteins encoded by these genes whose human homologs are targets of different drugs used for other diseases. Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested. Sublethal doses of PZQ combined with OMP significantly increased worm mortality in vitro when compared with PZQ or OMP alone, thus evidencing a synergistic effect. CONCLUSIONS: Functional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.

Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq.

BACKGROUND: Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is a public health problem. Schistosoma mansoni is the most widespread species responsible for schistosomiasis in the Americas, Middle East and Africa. Adult female worms (mated to males) release eggs in the hepatic portal vasculature and are the principal cause of morbidity. Comparative separate transcriptomes of female and male adult worms were previously assessed with using microarrays and Serial Analysis of Gene Expression (SAGE), thus limiting the possibility of finding novel genes. Moreover, the egg transcriptome was analyzed only once with limited bacterially cloned cDNA libraries. METHODOLOGY/PRINCIPAL FINDINGS: To compare the gene expression of S. mansoni eggs, females, and males, we performed RNA-Seq on these three parasite forms using 454/Roche technology and reconstructed the transcriptome using Trinity de novo assembly. The resulting contigs were mapped to the genome and were cross-referenced with predicted Smp genes and H3K4me3 ChIP-Seq public data. For the first time, we obtained separate, unbiased gene expression profiles for S. mansoni eggs and female and male adult worms, identifying enriched biological processes and specific enriched functions for each of the three parasite forms. Transcripts with no match to predicted genes were analyzed for their protein-coding potential and the presence of an encoded conserved protein domain. A set of 232 novel protein-coding genes with putative functions related to reproduction, metabolism, and cell biogenesis was detected, which contributes to the understanding of parasite biology. CONCLUSIONS/SIGNIFICANCE: Large-scale RNA-Seq analysis using de novo assembly associated with genome-wide information for histone marks in the vicinity of gene models constitutes a new approach to transcriptome analysis that has not yet been explored in schistosomes. Importantly, all data have been consolidated into a UCSC Genome Browser search- and download-tool (http://schistosoma.usp.br/). This database provides new ways to explore the schistosome genome and transcriptome and will facilitate molecular research on this important parasite.

Interference with hemozoin formation represents an important mechanism of schistosomicidal action of antimalarial quinoline methanols.

BACKGROUND: The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11(th) to 17(th) day after infection caused significant decreases in worm burden (39%-61%) and egg production (42%-98%). Hz formation was significantly inhibited (40%-65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms. CONCLUSIONS: The overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.

Schistosoma mansoni tegument protein Sm29 is able to induce a Th1-type of immune response and protection against parasite infection.

BACKGROUND: Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. METHODS AND FINDINGS: We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-gamma, TNF-alpha and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. CONCLUSION: This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

Analysis of Schistosoma mansoni genes shared with Deuterostomia and with possible roles in host interactions.

BACKGROUND: Schistosoma mansoni is a blood helminth parasite that causes schistosomiasis, a disease that affects 200 million people in the world. Many orthologs of known mammalian genes have been discovered in this parasite and evidence is accumulating that some of these genes encode proteins linked to signaling pathways in the parasite that appear to be involved with growth or development, suggesting a complex co-evolutionary process. RESULTS: In this work we found 427 genes conserved in the Deuterostomia group that have orthologs in S. mansoni and no members in any nematodes and insects so far sequenced. Among these genes we have identified Insulin Induced Gene (INSIG), Interferon Regulatory Factor (IRF) and vasohibin orthologs, known to be involved in mammals in mevalonate metabolism, immune response and angiogenesis control, respectively. We have chosen these three genes for a more detailed characterization, which included extension of their cloned messages to obtain full-length sequences. Interestingly, SmINSIG showed a 10-fold higher expression in adult females as opposed to males, in accordance with its possible role in regulating egg production. SmIRF has a DNA binding domain, a tryptophan-rich N-terminal region and several predicted phosphorylation sites, usually important for IRF activity. Fourteen different alternatively spliced forms of the S. mansoni vasohibin (SmVASL) gene were detected that encode seven different protein isoforms including one with a complete C-terminal end, and other isoforms with shorter C-terminal portions. Using S. mansoni homologs, we have employed a parsimonious rationale to compute the total gene losses/gains in nematodes, arthropods and deuterostomes under either the Coelomata or the Ecdysozoa evolutionary hypotheses; our results show a lower losses/gains number under the latter hypothesis. CONCLUSION: The genes discussed which are conserved between S. mansoni and deuterostomes, probably have an ancient origin and were lost in Ecdysozoa, being still present in Lophotrochozoa. Given their known functions in Deuterostomia, it is possible that some of them have been co-opted to perform functions related (directly or indirectly) to host adaptation or interaction with host signaling processes.

Use of a 44k oligoarray to explore the transcriptome of Schistosoma mansoni adult worms.

Recent advances in the study of Schistosoma mansoni genome and transcriptome have led to a better description of the S. mansoni gene complement. In this work, we report the design and use of a new S. mansoni 60-mer oligonucleotide microarray platform with approximately 44,000 probes, based on all publicly available cDNA sequence data for S. mansoni and Schistosoma japonicum. The large number of probes combined with the extensive sequence annotation available allowed a comprehensive approach, where most of the S. mansoni transcriptome is represented. Hybridization with adult worm RNA pointed to a set of genes transcriptionally active in this stage of the parasite's life cycle. Interestingly, a large proportion (43%) of genes for which transcription was detected in adults is comprised of "no match" genes, i.e. S. mansoni genes with unknown function and no identifiable orthologs in GenBank. Moreover, detection of bi-directional transcription for 7% of the active "no match" genes in adults leads us to hypothesize a widespread production of antisense RNA in S. mansoni, with possible regulatory roles.

Antisense intronic non-coding RNA levels correlate to the degree of tumor differentiation in prostate cancer.

A large fraction of transcripts are expressed antisense to introns of known genes in the human genome. Here we show the construction and use of a cDNA microarray platform enriched in intronic transcripts to assess their biological relevance in pathological conditions. To validate the approach, prostate cancer was used as a model, and 27 patient tumor samples with Gleason scores ranging from 5 to 10 were analyzed. We find that a considerably higher fraction (6.6%, [23/346]) of intronic transcripts are significantly correlated (P< or =0.001) to the degree of prostate tumor differentiation (Gleason score) when compared to transcripts from unannotated genomic regions (1%, [6/539]) or from exons of known genes (2%, [27/1369]). Among the top twelve transcripts most correlated to tumor differentiation, six are antisense intronic messages as shown by orientation-specific RT-PCR or Northern blot analysis with strand-specific riboprobe. Orientation-specific real-time RT-PCR with six tumor samples, confirmed the correlation (P=0.024) between the low/high degrees of tumor differentiation and antisense intronic RASSF1 transcript levels. The need to use intron arrays to reveal the transcriptome profile of antisense intronic RNA in cancer has clearly emerged.