Synergy of Omeprazole and Praziquantel <i>In Vitro</i> Treatment against <i>Schistosoma mansoni</i> Adult Worms

<div><p>Background</p><p>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.</p><p>Methodology</p><p>We used a custom-designed <i>Schistosoma mansoni</i> 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 <i>S</i>. <i>mansoni</i> adult worms with a survival <i>in vitro</i> assay.</p><p>Principal Findings</p><p>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 <i>in vitro</i> when compared with PZQ or OMP alone, thus evidencing a synergistic effect.</p><p>Conclusions</p><p>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 <i>S</i>. <i>mansoni</i> adult worms <i>in vitro</i> when compared with either drug alone.</p></div

General information about gene expression of paired or unpaired mature females exposed to PZQ compared with no-drug controls.

<p>^a Number of genes expressed in at least one condition, <i>i</i>.<i>e</i>. in no-drug control or in treated worms of either the paired or the unpaired worm groups.</p><p>^b q-value < 5%</p><p>^c Either predicted in <i>S</i>. <i>japonicum</i>, present in other species, or detected only in <i>S</i>. <i>mansoni</i> without close homologs in other species (no match).</p><p>General information about gene expression of paired or unpaired mature females exposed to PZQ compared with no-drug controls.</p

Enriched gene interaction network detected with similar expression pattern in PZQ-treated paired males and females.

<p>The gene interaction network is related to carbohydrate metabolism, molecular transport and small molecule biochemistry. The shapes of elements correspond to different types of molecules, as indicated in the inset box. Arrows indicate the relationship between the elements: dashed or solid lines indicate indirect or direct interactions, respectively. The color intensity is proportional to expression value, computed as log2 [PZQ/Control]; red corresponds to positive log-ratios, <i>i</i>.<i>e</i>. genes up-regulated in paired males and females treated with PZQ when compared with their respective no-drug controls, grey corresponds to those genes present in the analysis but not differentially expressed. In humans, one gene homolog in this network encodes a protein that is a known drug target and the corresponding drugs (Rx) are indicated.</p

Most significantly enriched interaction network of differentially expressed genes in PZQ-treated paired males.

<p>The gene interaction network is related to cellular assembly and organization, cell-to-cell signaling and interaction. The shapes of elements correspond to different types of molecules, as indicated in the inset box. Arrows indicate the relationship between the elements: dashed or solid lines indicate indirect or direct interactions, respectively. The color intensity is proportional to expression value, computed as log2 [PZQ/Control]; red corresponds to positive log-ratios, <i>i</i>.<i>e</i>. genes up-regulated in paired males treated with PZQ when compared with no-drug controls, green corresponds to negative log-ratios, <i>i</i>.<i>e</i>. down-regulated genes, and grey corresponds to those genes present in the analysis but not differentially expressed. Two genes from this network encode human proteins that are known drug targets and the corresponding drugs (Rx) are indicated.</p

Validation by RT-qPCR of expression changes induced by PZQ treatment in <i>S</i>. <i>mansoni</i> adult worms.

<p>The expression levels of genes were measured by RT-qPCR in parasites treated with PZQ (black bars) relative to the levels in the respective no-drug control parasites (gray bars). Genes were measured (A) in paired females; (B) in unpaired mature females; (C) in paired males; and (D) in paired females. Genes with significant difference (t-test, p < 0.05) are marked with asterisk. Next to each gene name, the gene expression fold-change induced by PZQ and detected by microarray is shown for comparison.</p

Global transcriptional changes driven by PZQ on <i>S</i>. <i>mansoni</i> adult worms.

<p>Overall summary with the names of the associated functional interaction networks identified as significantly enriched with differentially expressed <i>S</i>. <i>mansoni</i> genes in different comparisons: (A) genes affected by PZQ in opposite directions in paired or unpaired mature females, (B) all genes affected by PZQ in paired females, (C) all genes affected by PZQ in unpaired mature females, (D) all genes affected by PZQ in paired males, (E) genes affected in common in paired males and females. Color arrows indicated the direction of change in expression of the majority of the genes in each network: green implies a down-regulated expression of the majority of the genes in PZQ-treated worms, and red an up-regulated expression of the majority of the genes in PZQ-treated worms when compared with their respective no-drug controls. Rx corresponds to the identification of new candidate drugs known to act on the encoded protein products of the homolog human genes present in the indicated network, as described in the main text.</p

Enriched gene interaction network detected with opposite expression patterns in PZQ-treated paired or unpaired mature females.

<p>This gene interaction network is significantly enriched (p-value = 10⁻⁵²) with differentially expressed genes related to cellular development and drug metabolism whose expression was affected by PZQ in paired as well as in unpaired mature females (with opposite patterns). The shapes of elements correspond to different types of molecules, as indicated in the inset box. Arrows indicate the relationship between the elements: dashed or solid lines indicate indirect or direct interactions, respectively. The color intensity is proportional to the expression value, computed as log2 [PZQ/Control]; red corresponds to positive log-ratios, <i>i</i>.<i>e</i>. these genes were up-regulated in paired females treated with PZQ when compared with the no-drug controls; grey corresponds to a gene present in the analysis but not differentially expressed. In humans, a number of gene homologs in this network encode proteins that are known drug targets and the corresponding drugs (Rx) are indicated.</p

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

<div><p>Background</p><p>Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is a public health problem. <i>Schistosoma mansoni</i> 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.</p><p>Methodology/Principal findings</p><p>To compare the gene expression of <i>S</i>. <i>mansoni</i> eggs, females, and males, we performed RNA-Seq on these three parasite forms using 454/Roche technology and reconstructed the transcriptome using Trinity <i>de novo</i> 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 <i>S</i>. <i>mansoni</i> 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.</p><p>Conclusions/Significance</p><p>Large-scale RNA-Seq analysis using <i>de novo</i> 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 (<a href="http://schistosoma.usp.br/" target="_blank">http://schistosoma.usp.br/</a>). This database provides new ways to explore the schistosome genome and transcriptome and will facilitate molecular research on this important parasite.</p></div

General information about gene expression in paired males exposed to PZQ compared with no-drug controls.

<p>^a Number of genes expressed in at least one condition, <i>i</i>.<i>e</i>. in control or in treated paired male adult worms.</p><p>^b q-value < 5%</p><p>^c Either predicted in <i>S</i>. <i>japonicum</i>, present in other species, or detected only in <i>S</i>. <i>mansoni</i> without close homologs in other species (no match).</p><p>General information about gene expression in paired males exposed to PZQ compared with no-drug controls.</p

Kaplan-Meier survival curves for adult <i>S</i>. <i>mansoni</i> worms treated <i>in vitro</i> with PZQ+OMP, PZQ or OMP.

<p>Adult worm couples were treated with a synergistic combination of PZQ+OMP (blue line), with PZQ alone (red line) or with OMP alone (black line). The assays were performed with paired adult worm couples (20 couples for each of the three conditions in each of the two biological replicates) and the fraction of surviving worms was recorded after 2, 24, 48, 72, 96 and 120 hours; the Kaplan-Meier survival curve was calculated using all the events of the two biological replicas together, and the Log-rank Mantel-Cox statistical test was used to calculate the significance of the Hazard Ratio between OMP+PZQ and PZQ alone. The effect of the synergistic combination was analyzed for each gender separately: (A) for scoring males percent survival, couples were incubated in the presence of 150 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone; and (B) for scoring females percent survival, couples were incubated in the presence of 532 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone.</p