Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Interference of hemozoin formation in Schistosoma mansoni by quinine reduces experimental schistosomiasis

ABSTRACT: Background: The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel is still the only drug used for schistosomiasis treatment and reduction in drug efficiency has prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within S. mansoni gut is a major heme detoxification route involving lipid droplets in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of the antimalarial quinine (QN) 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 (75mg/kg/day) from 11th to 17th day after infection decreased not only total, males and females worm burden (46.2 %-51.0 %), eggs production, but also the granulomatous reaction to parasite eggs trapped in the liver. These effects correlated with a significant impairment of Hz production (40%) specifically in S. mansoni females, being parallel to remarkable ultrastructural changes in female worms, particularly in the gut epithelium. 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 QN treatment indicates that interference of Hz formation in S. mansoni is a valid chemotherapeutical target for development of new antischistosomal drugs. The perfused adult female worms were incubated with cold RNAlater solution (Ambion) and kept at 4 0C until RNA extraction. Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacture's instructions. RNA was quantified using Nanodrop ND-1000 UV/Vis spectrophotometer and its quality was assured with Agilent 2100 Bioanalyzer, a micro fluidics-based electrophoresis platform. 1 Î¼g total RNA from each sample was amplified using the T7-RNA polymerase based SuperScript Indirect RNA amplification system (Invitrogen) and subsequently 3 Î¼g of aminoallyl-modified amplified RNA was labeled using Cy3 or Cy5 by indirect labeling (General Electric). Amplification and labeling were done according to manufacturer specifications. The Cy3 and Cy5 labeled samples were then combined, dried and re-suspended in hybridization buffer (50 % formamide, 25 % RNase free water and 25 % Microarray Hybridization buffer 4x) (General Electric). Pools of RNA from worms treated with QN were hybridized against RNA from control worms. In order to ensure the uniformity of data, all experiments were performed using slides from the same printing batch and dyes from the same lot. The same washing protocol was used and immediately after washing we scanned the slides using the same parameters. Technical and biological replicates were performed. Dye swap was employed in order to account for dye biases. A total of eight different replicated data values were obtained for each probe on the array. The samples were hybridized overnight to cDNA microarray slides containing 4,000 elements in duplicate (GEO accession: GPL3929) using ASP hybridization chambers (GE, USA) at 42 Â°C. Slides were washed once for 3 minutes in low stringency wash solution at 45 Â°C (1 x SSC + 0.2 % SDS), followed by one wash for 5 minutes in medium stringency wash solution (0.1 x SSC + 0.2%SDS) and one wash of 1 minute in high stringency wash solution (0.1 x SSC). Moreover, slides were air dried and scanned using a microarray dual channel laser scanner (GenePix 4000B, Molecular Devices, USA) at 5 Î¼m resolution, 100 % laser power and PMT levels were adjusted in order to obtain similar average intensities of red and green signal. Data were extracted using Array Vision 8.0 program. To correct for systematic biases on the data originated from small differences in the labeling and/or detection efficiencies between the fluorescent dyes, expression ratios were logged (base 2) and normalized using a locally weighted linear regression (LOWESS) algorithm [60]. Normalized log2 (ratios) were further analyzed with the Significance Analysis of Microarrays tool (SAM) [61] using a 0.1 % false discovery rate (FDR) to find differentially expressed genes. The genes identified in the previous step were filtered using a 1.7 fold change cut-off in at least 4 out of the 8 data points. This step is critical in identifying more biological relevant changes. It is important to stress that the fold change filter was used after the identification of differentially expressed genes, and our results are not influenced by the potentially misleading effects caused by the use of arbitrary thresholds to trim data sets before significance testing .

ORGANISM(S): Schistosoma mansoni

SUBMITTER: Marcus Oliveira

PROVIDER: E-GEOD-14751 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

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

Corrêa Soares Juliana B R JB Menezes Diego D Vannier-Santos Marcos A MA Ferreira-Pereira Antonio A Almeida Giulliana T GT Venancio Thiago M TM Verjovski-Almeida Sergio S Zishiri Vincent K VK Kuter David D Hunter Roger R Egan Timothy J TJ Oliveira Marcus F MF

PLoS neglected tropical diseases 20090714 7

<h4>Background</h4>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 l ...[more]

PMID: 19597543

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Project description:Schistosomiasis continues to be a significant public health problem1. 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 S. mansoni. Our group recently identified Sm29, an antigen that is predominantly recognized by IgG1 and IgG3 antibodies of resistant patients2. In the present study, we show that Sm29 is located on the surface of adult worms and lung-stage schistosomula. Immunization of mice with recombinant (r) Sm29 engendered 51% reduction in adult worm burdens, 60% reduction in intestinal eggs and 55% reduction in liver granulomas. Protective immunity in mice was associated with high titers of specific IgG1 and IgG2a and elevated production of IFN-Î³, TNF-Î± and IL-12. Further, cellular responses of infected schistosomiasis patients to rSm29 consisted of elevated IFN-Î³ and an absence of IL-5. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to control mice revealed a significant (q< 0.01) down-regulation of 498 genes, while no up-regulation was detected. 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. This study demonstrates that the membrane-bound Sm29 protein is a new molecule that has great potential as a vaccine candidate against schistosomiasis. Keywords: Schistosoma mansoni gene expression in vaccinated mice Two biological samples were used in the study, each used in two arrays (technical replicates with dye swap) and each array containing two replicates of each spot. Overall, there are eight data points data points for each spot. Only cases with more than 4 of 8 points are valid ratios were considered. Value and its respective dye swap value [after changing the log(ratio) signal] were averaged, reducing the data point to four. The average values were than used in the significance testing using SAM (Tusher et al., 2001). The samples were used to get average values using the following combination: 3000139790L with 3000139792L; 3000139790R with 3000139792R; 3000139791L with 3000139795L; 3000139791R with 3000139795R The normalized, averaged dyeswap values are provided in a supplementary file at the foot of the record.

Project description:Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to the Brazilian citrus industry. Genome sequences of several strains of this phytopathogen are accessible, enabling large-scale functional studies. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. When treated with the iron chelator 2,2-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. In the presence of 100uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription - quantitative PCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron regulon of X. fastidiosa. The promoter regions of strain 9a5c genome were screened for putative Fur boxes and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron regulon of X. fastidiosa and present novel evidence for iron regulation of pathogenicity determinants. Keywords: stress response; response to iron-depleted condition Direct comparison between low iron content (200uM 2,2-dipyridyl) and control condition. Hybridizations are dye-swaped. There are 2 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).

Project description:Xylella fastidiosa is the etiologic agent of a wide range of plant diseases including citrus variegated chlorosis (CVC), a major threat to the Brazilian citrus industry. Genome sequences of several strains of this phytopathogen are accessible, enabling large-scale functional studies. Transcript levels in different iron availabilities were assessed with DNA microarrays representing 2608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c. When treated with the iron chelator 2,2-dipyridyl, 193 CDS were considered as up-regulated and 216 as down-regulated. In the presence of 100uM of ferric pyrophosphate, 218 and 256 CDS were considered as up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription - quantitative PCR that showed a Pearson correlation of 0.77 with array results. The CDS differentially expressed upon the iron concentration shift participate in diverse cellular functions. Many CDS involved with regulatory functions, pathogenicity and cell structure, were modulated in both conditions tested suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to pili/fimbriae functions. We also investigated the contribution of the ferric uptake regulator Fur to the iron regulon of X. fastidiosa. The promoter regions of strain 9a5c genome were screened for putative Fur boxes and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron regulon of X. fastidiosa and present novel evidence for iron regulation of pathogenicity determinants. Direct comparison between high iron content (100uM ferric pyrophosphate) and control condition. Hybridizations are dye-swaped. There are 2 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).

Project description:Background: Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel. Therefore, there is a pressing need to explore the effects of PZQ on the parasites at the molecular level and to pursue alternative and/or or synergistic drugs against schistosomiasis. Methodology: We used a custom-designed Schistosoma mansoni oligo-microarray to explore the effects of a sublethal dose of praziquantel on S. mansoni adult worms’ gene expression. We used functional analysis of gene interaction networks to identify differentially expressed genes that are known targets of other drugs already tested in humans for diverse disease conditions. Omeprazole, a proton pump inhibitor drug that is widely prescribed, was tested in combination with praziquantel. We comparatively assessed the efficacy of praziquantel or omeprazole alone and in combination against S. mansoni adult worms in vitro over a period of 120 hours. Principal Findings: We identified sets of genes that were affected by praziquantel on both paired and unpaired females, however with opposite gene expression patterns (up-regulated in paired and down-regulated in unpaired females), indicating that the transcriptomics changes induced by praziquantel are heavily influenced by the mating status. We also identified genes that were similarly affected by praziquantel in males and females. The use of sublethal doses of praziquantel together with omeprazole showed a significantly enhanced worm mortality in vitro compared to praziquantel alone, thus evidencing a synergic effect. Conclusions: Functional analysis of gene interaction networks is an important approach to identify genes whose expression is affected by one drug and that are at the same time known targets of other drugs already tested in humans for diverse disease conditions, thus pointing the latter as possible synergic drugs. Combined treatment with omeprazol increases the efficacy of praziquantel against S. mansoni adult worms in vitro, and additional in vivo studies are warranted. Two-condition experiment, PZQ-treated vs. untreated . Biological replicates: 2 with dye-swap

Dataset Information

Interference of hemozoin formation in Schistosoma mansoni by quinine reduces experimental schistosomiasis

Publications

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

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