Project description:Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows exclusively in the xylem of several plants, causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time-course experiment (2, 8 and 12 hours) revealed many differentially expressed genes under nitrogen starvation, such as genes related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. A more complete picture of the ?54 regulon was achieved by combining the transcriptome data with an in silico search for potential ?54-dependent promoters, using a position weight matrix approach. One of these ?54-predicted binding sites, located upstream of the glnA gene (encoding a glutamine synthetase), was validated by primer extension assays, confirming that this gene has a ?54-dependent promoter and contains a predicted NtrC binding site. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the ?54 regulon. For time-course studies, cells cultivated at late-exponential phase in PWG medium were used to inoculate a culture in 100 ml XDM2 medium to an optical density at 600 nm (OD600 nm) of 0.1. Cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Then, the culture was divided into two 6 portions and cells were washed with XDM2 medium (zero time) or XDM2 medium lacking all nitrogen sources (XDM0), respectively. The cultivation was continued for 2h, 8h and 12h in XDM0 to establish nitrogen starvation conditions. For each time point, cells in a 25-ml culture were collected by centrifugation and rapidly frozen in dry ice, until RNA isolation. Three RNA samples isolated from independently grown cultures of the cells at each starvation period (2h, 8h and 12h) were examined, and each preparation was subjected to microarray analysis. As the genes were spotted at least in duplicate, we obtained six replicates for each gene from three independent data sets per gene per starvation period. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes induced by nitrogen starvation in a ?54-dependent manner. To determine the effect of rpoN inactivation on gene expression after nitrogen starvation, the transcriptomes of the wild type and the rpoN strains were compared using DNA microarrays, with both strains grown on XDM2 medium and submitted to nitrogen starvation (XDM0) during 2 hours. Three RNA samples isolated from independently grown cultures of the cells were examined. Due to the platform design, each microarray slide was divided into "LEFT" and "RIGHT", allowing the probing of two technical replicates per slide.

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:A mouse hepatoma cell line (Hepa1-6 cells, ATCC) was grown in T-25 tissue culture flasks (Corning) at 37ÂºC in 5% CO2 under two conditions. The first condition was the control, which contained DMEM medium (Gibco BRL) with 25 mM glucose, 4 mM glutamine, 1% penicillin-streptomycin (Sigma), and 10% CPSR-3 (Sigma). The second condition was identical to the first, however the glutamine concentration was altered periodically. Once the cells had grown to confluency, the experiment began at 9:30 PM by exchanging the medium in all flasks. The control flasks received the same medium, containing 4 mM glutamine, while the experimental flasks received identical medium with no glutamine. Twenty-four hours later, the medium was exchanged again, however this time all flasks received medium containing 4 mM glutamine. The first medium exchange was repeated at 48 hours into the experiment, while the second was repeated at 72 hours into the experiment. Hence the glutamine concentration of the experimental flasks oscillated between 0 mM and 4 mM glutamine over 96 hours, while the glutamine concentration of the control flasks remained constant at 4 mM glutamine. At regular intervals, the flasks were sampled. Total RNA was isolated, medium samples were retained, and isotopic measurements were performed from flasks fed different labelled compounds. All samples taken during the experiment were analyzed. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60ÂºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42ÂºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ÂºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples from identical time points that were to be compared on DNA microarrays were then mixed. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65ÂºC. Following concentration, the sample was resuspended in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), 1 uL was removed for quantitation, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 ÂºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride, 239 mL 1-methyl-2-pyrrolidinone, and 10.7 mL of 1 M boric acid, pH 8.0. They were cleaned by rinsing in 250 mL of milli-Q water (X2), followed by 250 mL of 95% ethanol. They were finally dried using filtered air and stored in the dark at room temperature. Keywords = mouse, liver, hepatoma, cell culture, glutamine, carbon metabolism

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: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 .

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