Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Microarray analysis for differential gene expression in M1-SDHHbtail

ABSTRACT: A total of 215 gene were found to be differentially expressed; 90 of them were up regulated and 125 genes were down regulated. Most notably the functional categories that were affected include Virulence genes, carbohydrate genes and lipid synthesis genes. Some of the virulence genes were down regulated by more than 16-32 fold. These results indicate that SDH plays an important role in the regulation of virulence. S.pyogenes strain SF370 (wild-type) was procured from ATCC (ATCC 700294). M1-SDHHbtail (SDH cytoplasmically retained) mutant strain PGB-1 was obtained using pFW5 vector as described before ( Boel G. Jin, H. and Pancholi, V. (2005). Inhibition of cell surface export of groupA streptococcal anchorless surface dehydrogenase (SDH) affects bacterial adherence and antiphagocytic properties. Infect. Immun. 73:6237-6248). Both strains (M1-Wild-type and PGB1(M1-SDHHbtail) were grown in Todd-Hewitt broth to their late log phase. Bacteria were harvested by centrifugation and washed twice with sterile PBS. Total RNA was isolated from these washed streptococci using Qiagen RNeasy Mini kit. For synthesis and labeling of cDNA, 20 ug of total RNA, 1.5 ug of random hexamer, 0.5mM dNTPs (except that 0.2mM of dTTP was replaced by the same amount of amino-allyl dUTP to incorporate dUTP into first-strand cDNA) were used in each reverse transcriptase reaction (Superscript II Reverse Transcriptase, Invitrogen). Purified cDNA preparations from the wild-type and PGB1 mutant strains were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using three independently isolated RNA preparations (biological replicates), a total of 8 experiments (incorporating dye swaps) were performed. Accordingly eight hybridization measurements for this mutant were performed. Thus Exp-1 and -2 (GSM380310, GSM380320) are the technical replicates of the biological sample-1, Exp-3,-4 and -5 (GSM380321, GSM380322, GSM380331) are technical replicates of biological sample-2 and Exp-6,-7, and -8 (GSM380332,GSM380333, GSM380334) are technical replicates of the biological sample-3. Signals of the bound reagents on the microarray spots in terms of relative fluorescence values were measured and quantified by a laser scanner (GenePix 4100 ) at 10um/pixel resolution. The resulting images were processed using Gene Pix Pro software (version 4.0, Axon instruments). The raw data were obtained in the form of GenPix *.gpr output files. The web application CARMAweb (Comprehensive R based microarray analysis web service) was used for the normalization and analysis of microarray data. All raw data (in the form of *. GPR files) were uploaded to the web application in the data directory. Using appropriate navigation tree, background correction from the foreground signal was applied and within microarray normalization was achieved using the Lowess method. Genes flagged as bad spots by the scanning software were excluded from the analysis and all flagged spots were given a weight of zero. The normalized data were then subjected to fold-change analysis and t-statistics using Bioconductor multtest package. CARMAweb allows to set Log2 cut-off values for both the M (regulation) and the A (average expression) values. Differentially expressed genes (Log2 values of Mutant647-red vs. wild-type555-green or Log2 values Mutant-555(green) vs Wild-type 647-red) were defined based on cut-off value >1 Log2< i.e. all genes that show a two or more-fold up- or down-regulation. Bioconductors Multtest package provided suitable method to adjust P values according to multiple hypothesis testing problems.

ORGANISM(S): Streptococcus pyogenes

SUBMITTER: Vijay Pancholi

PROVIDER: E-GEOD-15231 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence.

Jin Hong H Agarwal Shivangi S Agarwal Shivani S Pancholi Vijay V

mBio 20110531 3

<h4>Unlabelled</h4>Streptococcal surface dehydrogenase (SDH) (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) is an anchorless major multifunctional surface protein in group A Streptococcus (GAS) with the ability to bind important mammalian proteins, including plasmin(ogen). Although several biological properties of SDH are suggestive of its possible role in GAS virulence, its direct role in GAS pathogenesis has not been ascertained because it is essential for GAS survival. Thus, it has remain ...[more]

PMID: 21628503

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Project description:A total of 192 genes were found to be differentially expressed; 38 genes of them were up regulated and 154 genes were down regulated. Most notably the functional categories that were affected include carbohydrate metabolism and cell envelop realted genes ( 56, 29.1% of the total),Virulence genes (12 genes 6.25% of the total significantly differentiated genes), and aminoacid transport genes (7 genes, 3.6 %). Among the virulence-related genes, the most notable ones were those belong to the production of capsule, the M protein, exotoxin, NAD glycohydrolase and Streptolysin S. Some of the capsule related genes were down regulated by more than 64 fold. Additionally 11 differentially expressed lipid metabolism related genes and four aminoacid transport and metabolism-related genes were upregulated. These results indicate that the C-terminal CHAP domain of CdhA (group A Streptococcal Cell division controlling and Chain-forming cell wall hydrolase) plays an important role in the regulation of virulence. Since these mutant lack cell wall hydrolase activity but are not defective in cell division or growth, we believe that CdhA isa multifunctional protein with N-terminal region controlling cell division and c-terminal region responsible for regulating bacterial virulence. S. pyogenes strain SF370 (wild-type) was procured from ATCC (ATCC 700294). M1-CdhA55(-) mutant strain was obtained using pFW5 vector containing spectinomycin resistance marker (aad9). The mutant expressing truncated CdhA that lack C-terminal 55 residues constituting catalytic site of the CHAP domain is not defective in cell division and growth, but lacks antiphagocytic property and attenuated for virulence. Both strains (M1-Wild-type and M1-CdhA55(-) were grown in Todd-Hewitt broth to their late log phase. Bacteria were harvested by centrifugation and washed twice with sterile PBS. Total RNA was isolated from these washed streptococci using Qiagen RNeasy Mini kit. For synthesis and labeling of cDNA, 20 ug of total RNA, 1.5 ug of random hexamer, 0.5mM dNTPs (except that 0.2mM of dTTP was replaced by the same amount of amino-allyl dUTP to incorporate dUTP into first-strand cDNA) were used in each reverse transcriptase reaction (Superscript II Reverse Transcriptase, Invitrogen). Purified cDNA preparations from the wild-type and M1-CdhA55(-) mutant strains were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using three independently isolated RNA preparations (biological replicates), a total of 7 experiments (incorporating dye swaps) were performed. Accordingly 7 hybridization measurements for this mutant were performed. Thus Exp-1 and 2 (GSM389993, GSM389994) are the technical replicates of the biological sample-1, Exp-3 and 4 (GSM389995, GSM390054) are technical replicates of biological sample-2 and Exp-5,-6, and -7 (GSM390055,GSM390056, and GSM390057) are technical replicates of the biological sample-3. Signals of the bound reagents on the microarray spots in terms of relative fluorescence values were measured and quantified by a laser scanner (GenePix 4100 ) at 10um/pixel resolution. The resulting images were processed using Gene Pix Pro software (version 4.0, Axon instruments). The raw data were obtained in the form of GenPix *.gpr output files. The web application CARMAweb (Comprehensive R based microarray analysis web service) was used for the normalization and analysis of microarray data. All raw data (in the form of *. GPR files) were uploaded to the web application in the data directory. Using appropriate navigation tree, background correction from the foreground signal was applied and within microarray normalization was achieved using the Lowess method. Genes flagged as bad spots by the scanning software were excluded from the analysis and all flagged spots were given a weight of zero. The normalized data were then subjected to fold-change analysis and t-statistics using Bioconductor multtest package. CARMAweb allows to set Log2 cut-off values for both the M (regulation) and the A (average expression) values. Differentially expressed genes (Log2 values of Mutant647-red vs. wild-type555-green or Log2 values Mutant-555(green) vs Wild-type 647-red) were defined based on cut-off value >1 Log2< i.e. all genes that show a two or more-fold up- or down-regulation. BioConductors Multtest package provided suitable method to adjust P values according to multiple hypothesis testing problems.

Project description:A total of 163 genes were found to be differentially expressed; 38 genes of them were up regulated and 125 genes were down regulated. Most notably the functional categories that were affected include Virulence genes (18 genes 11% of the total significantly differentiated genes), carbohydrate metabolism and cell envelop realted genes ( 31, 19.1% of the total), and aminoacid transport genes (21genes, 12.9%). Among the virulence-related genes, the most notable one were those belong to the production of capsule, the M protein, exotoxin, NAD glycohydrolase and Streptolysin S. Some of the capsule related genes were down regulated by more than 64 fold. These results indicate that CdhA (group A Streptococcal Cell division controlling and Chain-forming cell wall hydrolase) plays an important role in the regulation of virulence. S.pyogenes strain SF370 (wild-type) was procured from ATCC (ATCC 700294). M1-CdhA(-) mutant strain was obtained using pFW5 vector containing spectinomycin resistance marker (aad9). The mutant lacking CdhA is highly defective in cell division and growth, lacks antiphagocytic property and attenuated for virulence. Both strains (M1-Wild-type and M1-CdhA(-) were grown in Todd-Hewitt broth to their late log phase. Bacteria were harvested by centrifugation and washed twice with sterile PBS. Total RNA was isolated from these washed streptococci using Qiagen RNeasy Mini kit. For synthesis and labeling of cDNA, 20 ug of total RNA, 1.5 ug of random hexamer, 0.5mM dNTPs (except that 0.2mM of dTTP was replaced by the same amount of amino-allyl dUTP to incorporate dUTP into first-strand cDNA) were used in each reverse transcriptase reaction (Superscript II Reverse Transcriptase, Invitrogen). Purified cDNA preparations from the wild-type and M1CdhA(-) mutant strains were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using three independently isolated RNA preparations (biological replicates), a total of 11 experiments (incorporating dye swaps) were performed. Accordingly eleven hybridization measurements for this mutant were performed. Thus Exp-1 to -4 (GSM388703, GSM388717, GSM388718 and GSM388719) are the technical replicates of the biological sample-1, Exp-5 to 8(GSM388732, GSM388733, GSM388734, and GSM388735) are technical replicates of biological sample-2 and Exp-9,-10, and -11 (GSM388736,GSM388737, and GSM388738) are technical replicates of the biological sample-3. Signals of the bound reagents on the microarray spots in terms of relative fluorescence values were measured and quantified by a laser scanner (GenePix 4100 ) at 10um/pixel resolution. The resulting images were processed using Gene Pix Pro software (version 4.0, Axon instruments). The raw data were obtained in the form of GenPix *.gpr output files. The web application CARMAweb (Comprehensive R based microarray analysis web service) was used for the normalization and analysis of microarray data. All raw data (in the form of *. GPR files) were uploaded to the web application in the data directory. Using appropriate navigation tree, background correction from the foreground signal was applied and within microarray normalization was achieved using the Lowess method. Genes flagged as bad spots by the scanning software were excluded from the analysis and all flagged spots were given a weight of zero. The normalized data were then subjected to fold-change analysis and t-statistics using Bioconductor multtest package. CARMAweb allows to set Log2 cut-off values for both the M (regulation) and the A (average expression) values. Differentially expressed genes (Log2 values of Mutant647-red vs. wild-type555-green or Log2 values Mutant-555(green) vs Wild-type 647-red) were defined based on cut-off value >1 Log2< i.e. all genes that show a two or more-fold up- or down-regulation. Bioconductors Multtest package provided suitable method to adjust P values according to multiple hypothesis testing problems.

Project description:RNA from the aerial parts of 15-day-old eceriferum cer6-2 mutant and control plants were used in microarray experiments to screen for differentially expressed genes. Among those which were down regulated in the mutant, several were known to be involved in various plant defense mechanisms. The most remarkable difference was measured for the transcripts encoding the pathogenesis-related protein1 (PR-1), which is a marker of the systemic acquired resistance (SAR) pathway, with a 100-fold decrease in the cer6-2 mutant. Further Q-PCR experiments showed that the PR-1 transcript levels were low in two other eceriferum mutants, representing 1/200th and 1/25th of the normal values in the aerial part of 15-day-old cer2 and cer1-1 mutants, respectively. Interestingly, PR-1 mRNA levels were closed to the normal values in the cer3-1 and cer6-2R mutant, the latter being a plant where the synthesis of the very-long chain fatty acids, which is deficient in the cer6-2 plants, is partially restored. Altogether, these results strongly suggested that the amount of PR1-mRNA was not directly correlated to the amount of epicuticular wax on the leaves, but was rather correlated to the presence or absence of some particular lipid-constituents in the epicuticular wax layer. Related publication:; Garbay B, Tautu MT, Costaglioli P. Low level of pathogenesis-related protein 1 mRNA expression in 15-day-old Arabidopsis cer6-2 and cer2 eceriferum mutants. Plant Science 2007; Volume 172, Issue 2, Pages 299-305 Experiment Overall Design: Total RNA was purified from the aerial parts of 2-week-old plants (20 plants were pooled for each genotype). Arabidopsis plants were grown under long-day conditions (16 h light:8 h dark) at 22Â°C and 80% relative humidity. The control plants and the cer mutant were cultivated in the same phytotron, and the Petri dishes were placed randomly on the same shelf. This was done to avoid any difference in plant growth conditions between the mutants and the control plants. The microarrays used were Agilent Arabidopsis2. GSM87735 represents the direct experiment (cer6-2 Cy3, Ler-0 Cy5), and GSM88340 represents the dye-swap (Ler-0 Cy3, cer6-2 Cy5)

Project description:Abstract of associated manuscript: The Bacillus subtilis extracytoplasmic function (ECF) sigma(M) factor is activated by cell envelope stress elicited by antibiotics, and by acid, heat, ethanol and superoxide stresses. Here, we have used several complementary approaches to identify genes controlled by sigma(M). In many cases, expression is only partially dependent on sigma(M) because of both overlapping promoter recognition with other ECF sigma factors and the presence of additional promoter elements. Genes regulated by sigma(M) have a characteristic pattern of induction in response to cell envelope-acting antibiotics as evidenced by hierarchical clustering analysis. sigma(M) also contributes to the expression of the Spx transcription factor and thereby indirectly regulates genes of the Spx regulon. Cell envelope stress responses also include regulons controlled by sigma(W), sigma(B) and several two-component regulatory systems (e.g. LiaRS, YycFG, BceRS). Activation of the sigma(M) regulon increases expression of proteins functioning in transcriptional control, cell wall synthesis and shape determination, cell division, DNA damage monitoring, recombinational repair and detoxification. WT (-van) vs. WT (+van), sigM (-van) vs. sigM (+van), WT (-van) vs. sigM (-van), WT (+van) vs. sigM (+van), WT (-van) vs. spx (-van), WT (+van) vs. spx (+van). Each experiment was conducted at least twice using two independent total RNA preparations. For vancomycin untreated and treated experiments, untreated samples were labeled with Alexa Fluor 555 and treated samples with Alexa Fluor 647. For WT vs. mutant experiments, wild type was labeled with Alexa Fluor 555 and mutants with Alexa Fluor 647. For dye swap experiment, wild-type was labeled with Alexa Fluor 647 and mutant with Alexa Fluor 555. Bacillus subtilis CU1065, WT (-van) vs. WT (+van), sigM (-van) vs. sigM (+van), WT (-van) vs. sigM (-van), WT (+van) vs. sigM (+van), WT (-van) vs. spx (-van), WT (+van) vs. spx (+van)

Dataset Information

Microarray analysis for differential gene expression in M1-SDHHbtail

Publications

Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence.

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