Microarray analysis of S. pyogenes Type M1-CdhA55(-) mutant
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ABSTRACT:
ORGANISM(S): Streptococcus pyogenes
PROVIDER: GSE15598 | GEO | 2010/10/01
SECONDARY ACCESSION(S): PRJNA115717
REPOSITORIES: GEO
Similar Datasets
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.
2010-10-01 | GSE15546 | GEO
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.
2010-10-01 | E-GEOD-15598 | biostudies-arrayexpress
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.
2010-10-01 | E-GEOD-15546 | biostudies-arrayexpress
Project description:Pasteurella multocida is a Gram-negative capsulated bacterium responsible for a range of diseases that cause severe morbidity and mortality in livestock animals. The hyaluronic acid (HA) capsule produced by P. multocida serogroup A strains is a critical virulence factor. In this study, we utilised transposon-directed insertion site sequencing (TraDIS) to identify genes essential for in vitro growth of P. multocida, and combined TraDIS with discontinuous density gradients (TraDISort) to identify genes required for HA capsule production and regulation in this pathogen. Analysis of mutants with a high cell density phenotype, indicative of the loss of extracellular capsule, led to the identification of 69 genes important for capsule production. These genes included all previously characterized genes in the capsule biosynthesis locus, and fis and hfq that encode known positive regulators of P. multocida capsule. Many of the other capsule-associated genes identified in this study were involved in regulation or activation of the stringent response, including spoT and relA that encode proteins that regulate the concentration of guanosine alarmones. Disruption of the autoregulatory domains in the C-terminal half of SpoT using insertional mutagenesis resulted in reduced expression of capsule biosynthesis genes and an acapsular phenotype. Overall, these findings have greatly increased the understanding of hyaluronic acid capsule production and regulation in P. multocida.
2022-03-14 | GSE198087 | GEO
Project description:Cryptococcus neoformans is a fungal pathogen responsible for hundreds of thousands of deaths per year. Its critical virulence factor is a polysacharride capsule which grows large upon entry into a mammalian host. We previously identified USV101 as a transcription factor whose deletion results in enlarged capsules. Here, we characterize strains lacking or overexpressing USV101 in terms of their virulence-related phenotypes, the altered course of infection by them and immune response to them in a mouse model, the relationship of Usv101 to other transcription factors involved in capsule regulation, and the changes in Usv101 activity during capsule induction. To study the function of Usv101, a key C. neoformans regulator of virulence, and its interactions with downstream capsule-regulating TFs GAT201 and SP1, expression profiles were generated by RNA-seq of a usv101 deletion mutant, a USV101 overexpression mutant, strains expressing GAT201 under various promoters, various double mutants, and wildtype cells grown in a capsule non-inducing condition, a capsule inducing condition for 90 minutes, or a capsule inducing condition for 24 hours.
2016-03-31 | E-GEOD-69532 | biostudies-arrayexpress
Project description:Rggs are a group of transcriptional regulators found commonly in multiple homologous in a given species of streptococci, and they play diverse roles in metabolism and virulence. Here we studied Rgg1518’s (SPD_1518) role in sugar metabolism, and in vivo survival and virulence of Streptococcus pneumoniae. In vitro analysis showed that Rgg1518 is highly induced by galactose and mannose, and its isogenic mutant is attenuated in growth on these sugars compared to the wild type. The regulon information obtained by microarray analysis showed that Rgg1518 has by far the largest regulon on galactose compared to the other Rggs and controls the expression of functionally diverse set of genes involved in galactose uptake and catabolism, capsule biosynthesis, protein translation as well as those involved in other metabolic functions. We showed that Rgg1518 is the repressor of capsule biosynthesis, and identified Rgg1518 binding motif in the regulatory region of capsule locus. We demonstrated an interregulatory interactions among Rggs. Furthermore, the rgg1518 mutant was found to be attenuated in colonization and virulence in a mouse model of colonisation and pneumonia.
2021-06-01 | GSE154887 | GEO
Project description:* Bone signaling in middle ear development * <br/>Common middle ear diseases, such as chronic suppurative otitis media and cholesteatoma, may affect bone behavior in the middle ear air cell system. <br/> This study analyzed gene expression of bone-related signaling factors and gene sets from lining tissues in the developing middle ear of the rat. Candidate gene products were compared with previously published data on middle ear bone metabolism. <br/> Microarray technology was used to identify bone-related genes and gene sets, which were differentially expressed between the adult (quiescent) bulla and young (resorbing/forming) bulla. <br/><br/> * Gene expression of the otic capsule *<br/>The behavior of bone cells within the otic capsule is unique. After occification localized bone remodeling is virtually absent. Human otosclerosis is a localized disease within the otic capsule where the bone starts to remodel pathologically. Disease etiology is unknown and the pathogenesis is only partially elucidated.<br/><br/> The objective of this study is to measure bone-related gene expression of the otic capsule in order to reveal additionally signaling factors responsible for the absent bone remodeling within the otic capsule.<br/><br/> Microarray technology was used to determine genes involved in the bone metabolism, which were differentially expressed between lining tissues from the otic capsule and lining tissues from the middle ear of the rat.
2014-02-20 | E-MTAB-2336 | biostudies-arrayexpress
Project description:Cryptococcus neoformans is a fungal pathogen responsible for hundreds of thousands of deaths per year. Its critical virulence factor is a polysacharride capsule which grows large upon entry into a mammalian host. We previously identified USV101 as a transcription factor whose deletion results in enlarged capsules. Here, we characterize strains lacking or overexpressing USV101 in terms of their virulence-related phenotypes, the altered course of infection by them and immune response to them in a mouse model, the relationship of Usv101 to other transcription factors involved in capsule regulation, and the changes in Usv101 activity during capsule induction.
2016-03-31 | GSE69532 | GEO
Project description:Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The activity of several enzymes involved in PG synthesis is regulated by serine/threonine kinase (STKs). In firmicutes, inactivation of genes encoding STKs is associated with a range of phenotypes including cell division defects and changes in cell wall metabolism, but only a few kinase substrates have been identified. We previously demonstrated that the STK PrkC plays an important role in cell division, cell wall metabolism and drug resistance in Clostridioides difficile. In this work, we identified and characterized a PGH, CD1135 that is a PrkC substrate. We showed that CD1135 hydrolyses PG between daughter cells to allow cell separation. We demonstrated that CD1135 phosphorylation inhibits CD1135 its export, therefore controlling the hydrolytic activity in the cell wall. High level of CD1135 in the cell wall leads to cell elongation, whereas low level causes cell separation defects. We provided evidences that the STK signaling pathway regulates PGHs homeostasis to control PG hydrolysis during growth and cell division.
2021-04-08 | PXD021541 | Pride
Project description:Purpose: Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying TFs involved in a specific process and used it to map regulation of the key virulence factor of Cryptococcus neoformans, its capsule. Results: The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, mid-level, and “foreman” TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of 41 TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. Conclusions: These data resources and analyses provide the first integrated, systems level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to labs focused on specific physiological processes. A total of 288 expression profiles were generated by RNA-Seq analysis of Mutant and wildtype cells grown in capsule inducing conditions for 90 minutes. In addition, 3 replicate expression profiles were generated for wildtype cells grown in : a capsule non-inducing condition, a capsule inducing condition for 180 minutes, a capsule inducing condition for 480 minutes, and a capsule inducing condition for 1440 minutes. In addition, ChIP-seq of NRG1(CNAG_05222) in capsule inducing conditions, and USV101(CNAG_05420) in both capsule inducing and non-inducing conditions were generated.
2015-01-14 | E-GEOD-60398 | biostudies-arrayexpress