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M1 serotype (strain SF370): pharyngeal cell adherence vs. association

ABSTRACT: We developed spotted oligonucleotide arrays of the S. pyogenes SF370 (an M1 serotype) genome and compared the transcriptomes of streptococci that adhere to Detroit 562 human pharyngeal cells to non-adherent (“associated”) streptococci within the same experiment. We replicated experiments independently and used dye-swaps to incorporate biological and technical variation, biological replicate experiments. Following filtering and normalization, we analyzed data from four biological replicates with robust summary statistics, Bayesian statistics and permutation algorithms to identify genes differentially expressed with significance during pharyngeal cell adherence. This analysis identified 79 genes (4% of the genome) exhibiting statistically significant fold changes in expression (PF value < 0.05) during adherence from 1769 open reading frames represented on the array. The genes encoding several known virulence factors (e.g. cysteine protease SpeB and streptolysin O) were differentially expressed with significance; however, many were either never before associated with adherence (e.g. phage-encoded genes including speH), or of undefined function. Genes demonstrating up-regulation (n= 45) and down-regulation (n= 34) included virulence factors, prophage-encoded transcripts, metabolic genes and transcriptional regulators (Table 2). Undefined or hypothetical genes comprised 27% of differentially expressed genes (n= 21; 11 chromosomally-encoded, 10 phage-encoded). We conducted TaqMan (qRT-PCR) analysis of 11 differentially expressed genes to validate selected microarray hybridization results. We further analyzed the data with neighbor clustering, using a set of novel computational algorithms (termed “GenomeCrawler”) for evaluating bacterial microarray data, which couples the expression profiles of genes with their physical location on the chromosome. When applied to the microarray data from this study, neighbor clustering identified a greater number of differentially expressed genes, which facilitated the reconstruction of known multimeric proteins, complete metabolic pathways and intact virulence loci that would not have been possible without its application. These data sets and algorithms are freely available for download at www.rockefeller.edu/vaf. Keywords: Pharyngeal cell adherent vs. associated

ORGANISM(S): Streptococcus pyogenes

PROVIDER: GSE7620 | GEO | 2007/04/27

SECONDARY ACCESSION(S): PRJNA100147

REPOSITORIES: GEO

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Project description: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.

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M1 serotype (strain SF370): pharyngeal cell adherence vs. association

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