Comment[ArrayExpressAccession] E-GEOD-26655 Public Release Date 2011-04-01 Investigation Title Defining the serotype M3 RopB regulon Comment[Submitted Name] Defining the serotype M3 RopB regulon Experiment Description Recent whole-genome sequencing of large populations of the same bacterial species has revealed significant disparity among genes in the frequency of single nucleotide polymorphisms (SNPs). For example, a previous analysis of invasive serotype M3 group A streptococci (GAS) found the highest frequency of SNPs in the gene (ropB) encoding the regulator of proteinase B (RopB). This finding led us to hypothesize that RopB polymorphisms contribute to altered GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains from a surveillance study identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically different effects on GAS global gene expression. Further, analysis of laboratory-generated isoallelic GAS strains differing only by a single amino acid replacement in RopB confirmed that the variant protein affected the transcript level of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that RopB polymorphisms significantly influence GAS virulence and disease manifestations. These studies detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections. This study examined the effects of RopB inactivaiton on two distinct serotype M3 group A streptococcal strains with distinct forms of the RopB protein. RopB was inactivated in a strain with a wild-type RopB allele (strain MGAS10870) and in a strain with a RopB allele containing a C85Y polymorphism (strain MGAS9937). The wild-type and RopB inactivated strains were grown in duplicate to the early stationary growth phase in standard laboratory medium (THY). Total RNA was isolated, converted to cDNA, and hybridized to a custom-made Affymetrix GeneChip. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://bar.ebi.ac.uk:8080/trac/browser/branches/curator/ExperimentalFactorOntology/ExFactorInOWL/currentrelease/eforelease/efo.owl Person Last Name Shelburne Carroll Shelburne Musser Person First Name Samuel Ronan Samuel James Person Mid Initials K A M Person Email sshelburne@mdanderson.org Person Affiliation MD Anderson Cancer Center Person Phone 713-792-3629 Person Fax 713-794-4350 Person Address Infectious Diseases, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, USA Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE26655-1 P-GSE26655-5 P-GSE26655-4 P-GSE26655-2 P-GSE26655-3 P-GSE26655-6 Protocol Description ID_REF =
VALUE = signal intensity (spot density) Per Affymetrix protocol Per Affymetrix protocol Strain was grown to early stationary phase in THY. 2x RNAprotect (Qiagen) was added for 5 mins and cells were pelleted. Total RNA was extracted with RNeasy (Qiagen) and reverse transcribed to cDNA with SuperScriptIII (Invitrogen). cDNA was labeled and hybridized following Affymetrix instructions. Per Affymetrix protocol Probes corresponding to the serotype M3 strains used were extracted from the total data set. The total intensity of the array for each sample was then calculated by adding the values for each of the probe sets. The probe set values were then normalized such that the total intensity derived from each sample was equivalent. Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation image_aquisition hybridization nucleic_acid_extraction labeling feature_extraction Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name GENOTYPE/VARIATION STRAIN Experimental Factor Type genotype/variation strain Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE26655 Comment[GEOLastUpdateDate] 2011-04-01 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2011-03-31 Comment[ArrayExpressSubmissionDate] 2011-01-17 SDRF File E-GEOD-26655.sdrf.txt