Integrated Analysis of Population Genomics, Transcriptomics and Virulence Provides Novel Insights into Streptococcus pyogenes Pathogenesis [isogenic]

Purpose: We used GAS as a model bacterial pathogen to investigate the complex relationship between genome, transcriptome, and virulence in a large population of type emm28 strains recovered from humans over 26 years in geographically diverse locations. Methods: An isogenic mutant strain (MGAS27961-10T) with a 10-T homopolymeric tract was generated using allelic exchange. Primers were used to amplify a 2,690-bp fragment using genomic DNA of MGAS28085 an emm28 clinical isolate with the naturally occurring 10 “T” variant region. The resulting PCR product was cloned into suicide plasmid pBBL740 and transformed into the parental strain MGAS27961. RNA-seq was performed on the isogenic mutant strain MGAS27961-10T and parental strain (MGAS27961-9T). Quality of the total RNA, rRNA-depleted RNA, and cDNA libraries was evaluated with RNA Nano, RNA Pico, and DNA high-sensitivity kits (Agilent Technologies), respectively, using an Agilent 2100 Bioanalyzer. For each sample, the cDNA library concentration was measured fluorometrically with Qubit™ dsDNA HS assay kits (Invitrogen). The cDNA libraries were diluted, pooled, and sequenced with an Illumina NextSeq 500 instrument. Overall design: RNA-seq cDNA libraries for isogenic mutant strain (MGAS27961-10T) and parental strain (MGAS27961-9T), in triplicate, were generated at two phases of growth, mid-exponential (ME, OD600nm=0.5) and early-stationay (ES, OD600nm=1.65).