Genetic heterogeneity of the Spy1336/R28 – Spy1337 virulence axis in Streptococcus pyogenes and effect on gene transcript levels and pathogenesis

Purpose: We used RNA-seq to analyze the transcriptomes of serotype M28 S. pyogenes deletion and SNP isogenic mutants strains. We discovered that changes in the number of T residues in a homopolymeric tract located upstream of the divergently transcribed Spy1336/R28 and Spy1337 genes affected global gene expression and virulence. We performed RNA-seq comparing isogenic mutants grown in vitro containing either 9 or 11Ts, and isogenic deletion mutants, deleted for gene Spy1337, and deleted for both, Spy1336 and Spy1337. Methods: We grew isogenic strains in rich media, and collected samples in triplicate at both mid-exponential (ME) and early stationary (ES) phase to perform RNA-seq analysis. 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 550 instrument Strains were grown in vitro, in triplicate, and RNA-seq cDNA libraries were generated using Illumina Scriptseq for bacteria.