New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates

Purpose: We used dual RNA-seq to analyze the transcriptomes of serotype M1 S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates (NHPs). We identified genes important for necrotizing myositis, made isogenic deletion mutants and performed RNA-seq comparing one of those deletion mutant strains (dahA) grown in vitro and compared to its parental wild-type strain. Methods: We grew the emm1 reference strain MGAS2221 in rich media (growth in vitro), and collected samples in triplicate at both mid-exponential (ME), and early stationary (ES) phase, to perform RNA-seq analysis. In addition, three NHPs were injected in the quadriceps skeletal muscle with the same GAS strain, necropsied after 24 h, samples were excised in concentric sections (from 1, at the inoculation site, to 5 on the periphery), and analyzed by dual RNA-seq (growth in vivo). Mock-infected skeletal muscle tissue was collected as negative control. Isogenic mutant for dahA and its parental wild-type strain were grown under the same conditions. 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. .cDNA libraries for the dahA isogenic mutant and its parental wild-type strain, grown in vitro, were generated using Epicentre Scriptseq for bacteria and sequenced on Illumina NextSeq 500 instrument. Overall design: RNA-seq was performed on MGAS2221 grown in THY (triplicates) and dual RNA-seq was performed on the in-vivo samples (infected and mock infected NHP tissues). RNA-seq was also performed on the dahA isogenic mutant strain and its parental wild-type. Please note that the Samples 25-39 are mock-infected samples (separate sections from site of infection) have no processed data since they were combined to generate the samples 40-42. The raw files for all mock-infected samples (Samples 25-42) are included for users who may try to analyze the data with all the mock-samples as controls.