Analysis of Streptococcus dysgalactiae subspecies equisimilis gene transcripts during experimental primate necrotizing myositis

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is a Gram-positive bacterial pathogen capable of causing various infections in humans. Recently, isolates of SDSE emm type stG62647 have emerged as a cause of severe invasive infections, including necrotizing myositis. However, the molecular processes underlying these infections remain poorly understood. To address this gap, we performed RNAseq analysis to examine SDSE gene transcript levels during experimental necrotizing myositis infection in non-human primates, an animal phylogenetically closely related to humans. We analyzed the transcriptomes of two related SDSE stG62647 human isolates (MGCS36044 and MGCS36089) during necrotizing myositis infection in six non-human primates. The transcriptome from in vitro growth in nutrient-rich media differed considerably from that of SDSE bacteria grown in vivo in experimental necrotizing myositis, with 254 genes differentially expressed, indicating extensive genetic adaptation in infected skeletal muscle. Notably, we observed marked upregulation of ihk-irr genes encoding a two-component regulatory system that promotes evasion of phagocytosis and resistance to killing by human polymorphonuclear leukocytes in Streptococcus pyogenes. Similarly, genes comprising the sag operon encoding the streptolysin S cytolytic toxin virulence factor demonstrated very high transcript abundance in vivo. Additionally, we present evidence that a 40-nt deletion in fasB alters expression of ska, encoding streptokinase. Collectively, our data provide new insights into the SDSE genes transcribed in vivo, thereby enhancing our understanding of the molecular basis of pathogen and primate host interactions. The SDSE genes identified in this study offer promising targets for future studies on molecular pathogenesis and therapeutics interventions. Overall design: RNAseq was performed on SDSE MGCS36044 and MGCS36089 grown in vitro in THY (quadruplicates) that had been collected at mid-exponential (ME) (OD=1) and early stationary phase (ES) (OD=2). RNAseq was also performed on MGCS36044 and MGCS36089 isolated from skeletal muscle biopsies from six NHPs at 24h post-infection. Each NHP was concurrently infected with strain MGCS36044 in the right leg and strain MGCS36089 in the left leg. Muscle biopsies were obtained in five concentric layers (L1 to L5), with L1 corresponding to the inoculation site, and L5 to the outermost layer, and reads from all five biopsy layers per NHP were pooled.