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

Abstract

A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcriptome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro, with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 (Spy0281 [dahA]), ihk-irr, slr, isp, and ciaH, that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications. IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group AStreptococcus

“…al,. including sagA, arcA, and the cellobiose PTS operon, were directly bound by CcpA in our study, all of which we classified as HPr-dependent. Conversely, the transcript levels of the directly CcpA bound, HPr-independent genes of the sialic acid operon, prtS, ackA, and hpr showed no significant upregulation during necrotizing fasciitis (Kachroo et al, 2020). These findings are consistent with the concept that CcpA can maintain repression of these genes even under metabolically unfavorable conditions when HPrSer46~P levels would be expected to be low.…”

“…The dependence/independence of CcpA from HPr~P could assist with fine-tuning regulation of pertinent genes by CcpA such that site specific resources can be utilized optimally to aid the pathogen. For example, a recent study compared the GAS transcriptome during necrotizing fasciitis to growth in standard laboratory medium (Kachroo et al, 2020). Some of the most highly upregulated genes in Kachroo et.…”

Streptococcus pyogenes genes that promote pharyngitis in primates

“…The polycistronic expression and upregulation of these genes in whole blood and during necrotizing myositis suggest that HitABC and Irr-Ihk might be functionally related. In line with this idea, we reported that hitABC and irr-ihk are both crucial for GAS virulence in an NHP model of necrotizing myositis (32,98). Because the Irr-Ihk TCS contributes to GAS's ability to resist being killed by neutrophils and neutrophil-derived antimicrobial peptides (72), further functional studies are needed to determine whether the HitABC transporter serves as an efflux pump for neutrophil-derived antimicrobial peptides.…”

High Cell Density and Antimicrobial Persistence in Streptococcus Pyogenes 

“…34 The Ihk/Irr two-component system is involved in the regulation of various streptococcal processes, including virulence. 35,36 It is notable that ihk/irr were overexpressed in a non-human primate model of GAS necrotizing myositis, and these genes were implicated in GAS resistance to polymorphonuclear phagocytosis. 36 The fact that CCCP inhibited ihk/irr gene regulators and the RND family operon concomitantly, and that both the operon and regulators displayed increased expression in GAS-persistent cells, raises an interesting hypothesis about another possible role for the Ihk/Irr system besides virulence regulation.…”

“…35,36 It is notable that ihk/irr were overexpressed in a non-human primate model of GAS necrotizing myositis, and these genes were implicated in GAS resistance to polymorphonuclear phagocytosis. 36 The fact that CCCP inhibited ihk/irr gene regulators and the RND family operon concomitantly, and that both the operon and regulators displayed increased expression in GAS-persistent cells, raises an interesting hypothesis about another possible role for the Ihk/Irr system besides virulence regulation. However, despite the gene co-localization and the concomitant regulation of ihk/irr and the genes of the ABC transport operon by CCCP, molecular cloning strategies are needed to validate the hypothesis that ihk/irr may not only regulate GAS virulence but also this transport system.…”