New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates
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ABSTRACT:
PROVIDER: PRJNA602750 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10954321.fastq.gz | Fastqsanger.gz | |||
| SRR10954322.fastq.gz | Fastqsanger.gz | |||
| SRR10954323.fastq.gz | Fastqsanger.gz | |||
| SRR10954324.fastq.gz | Fastqsanger.gz | |||
| SRR10954325.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description: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.
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Project description:Complement proteins are deposited in the muscles of patients with myositis. However, the local expression and regulation of complement genes within myositis muscle have not been well characterized. In this study, bulk RNA sequencing (RNAseq) analyses of muscle biopsy specimens revealed that complement genes are locally overexpressed and correlate with markers of myositis disease activity, including the expression of interferon-gamma (IFN )-induced genes. Single cell and single nuclei RNAseq analyses showed that most local expression of complement genes occurs in macrophages, fibroblasts, and satellite cells, with each cell type expressing different sets of complement genes. Biopsies from immune-mediated necrotizing myopathy patients, who have the lowest levels of IFN -induced genes, also had the lowest complement gene expression levels. Furthermore, data from cultured human cells showed that IFN upregulates complement expression in macrophages, fibroblasts, and muscle cells. Taken together, our results suggest that in myositis muscle, IFN coordinates the local overexpression of complement genes that occurs in several cell types.
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