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.

中文翻译：

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通过灵长类坏死性肌炎的多维分析确定新的发病机制和转化线索。


当代生物医学研究的一个基本目标是了解疾病发病机制的分子基础，并利用这些信息来开发有针对性的、更有效的治疗方法。由细菌病原体化脓性链球菌引起的坏死性肌炎是一种毁灭性的人类感染，死亡率高，成功的治疗方案很少。我们使用双转录组测序（RNA-seq）来分析化脓性链球菌和同时从受感染的非人灵长类动物中恢复的宿主骨骼肌的转录组。与体外生长的生物体相比，体内细菌转录组发生了惊人的重塑，导致毒力的基因显着上调，并改变了代谢基因的调节。受感染的非人灵长类动物 (NHP) 的肌肉组织转录组与模拟感染动物的肌肉组织转录组存在显着差异，部分原因是导致炎症和宿主防御过程的基因发生了重大变化。我们发现 A 族链球菌 (GAS) 毒力因子转录本与参与宿主免疫反应和炎症的基因之间存在显着的正相关性。我们还发现体内细菌毒力基因表达量与病原体适应性之间存在显着相关性，正如之前进行的全基因组转座子定向插入位点测序（TraDIS）所评估的那样。通过将细菌 RNA-seq 数据与 TraDIS 生成的适应度数据整合，我们发现了 5 个新的病原体基因，即化脓性链球菌 0281 (Spy0281 [dahA])、ihk-irr、slr、isp 和 ciaH，它们有助于坏死性肌炎并使用等基因缺失突变株证实了这些发现。 总而言之，我们的研究结果提供了有关灵长类骨骼肌严重侵袭性感染中发生的分子事件的丰富新信息，具有广泛的转化研究意义。 重要性 由化脓性链球菌引起的坏死性肌炎具有很高的发病率和死亡率，成功的治疗选择相对较少。此外，还没有获得许可的人类化脓性链球菌疫苗。为了加深对这种感染的分子基础的了解，我们采用了多维分析策略，其中包括双 RNA 测序和来自非人类灵长类动物实验感染的其他数据。这些数据用于针对五个链球菌基因进行发病机制研究，从而明确证明这些基因有助于坏死性感染中病原体与宿主的分子相互作用。我们利用最近进行的全基因组转座子诱变研究中得出的适应性数据，发现体内细菌毒力基因表达量与病原体适应性之间的显着相关性。总的来说，我们的发现对转化研究具有重大影响，可能包括疫苗工作。