Gene analysis of intracellular and extracellular Streptococcus pyogenes in human macrophages. Streptococcus pyogenes

The objective of this study was to investigate which genes are important for Streptococcus pyogenes during intracellular survival in human macrophages. Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is yet unknown. To address this, gene expression profiles of S. pyogenes within human macrophages were determined and compared to those of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection and within 2 h post infection, the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key upregulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). We observed that an isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts as compared to wild-type bacteria following infection of macrophages. Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurrent with an upreguation of the well-studied covR/S two component regulator. In reinfection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems. Overall design: Five samples with three biological replicates are analysed. Each open reading frame in triplicate (three technical replicas per sample). Resulting in 6-9 data points per gene per condition. The extracellular bacteria are control samples and the internal control is the house-keeping gene gyrase.

本研究旨在探究化脓性链球菌（Streptococcus pyogenes）在人类巨噬细胞内存活所依赖的关键基因。化脓性链球菌是一类重要的人类致病菌，近年被证实可在坏死性筋膜炎等重症侵袭性感染进程中作为胞内微生物存在。尽管已明确表面锚定M蛋白是影响吞噬体成熟及化脓性链球菌在巨噬细胞内存活的关键因子，但该病原体适应并在胞内持续存活所需的整体转录调控谱仍未可知。
为解答这一科学问题，本研究采用定制化微阵列（customized microarrays）与实时定量逆转录PCR（real-time qRT-PCR），测定了人类巨噬细胞内化脓性链球菌的基因表达谱，并将其与胞外细菌的表达谱进行对比。为模拟感染早期适应胞内环境的阶段，研究人员于感染后2小时采集样本；在感染后2小时内，胞内环境中的145个链球菌基因的表达发生显著改变。其中大部分差异调控基因与代谢及能量依赖过程相关。早期胞内细菌中上调的关键基因为ihk与irr，二者编码双组分基因调控系统（two-component gene regulatory system, TCS）。
研究发现，相较于野生型菌株，ihk/irr缺陷的同基因化脓性链球菌突变株在感染巨噬细胞后的菌落计数显著降低。对比感染后2小时与6小时的选定基因表达谱，可见响应调控因子随时间发生显著变化：ihk/irr基因下调的同时，研究较为充分的covR/S双组分调控因子出现上调。在复感染实验中，感染6小时采集的胞内细菌在巨噬细胞内的存活能力显著高于感染2小时采集的细菌。上述研究结果阐明了化脓性链球菌在胞内生命周期中，如何通过特定双组分系统的时序表达精准调控基因表达。
实验设计：本研究共分析5份样本，每份设置3次生物学重复；每个开放阅读框（open reading frame, ORF）设置3次技术重复，最终每个实验条件下每个基因可获得6-9个数据点。胞外细菌作为对照样本，内参基因为持家基因旋转酶（gyrase）。