Dual RNA-seq of intracellular S. aureus pKikume with different metabolic states within macrophages

The overall aim of this study was to investigate the strategies used by S. aureus to survive and multiply within professional phagocytes (macrophages). For this purpose, an S. aureus strain encoding the reporter mKikumeGR that enables to monitor the metabolic activity and proliferation status of the internalized bacteria was used to simultaneously assess gene expression changes in host cells and pathogens in a dual RNA-seq approach and determine the correlation between these changes and the metabolic state of internalized bacteria. Therefore, macrophages were infected with S. aureus pKikume, sorted via FACS 4h p.I. into one subpopulation of macrophages harboring mainly metabolic active, green bacteria and one population harboring metabolic inactive, red bacteria, and dual RNA-seq was performed on both subpopulations.

本研究的总体目标是探究金黄色葡萄球菌（Staphylococcus aureus，简称S. aureus）在专职吞噬细胞（professional phagocytes，即巨噬细胞（macrophages））内存活与增殖的策略。为此，本研究选用携带报告基因mKikumeGR的金黄色葡萄球菌菌株，该报告基因可用于示踪内化细菌的代谢活性与增殖状态；同时采用双RNA测序（dual RNA-seq）技术同步分析宿主细胞与病原菌的基因表达变化，并解析此类变化与内化细菌代谢状态之间的相关性。实验中，研究人员以该菌株感染巨噬细胞，于感染后4小时通过荧光激活细胞分选（Fluorescence Activated Cell Sorting，简称FACS）将感染细胞分为两个亚群：其一为主要携带代谢活跃的绿色细菌的巨噬细胞亚群，其二为携带代谢失活的红色细菌的巨噬细胞亚群；随后对两个亚群均开展双RNA测序实验。