Paired single-cell host profiling with multiplex-tagged bacterial mutants reveals intracellular virulence-immune networks III

Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA-sequencing (scRNA-seq) are increasingly used to study the host factors underlying diverse cellular phenotypes. But current approaches do not permit the simultaneous unbiased study of both host and bacterial factors during infection. Here, we developed scPAIR-seq, an approach to analyze both host and pathogen factors during infection by combining multiplex-tagged mutant bacterial library with scRNA-seq to identify mutant-specific changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of Salmonella Typhimurium secretion system effector mutants. We developed a pipeline to independently analyze redundancy between effectors and mutant-specific unique fingerprints, and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome. Validating our procedure for mapping bacterial mutant identity within single host cell by quantification of tagged SPI-2 mutants from matched bulk infected samples

宿主细胞与胞内细菌病原体的相互作用会引发决定感染结局的复杂表型。单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）正日益广泛地用于探究多种细胞表型背后的宿主调控因子。然而当前的研究方法无法在感染过程中同时对宿主与细菌因子开展无偏分析。本研究开发了scPAIR-seq技术，该方法通过将多重标签突变体细菌文库与单细胞RNA测序相结合，以鉴定宿主转录组中突变体特异性的表达变化，从而实现在感染过程中同步分析宿主与病原体因子。我们将scPAIR-seq应用于感染鼠伤寒沙门氏菌分泌系统效应蛋白突变体文库的巨噬细胞。我们开发了一套分析流程，可独立分析效应蛋白间的功能冗余性以及突变体特异性的独特特征，并通过单个效应蛋白对宿主免疫通路的影响，绘制其全局毒力网络。scPAIR-seq是一款强大的工具，可用于解析细菌的毒力策略及其与驱动感染结局的宿主防御策略之间的复杂相互作用。我们通过从匹配的批量感染样本中定量标记的SPI-2突变体，验证了在单个宿主细胞内鉴定细菌突变体身份的实验流程。