Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here, researchers from the University of Würzburg used a generic ‘dual RNA-seq’ approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. They identified a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and they identified infection-specific alterations in multiple long noncoding RNAs.
Dual RNA-seq captures the full transcript repertoire of infected cells
a, Dual RNA-seq workflow. b, Representative mapping statistics from sorted Salmonella-infected HeLa-S3 cells. LB, Salmonella grown in medium. ‘miRNA’ includes primary and mature forms; ‘mitoRNA’ refers to mitochondrial transcripts. IGR, intergenic region; miscRNA, miscellaneous RNA. c, Compared to extracellular Salmonella (0 h), intracellular bacteria at 4 h p.i. repress SPI-1 and induce SPI-2 effector genes. d, Invaded (GFP+) host cells at 24 h p.i. activate NF-κB-associated immunity genes. Data in panels c and d represent fold changes calculated by edgeR over 3 biological replicates.
This study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.