An invasive bacterium must adapt to the environment of the host cell while the host cell reacts to the invader. However, the analysis of transcriptomes to understand changes in host and pathogen gene expression has typically been applied to a single species at a time. Here, Westermann et al. use the sensitivity of dual RNA sequencing (RNA-seq) technologies to tease apart the subtle interactions of pathogen and host.
“Dual RNA-seq captures both transcriptomes and uses in silico analysis to distinguish species-specific transcripts”
The authors infected HeLa cells with Salmonella enterica serovar Typhimurium (henceforth Salmonella) and sequenced the transcriptomes present in the infected cells at 4 and 24 hours post-invasion. Traditional RNA-seq focuses on one species at a time and uses mRNA as an indicator of gene expression, with the disadvantage of excluding non-protein-coding genes and requiring separation of pathogen and host. Dual RNA-seq captures both transcriptomes and uses in silico analysis to distinguish species-specific transcripts. Initial sequencing without enrichment or depletion of specific RNA types revealed an expected vast excess of human RNA sequences compared to bacterial. To heighten sensitivity, the team depleted the abundant rRNA transcripts from the data set and analysed small regulatory RNAs (sRNAs) over a time course up to 24 hours. They were able to identify 145 known and 189 putative Salmonella sRNAs that changed expression following invasion.
The most highly upregulated sRNA, PinT, has been suspected to be a virulence factor and here is shown to be regulated by the PhoP/Q system, which is crucial for the intracellular survival of Salmonella. However, PinT-knockout cells have a weak phenotype, and the molecular functions of this sRNA were unknown. Using dual RNA-seq along with overexpression or deletion of PinT in Salmonella, the authors show that PinT represses mediators of invasion such as the Salmonella pathogenicity island 1 (SPI-1) effectors SopE and SopE2 and the virulence activation genes CRP and GrxA, and thus, indirectly, the SPI-2 genes. This indicates a role for PinT in regulating the transition from invasion to intracellular replication. (read more…)
- Ross Cloney R. (2016) Dual RNA-seq for host–pathogen transcriptomics. Nature Rev Gen [Epub ahead of print. [abstract]
- Westermann AJ, Förstner KU, Amman F, Barquist L, Chao Y, Schulte LN, Müller L, Reinhardt R, Stadler PF, Vogel J. (2016) Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions. Nature [Epub ahead of print]. [abstract]