Our knowledge of the functions required by extracellular bacterial pathogens to grow in host tissues is still limited. Most available information refers to studies conducted under laboratory growth conditions that mimic host environments but exclude the influence of the host immune system. Now, researchers from the Helmholtz Centre for Infection Research developed an unbiased experimental approach that allows simultaneous monitoring of genome-wide infection-linked transcriptional alterations of the host and colonizing extracellular pathogens.
Tissue dual RNA sequencing allows simultaneous transcript profiling of a pathogen and its infected host. This sensitive approach led to the identification of host immune responses and virulence-relevant bacterial functions that were not previously reported in the context of a Yersinia infection. Application of this tool will allow transcript profiling of other pathogens to unravel concealed gene functions that are crucial for survival in different host niches and will improve identification of potential drug targets.
Tissue dual RNA-seq workflow and global reports
(A) For tissue dual RNA-seq, female BALB/c mice were orally infected with 2 × 108 cfu Y. pseudotuberculosis IP32953 (infected) or 1× PBS (uninfected). For in vitro RNA-seq, IP32953 was grown in LB to exponential or stationary phase at 25 °C or 37 °C. Total RNA was isolated from Peyer’s patches of mice or bacterial cultures processed for preparation of strand-specific barcoded cDNA libraries and sequenced. cDNA reads were separated in silico by mapping to the mm10 and the IP32953 genomes. TAP, tobacco acid pyrophosphatase. (B) Circos plot visualizing reads per kilobase transcript length per million mapped reads-normalized expression values of in vitro and in vivo RNA-seq data for the IP32953 chromosome (NC_006155.1) and pYV virulence plasmid (NC_006153.2). E25, exponential phase 25 °C; E37, exponential phase 37 °C; in vivo, infected Peyer’s patches; S25, stationary phase 25 °C; S37, stationary phase 37 °C. (C) Heat map of Euclidian sample distances of rlog-transformed read counts for triplicate mouse RNA-seq data from RNA pools of uninfected and infected Peyer’s patches. (D) Principal component (PC) analysis of mean centered and scaled rlog-transformed read count values of bacterial in vitro and in vivo RNA-seq data for the Y. pseudotuberculosis IP32953 core genome and the pYV virulence plasmid.