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Simultaneous Analysis of Host, Pathogen Transcripts Highlights Infection Features

NEW YORK (GenomeWeb) – A new Nature study suggests non-coding RNA produced by pathogenic bacteria can influence transcripts produced by both the microbe itself and the infected host.

Using an approach known as dual RNA-seq, researchers from Germany, Austria, and the US characterized host transcripts and pathogen messenger RNAs in tandem over time in a human cell line infected with Salmonella enterica from the Typhimurium serovar, an intracellular pathogen known for producing a large repertoire of small regulatory RNAs.

In the process, they narrowed in on a small RNA called PinT that is activated by Salmonella upon host cell infection. Subsequent analyses of the small RNA suggest it regulates S. enterica genes involved in cell invasion and survival. But PinT also appears to influence host transcriptional patterns, leading to shifts in representation by several long, non-coding RNAs as well as enhanced activation of JAK-STAT signaling pathways.

"Our 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," senior author Jörg Vogel, a molecular infection biology and infectious diseases researcher at the University of Würzburg, and colleagues wrote.

Although RNA sequencing is increasingly being used to explore host and pathogen features involved in infection, the team explained, most studies have focused on protein-coding transcripts, assessing bacterial RNA sequences and host RNAs separately.

"[A]s bacterial infections of eukaryotic cells involve two interacting organisms with profoundly different transcriptomes, RNA-seq studies are commonly restricted to either the pathogen or host after their physical separation," the authors wrote.

In an effort to simultaneously assess coding and non-coding transcripts in a bacterial pathogen and affected host, the researchers relied on dual RNA-seq, a strategy that uses green fluorescent-tagged Salmonella to identify infected human host cells from a HeLa cell line.

After isolating cells four hours and 24 hours post-infection, the team used the Illumina HiSeq 2000 instrument to generate tens of millions of reads from total RNA present in the samples.

Although human transcripts far exceeded those of the Salmonella, the approach made it possible to pick up transcripts representing messenger RNA and non-coding RNAs from both organisms.

To take a closer look at small RNAs with regulatory potential, the researchers added an extra step to deplete host and pathogen ribosomal RNAs. Based on dual RNA-seq experiments done at five time points during the first day of infection, they uncovered hundreds of new and known small RNAs from Salmonella.

One of these, PinT, showed the highest levels of activation after infection, the team reported. Through a series of follow-up experiments, the group demonstrated that the small RNA acts not only as a co-activator of Salmonella genes important to invasion, infection, and intracellular replication, but also as a regulator of coding and non-coding transcripts produced by infected host cells.

"Our findings with PinT and other intracellularly induced [small RNAs] illustrate how small perturbations in the infection process, such as dysregulation of a few Salmonella mRNAs, can propagate through the entire host system, potentially leading to different disease outcomes in the context of a whole organism," the researchers concluded.