Resolving host–pathogen interactions by dual RNA-seq

Abstract

The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables “dual RNA-seq” studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.

Functional convergence of gliP and aspf1 in Aspergillus fumigatus pathogenicity

“…Distinct features of the infection environment have been revealed by diverse approaches that include histological analysis [23,24], imaging mass spectrometry [25], and transcriptional profiling [26]. We have previously used transcriptional profiling to understand the genetic control of pathogenicity in infected tissue [8,27,28].…”

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

“…Moreover, as RNA-seq is sequencing-based and read mapping onto the respective genomes is performed in silico, cross-reactivity when studying different species, an important drawback of microarrays, does not any longer occur. More specifically, in recent years several studies made use of dual RNA-seq, a term coined by Westermann et al [43], when studying host-pathogen interactions using in vitro infection models (reviewed in [19]). When assuming 10 bacteria per infected cell, sequencing depth requirements to obtain correct reflections of both host and pathogen expression profiles were estimated to be 200 and 2,000 million total reads for rRNA depleted and total RNA samples, respectively [43].…”

“…More recently, dual RNAseq was also used to simultaneously study host and pathogen gene expression profiles in in vivo infection models (reviewed in [19]). Briefly, gene expression was analyzed in mice infected with…”

“…In both examples, infected tissues were isolated and homogenized prior to total RNA extraction for sequencing. Since multiple types of cells might be involved, this approach has also been generally referred to as multi RNA-seq [19].…”

“…Moreover, increased heterogeneity in bacterial pathogen populations might add another level of complexity to multi RNAseq studies, making it for example also necessary to differentiate between the intra-or extracellular origin of bacteria without a priori isolation of infected cells as for instance, Salmonella alternates between a proliferative intracellular stage and an extracellular disseminative stage [53]. Since efficient and cost-effective separation methods are still lacking, the use of multi RNA-seq to study hostpathogen interactions remains somewhat limited [19]. known as the Salmonella-containing vacuole (SCV).…”