Dual RNA-seq of pathogen and host

Abstract

SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

The importance of regulatory RNAs in Staphylococcus aureus

“…In addition, the expression of sRNA in S. aureus grown in rich medium culture does not reflect the in vivo conditions. Hence, a major challenge is to understand the contribution of S. aureus sRNAs in the human context and to gain knowledge about the sRNA roles and their co-factors during human colonization and infection (Westermann et al, 2012).…”

RNA-Seq unveils new attributes of the heterogeneous Salmonella-host cell communication

“…This communication has been approached by the so called "dual" RNA-Seq technology. 20 In a recent study, Vogel and colleagues afforded this goal by using GFP-expressing S. Typhimurium to infect HeLa epithelial cells. 21 Infected cells were sorted at different post-inoculation times (2, 4, 8, 16 and 24 h) and RNA-Seq was applied to get simultaneous information of transcripts from the infected eukaryotic cells and intracellular bacteria in a population of infected cells previously sorted by fluorescence-activated cell sorting (FACS).…”

Avian transcriptomics: opportunities and challenges

“…A small fraction of the sequences, however, might represent transcripts from a previously known or unknown pathogen from the host tissue sampled. RNA-seq thus enables the simultaneous study of gene expression in the host and its pathogens, a possibility which was limited when using pre-NGS techniques due to the need for specific primers and hence prior knowledge of the pathogens present in the sample (Westermann et al 2012). In a study on the effect of avian malaria infection on Eurasian Siskins Carduelis spinus the transcriptome was characterised from the host (Videvall et al 2015) and the parasite simultaneously.…”