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

Abstract

High-throughput RNA sequencing (RNA-Seq) has uncovered hundreds of small RNAs and complex modes of RNA regulation in every bacterium analyzed to date. This complexity agrees with the adaptability of most bacteria to varied environments including, in the case of pathogens, the new niches encountered in the host. Recent RNA-Seq studies have analyzed simultaneously gene expression in the intracellular pathogen Salmonella enterica and infected host cells at population and single-cell level. Distinct polarization states or interferon responses in the infected macrophage were linked to variable growth rates or activities of defined virulence regulators in intra-phagosomal bacteria. Intracellular Salmonella, however, exhibit disparate intracellular lifestyles depending the host cell, ranging from a hyper-replicative cytosolic state in epithelial cells to a non-replicative intra-phagosomal condition in varied host cell types. The basis of such diverse pathogen-host communications could be examined by RNA-Seq studies in single intracellular Salmonella cells, certainly a challenge for future investigations.

Keywords: Heterogeneity; RNA-Seq; Salmonella; intracellular lifestyle; single-cell.

Figure 1.

Disparity between RNA-Seq data and those obtained at the protein level for the ecgA (STM1940) gene of S. Typhimurium. (A) expression profiles obtained by RNA-Seq for the genomic region in which ecgA is located. These profiles were obtained from intracellular bacteria following infection of macrophages (see Srikumar et al.) and from bacteria grown in media mimicking different infection-relevant conditions (see Kröger et al.) The image was mounted from the publicly available S. Typhimurium gene expression compendium (http://bioinf.gen.tcd.ie/cgi-bin/salcom.pl?db = salcom_mac_HL); (B) EcgA protein levels detected in bacteria grown in LB medium up to late stationary phase (LSP) or medium exponential phase (MEP); (C) EcgA levels in bacteria grown in ISM minimal medium to LSP and MEP. Protein extracts were prepared from wild-type (WT) and a ΔphoP mutant. Note the absence of reads in the RNA expression profile for ecgA (STM1940) in the LB medium-LSP and in low Mg²⁺ medium, conditions in which the EcgA protein is produced (see also Rico-Pérez et al.).¹² In concordance with transcriptomic data obtained from intracellular S. Typhimurium isolated from fibroblasts and the RNA-Seq data obtained in bacteria infecting macrophages, EcgA protein levels increase notoriously in intracellular bacteria by a regulation mediated by the PhoP-PhoQ two-component system (see Rico-Pérez et al.).

Figure 2.

Experimental conditions in which dual RNA-Seq has been applied to analyze the communication of S. Typhimurium with eukaryotic cells. (A) studies in macrophages. The scheme highlights the experimental set-up used by Hung and colleagues based on the use of pH-rodo to detect dead and live intracellular bacteria and distinct cell sorting procedures (see text). See text for other studies in macrophages as those of Hinton and colleagues, based on dual-RNA-Seq in unsorted cultures exposed to the pathogen, and Vogel and colleagues, which applied RNA-Seq of eukaryotic poly-A⁺ transcripts in single cells from uninfected and infected populations. (B) studies in epithelial cells. In this case, Vogel and colleagues applied dual RNA-Seq to sorted populations of infected and uninfected HeLa epithelial cells exposed to the pathogen. Gene expression profiles have not yet been yet defined for cytosolic and intra-phagosomal bacterial populations colonizing epithelial cells.