New RNA-seq approaches for the study of bacterial pathogens

doi:10.1016/j.mib.2017.01.001

Current Opinion in Microbiology

Volume 35, February 2017, Pages 78-87

https://doi.org/10.1016/j.mib.2017.01.001 Get rights and content

Highlights

•
A surge of new RNA-seq based methods to study bacterial pathogenesis.
•
RIP-seq, CLIP-seq, CLASH, RIL-seq, GRIL-seq, MAPS and Term-seq discover new RNA-based regulations.
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Grad-seq guides the discovery of RNA-binding proteins.
•
Dual RNA-seq for simultaneous transcriptomics in host–pathogen interactions.
•
Single-cell RNA-seq addresses heterogeneity in infections.

Understanding how bacteria cause disease requires knowledge of which genes are expressed and how they are regulated during infection. While RNA-seq is now a routine method for gene expression analysis in bacterial pathogens, the past years have also witnessed a surge of novel RNA-seq based approaches going beyond standard mRNA profiling. These include variations of the technique to capture post-transcriptional networks controlled by small RNAs and to discover associated RNA-binding proteins in the pathogen itself. Dual RNA-seq analyzing pathogen and host simultaneously has revealed roles of noncoding RNAs during infection and enabled the correlation of bacterial gene activity with specific host responses. Single-cell RNA-seq studies have addressed how heterogeneity among individual host cells may determine infection outcomes.

Graphical abstract

Section snippets

Target predictions of individual sRNAs

Regulatory RNAs exert post-transcriptional control in many pathways of bacterial physiology and virulence [9^••]. A primary example are the sRNAs, noncoding transcripts in the 50–250 nucleotide range, the majority of which regulate trans-encoded mRNAs by base-paring mechanisms [9^••] and in, Gram-negative pathogens, typically depend on the RNA chaperones Hfq and ProQ [10, 11••, 12]. By contrast, the ubiquitous CsrB-like sRNAs control mRNAs indirectly by antagonizing CsrA-like translational

RNA inventories

Target searches for individual sRNAs seek to answer-specific physiological questions, but it is advisable to understand early on in what global context these sRNAs act. Which RBP does a given sRNA interact with? Are the candidate targets of an Hfq-associated sRNA also bound by Hfq?

RIP-seq offers a cost-effective approach to obtaining a quick overview of major RNA regulons in a pathogen of interest; it applies RNA-seq to analyse transcripts obtained by co-immunoprecipitation (coIP) with an RBP

Global sRNA interactomes in one go

The pace of discovery and vast number of sRNAs in bacterial pathogens [8^•] demands the development of methods that go beyond target inventories of individual sRNAs or RBPs. Two recent studies have progressed to reporting global sRNA–mRNA interactomes in one go [29••, 30••]. Common to both studies is the use of in vivo UV crosslinking and ligation of sRNA–mRNA pairs during purification with central proteins, either Hfq itself [30^••] or endoribonuclease RNase E [29^••], which degrades mRNAs upon

Discovery of novel RBPs with Grad-seq

It is emerging from RNA profiling of Hfq and CsrA that these RBPs interact with but a subset of the cellular sRNAs and mRNAs. Moreover, many bacteria lack a functional Hfq protein, suggesting that other global sRNA-binding proteins must exist. Many new RBPs have been discovered in eukaryotes, but the underlying methods [2] are not transferable to bacteria whose transcripts lack a functional poly(A) tail. The new Grad-seq approach [11^••] (Figure 2a) overcomes this limitation by defining major

Dual RNA-seq for simultaneous analysis of pathogen and host

The lifestyle of bacterial pathogens involves interactions not only with host cells but also with other microbes. On the latter, RNA-seq is heralding a new era of microbiome research [36]. A recently developed approach called Term-seq (Figure 3a), which enables genome-wide mapping of transcript termination, has proven successful at discovering antibiotic-responsive riboswitches from human microbiome samples [37^••].

For pathogen interactions with eukaryotic cells, transcriptomic studies were long

Single-cell RNA-seq reveals heterogeneous host responses

Host–pathogen interactions are increasingly found to involve a high degree of cellular heterogeneity that impacts disease progression, pathogen dissemination and antimicrobial treatment [42^•]. New fluorescent reporter systems have revealed considerable heterogeneity in bacterial intracellular growth rate or stress response activity [43, 44], pinpointing persister cells with a non/slow growing phenotype that resist antibiotic treatment [42^•]. Host immune cells exposed to a homogeneous stimulus

Future perspectives

The ‘–seq’ methods have become important tools in understanding “how, when, and why sRNA-mediated regulation is of such importance to bacterial lifestyles” [55] and resolving RNA expression changes important for the success of a bacterial pathogen. For space reasons, we are unable to cover several additional variations of RNA-seq measuring protein synthesis [56] or illuminating RNA processing, structure changes and modification [26, 57, 58]. Another largely unresolved question is ‘where’

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

We thank members of the Vogel group for comments on the manuscript. The Vogel lab receives relevant funds from DFG (Graduate programme GRK 2157/1) and the Bavarian BioSysNet Program.

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View full text

New RNA-seq approaches for the study of bacterial pathogens

Highlights

Graphical abstract

Section snippets

Target predictions of individual sRNAs

RNA inventories

Global sRNA interactomes in one go

Discovery of novel RBPs with Grad-seq

Dual RNA-seq for simultaneous analysis of pathogen and host

Single-cell RNA-seq reveals heterogeneous host responses

Future perspectives

References and recommended reading

Acknowledgements

Curr Opin Microbiol

RNA

Mol Microbiol

RNA Biol

Science

Cell Microbiol

Cell

Science

Mol Cell

Nat Rev Microbiol

RNA

Nature

Curr Opin Microbiol

Coming of age: ten years of next-generation sequencing technologies

Nat Rev Genet

Comprehensive identification of RNA-binding proteins by RNA interactome capture

Methods Mol Biol

Dual RNA-seq of pathogen and host

Nat Rev Microbiol

Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq

PLoS Genet

Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing

Proc Natl Acad Sci U S A

The primary transcriptome of the major human pathogen Helicobacter pylori

Nature

Accelerating discovery and functional analysis of small RNAs with new technologies

Annu Rev Genet

Small RNAs in bacteria and archaea: who they are, what they do, and how they do it

Adv Genet

Grad-seq guides the discovery of ProQ as a major small RNA-binding protein

Proc Natl Acad Sci U S A

Silencing of natural transformation by an RNA chaperone and a multitarget small RNA

Proc Natl Acad Sci U S A

Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins

Nucleic Acids Res

Small RNA functions in carbon metabolism and virulence of enteric pathogens

Front Cell Infect Microbiol

Unexpected versatility in bacterial riboswitches

Trends Genet

Bacterial RNA thermometers: molecular zippers and switches

Nat Rev Microbiol

Effect of RyhB small RNA on global iron use in Escherichia coli

J Bacteriol

Pervasive post-transcriptional control of genes involved in amino acid metabolism by the Hfq-dependent GcvB small RNA

Mol Microbiol

Dual RNA-seq unveils noncoding RNA functions in host–pathogen interactions

Nature

MicL, a new sigmaE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein

Genes Dev

Identification of bacterial sRNA regulatory targets using ribosome profiling

Nucleic Acids Res

A 3′ external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise

Mol Cell