New RNA-seq approaches for the study of bacterial pathogens
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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