Small RNAs in major foodborne pathogens: from novel regulatory activities to future applications

Small regulatory RNAs (sRNAs) are involved in post-transcriptional control of important cellular processes and contribute to the success of a pathogen. Here, we use studies primarily selected from Salmonella enterica and Listeria monocytogenes to illustrate the current status of sRNA biology in important foodborne pathogens. We discuss how the regulatory activities of sRNAs can be affected by base pairing RNAs known as 'sponge RNAs', or by RNA-binding proteins, such as the newly discovered sRNA chaperone ProQ. Furthermore, we highlight recent findings for sRNAs with regulatory roles during infection, some of which are present in multiple copies, designated 'sibling sRNAs'. Importantly, knowledge on sRNA-mediated regulation can be exploited for biotechnological applications, such as in generating gene knockdowns to promote desired traits.


Introduction
Non-coding RNAs serve as regulators of gene expression in bacteria, most often through interactions with other RNA molecules, and influence important cellular processes such as metabolism, stress responses and virulence [1]. In the major foodborne pathogens Salmonella enterica serovar Typhimurium and Listeria monocytogenes, one class of non-coding RNAs, the small regulatory RNAs (sRNAs), has been studied intensively, predominantly in the context of bacterial infection, and several examples of sRNAs that control the expression of virulence genes at the post-transcriptional level are known [2]. Here, we review the latest discoveries in sRNA biology in important foodborne pathogens, with special emphasis on regulatory functions of sRNAs that aid adaptation to host-specific niches.
Additionally, we highlight regulatory mechanisms employed by sRNAs, as well as accessory interacting factors, which hold potential for use in synthetic biology and other areas of biotechnology.

Hfq-binding sRNAs -recent discoveries
The largest group of sRNAs in bacteria acts by direct base pairing to specific mRNAs, leading to either inhibition or enhancement of protein expression [1]. Cis-acting sRNAs form fully complementary interactions with their target mRNAs, whereas trans-acting sRNAs are only partly complementary to their partner mRNAs. In Gram-negative bacteria, the interaction between a trans-acting sRNA and its targets often relies on an RNA chaperone, such as Hfq, which promotes sRNA-mRNA duplex formation, whereas in Gram-positive bacteria, a role for Hfq in sRNA-mediated control is less clear [3].
Yet, Hfq contributes to stress tolerance and virulence in both Gram-positive L. monocytogenes [4] and Gram-negative Salmonella [5]. In early studies, the RNA-binding property of Hfq was successfully used 4 as means to identify sRNAs, such as LhrA in L. monocytogenes and GcvB in Salmonella [6,7]. Although LhrA and GcvB differ with respect to origin, size and nucleotide sequence, they both rely on Hfq for stability and regulatory activity [8,9]. During growth in rich medium, LhrA accumulates upon entry into stationary growth phase and affects the expression of nearly 300 genes, half of which belong to the regulon of the general stress sigma factor, σ B [10]. GcvB, on the other hand, is mainly expressed during exponential growth in rich medium and its regulon is highly enriched with genes encoding amino acid-and peptide transporters and amino acid biosynthesis proteins [11]. Both sRNAs use specific seed sequences to pair with complementary sites within the 5´-untranslated region (5´ UTR) of their target mRNAs (Figure 1a and 1b). Intriguingly, recent findings demonstrate that sRNAs themselves are targets of regulation by other transcripts acting as "RNA sponges" or "anti-sRNAs".
One example from Salmonella involves a small Hfq-binding RNA, SroC, that derives from processing of the gltIJKL mRNA and antagonizes the activity of GcvB by direct base pairing ( Figure 2) [12 • ]. The gltIJKL mRNA itself is a target of GcvB, thus, SroC and GcvB together form a feed-forward loop that increases the expression of gltIJKL, and moreover, de-represses other targets in the GcvB regulon [12 • ]. A search for Hfq-binding sRNAs in the enterohemorrhagic Escherichia coli (EHEC) transcriptome identified the anti-sRNA AgvB, which is encoded from a bacteriophage-derived region and targets an EHEC sRNA homologous to GcvB [13 • ]. In an Hfq-dependent manner, AgvB base pairs with the seed region of GcvB, blocking the interactions between GcvB and its target mRNAs [13 • ]. Interestingly, a growth experiment in bovine terminal rectal mucus, mimicking the preferred colonization site of EHEC in cattle, showed that a strain lacking the anti-sRNA had reduced competitive fitness compared to wild-type, confirming that AgvB is important for niche adaptation in EHEC [13 • ]. Transcripts acting to 5 regulate the stability and/or activity of sRNAs are likely to be discovered in other foodborne pathogens as well; indeed, several pairs of interacting sRNAs have been predicted in L.
monocytogenes [14], however, the functional relevance (if any) of such sRNA-sRNA interactions remains to be investigated.

Expanding the family of global sRNA chaperones
With the technological advance in large-scale transcriptomics, the number of published sRNA candidates has increased tremendously, now counting several hundred in both L. monocytogenes and Salmonella [14][15][16][17]. Curiously, RaiZ also interacts with Hfq, but this interaction has no effect on sRNA stability or RaiZ-  [34]. Their exact intracellular function is yet to be elucidated, but extracellular studies have implicated RyhB-1 and -2 in growth cessation during iron 7 starvation and in survival under acid shock, oxidative or nitrosative stress [30, [35][36][37]. As similar stress conditions are encountered inside macrophages, RyhB-1 and -2 may contribute to intracellular survival by aiding the adaptation to these hostile environmental conditions. The E. coli RyhB homologue contributes to iron homeostasis when iron is limiting by repressing the translation of several non-essential, iron-containing proteins and enhancing the expression of siderophore synthesis [38]. Several lines of evidence indicate that RyhB-1 and -2 function in a similar manner in Salmonella [35,39,40], still, direct base pairing has only been confirmed for the iroN mRNA target, encoding a receptor for the Fe 3+ -bound siderophore salmochelin, which is positively regulated by the RyhB siblings [41 • ]. Typically, sRNA-mediated translational activation involves a structural change in the 5' UTR, which renders the RBS accessible, and thus facilitates translation initiation [42], as exemplified monocytogenes. The LhrC family of sRNAs includes the highly homologous LhrC1-5 [7] as well as Rli22 and Rli33-1, which are both structurally and functionally related to the LhrCs, but present lower homology [44]. The seven siblings are expressed from individual promoters, of which rli22 and lhrC1-5 are positively regulated by the two-component system LisRK that responds to cell envelope stress 44], whereas σ B controls the expression of rli33-1 (Figure 3b) [15,44]. All seven sRNAs are induced upon exposure to whole human blood [14], and six of them are highly expressed during intracellular replication in macrophages [15]. Strikingly, Rli22 was the only member found to be expressed when L. monocytogenes resides in the intestinal lumen of mice [14], pointing out that additional factor(s) may contribute to the expression of this sRNA [44]. The differential expression of sRNA siblings may enable the bacterium to integrate numerous stimuli into the control of the LhrC regulon, and further suggests What is the role of LhrC regulation during infection? Presently, there is no simple answer to this question, but it should be noted that the seven siblings act as repressors of cell envelope-associated proteins with virulence functions. Since surface proteins are recognized by the immune system, the 9 regulatory action by the LhrCs may be seen as an attempt to evade immune detection. In addition, LhrC1-5 and Rli33-1 contribute to infection of macrophages [15,26], suggesting a role for these sRNAs in the intracellular environment. Curiously, the LhrC regulon comprises genes encoding amino acid and oligopeptide transporters, and branched chain amino acid biosynthesis proteins, indicating that upon phagocytosis, a transient decrease in the uptake and biosynthesis of amino acids may be beneficial to L. monocytogenes [26]. Notably, recent studies demonstrated that a decrease in branched chain amino acid availability leads to an increase in virulence gene expression [45,46].

Biotechnological applications of "basic knowledge" on bacterial sRNAs
sRNAs are not limited to pathogenic species and numerous candidates are indeed found in biotechnologically relevant species such as Clostridium acetobutylicum [49] and Lactococcus lactis [50], in which they may be involved in controlling stress responses and metabolism. Additionally, sRNAs may also be considered a great biotechnological toolbox in synthetic biology. For instance, base pairing antisense RNAs have successfully been employed to knockdown selected genes in organisms such as the wine-associated lactic acid bacterium Oenocuccus oeni [51] and the butanolproducing C. acetobutylicum [52], which are difficult to genetically engineer. Under some conditions, it might even be advantageous to knockdown, rather than knockout, gene expression; for example, a double knockdown strategy was used to screen 18 E. coli strains for the influence of genetic background on phenol production and tolerance [53], a task that would have been very timeconsuming using knockouts. Moreover, antisense knockdown allows for transient target regulation by controlling the sRNA expression, or partial target knockdown by controlling the strength of interaction 11 between an sRNA and its target. The latter naturally occurs in E. coli where RyhB partially represses cysE, encoding an essential serine acetyltransferase, thereby redirecting serine flux into siderophore synthesis when iron is limiting [54]. Compared to protein-based regulation of pathway fluxes, RNAbased regulation may have a faster recovery time [55]. Besides, when considering base pairing sRNAs, the relationship between sequence and function is often more predictable. Based on knowledge about sRNA regulation in model bacteria such as E. coli, Cho and Lee [52] rationally designed a synthetic sRNA based on the scaffold of an E. coli sRNA to knockdown translation of a target gene in C.
acetobutylicum as a proof-of-concept. Even synthetic sRNAs targeting multiple mRNAs simultaneously are being developed to coordinate the expression of key enzymes in a pathway [56]. Thus, studying the diverse regulatory mechanisms employed by sRNAs and the multiple contributing factors in different species, lays the foundation for future utilization of RNA-based regulation in biotechnological processes.

Conclusions
Most of our current knowledge on sRNAs in foodborne pathogens derives from studies in L. monocytogenes and Salmonella, aiming to explore the importance of sRNAs during infection. Indeed, several examples of sRNAs with roles in virulence control have been uncovered, some of which are highlighted in this review. In future studies, we suggest that functional analyses of sRNAs are extended to include conditions encountered by foodborne pathogens in the external environment (e.g. in soil [57]) or during food production and storage. Furthermore, hundreds of sRNAs are waiting to be characterized in other important foodborne pathogens, such as Campylobacter jejuni [58,59].
The use of RNA-seq-based methodologies promises to provide rapid and global insights into the 12 regulatory functions of these sRNAs. Ultimately, such studies would stimulate the discovery of novel players in RNA-based regulation in bacteria, which could be exploited for biotechnological applications.     Homologous sRNAs may be differentially expressed due to nucleotide differences in their promoter regions. External stimuli known to induce the expression of the RyhB siblings in Salmonella (a) and LhrC siblings in L. monocytogenes (b) are shown together with the transcriptional regulators (yellow ovals) involved; a question mark indicates that the signaling pathway responsible for the induction is presently unknown.
(a) Transcription of the RyhB siblings in Salmonella is controlled by a common set of transcriptional regulators; however, their strength of regulation is not identical for the two promoters (relative strength at each promoter is indicated by the thickness of the arrow). The RyhB siblings share a 33 nt sequence with perfect homology, which forms the basis of the regulation of shared targets [35,40,41 • ], whereas differences in the flanking sequences might explain the regulation of unique targets by either sRNA [43]. The thickness of the lines indicate the observed relative strength of regulation by the sibling sRNAs, which might reflect both differences in sRNA expression as well as the strength of interaction with mRNA targets. Dashed lines indicate that RyhB expression affects target mRNA level, but dependence on direct base pairing has not been examined further. Selected target 22 genes are shown to illustrate the differences, but mRNA levels of more genes are known to be affected by the RyhB siblings [35,37,43].  [44].