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RNAs: regulators of bacterial virulence

Jonas Gripenland*, Sakura Netterling*, Edmund Loh*, Teresa Tiensuu*, Alejandro ToledoArana and Jrgen Johansson*

Abstract | RNA-based pathways that regulate protein expression are much more widespread than previously thought. Regulatory RNAs, including 5 and 3 untranslated regions next to the coding sequence, cis-acting antisense RNAs and trans-acting small non-coding RNAs, are effective regulatory molecules that can influence protein expression and function in response to external cues such as temperature, pH and levels of metabolites. This Review discusses the mechanisms by which these regulatory RNAs, together with accessory proteins such as RNases, control the fate of mRNAs and proteins and how this regulation influences virulence in pathogenic bacteria.
To survive and thrive in an often hostile environment, a bacterium has to monitor its surroundings and adjust its gene expression and physiology accordingly. This is especially important for pathogenic bacteria, which continuously interact with the host during an infection. RNAs are excellent regulatory molecules that can carry out a plethora of regulatory tasks1 (FIG. 1). For instance, RNAs can directly sense environmental cues, such as differences in temperature, pH and nutrient availability, through regulatory regions that lie upstream of the coding sequence on the same transcript, leading to an altered transcriptional read-through or translation initiation of that downstream coding sequence. Furthermore, bacteria can regulate transcript expression through cisacting RNAs, which function in an antisense manner and control the expression of mRNAs encoded on the opposite DNA strand, or trans-acting small non-coding RNAs (sRNAs), which function at a distance to alter the expression of target RNAs through an antisense mechanism. The fate of RNA transcripts can also be controlled by proteins, including RNases and RNA chaperones, which can degrade both cis- and trans-acting antisense regulatory RNAs and their target mRNAs or can facilitate the interaction of trans-acting sRNAs with the target mRNAs, respectively. Because pathogenic bacteria encounter diverse environmental conditions, they require rapid regulatory circuits to survive, making regulatory RNAs particularly suitable for controlling bacterial virulence. Together with regulatory proteins and two-component systems, regulatory RNAs integrate environmental stimuli into outputs that are important for pathogenicity. In fact, there is evidence that regulatory RNAs are more suitable than proteins for controlling gene expression. First, the energy cost of transcription (that is, generating regulatory and target RNAs) is much lower than that of translating regulatory proteins. Second, regulatory RNAs can control gene expression much faster than regulatory proteins; this is especially true for 5 untranslated regions (UTRs), which directly dictate the expression of downstream mRNAs on sensing an environmental cue. Third, regulatory RNAs are generally much less stable than regulatory proteins, which allows their rapid clearance when they are no longer needed. Fourth, many regulatory RNAs act at the post-transcriptional level and can therefore modify mRNAs that have already been expressed; they can thereby dictate and possibly overcome effects at the transcriptional level. Studies in the past few years have shown that regulation mediated by RNAs and their associated proteins is more common than previously thought. Genome-wide analyses based on tiling arrays and high-throughput RNA-sequencing technologies (BOXes 1,2) have revealed the transcriptomes of several bacteria, including pathogenic bacteria2. Both techniques allow the unbiased visualization of all RNA molecules transcribed during specific conditions (for example, during stress, high osmolarity and low oxygen) without taking into account the positions of annotated ORFs. Therefore, the positions of all RNAs transcribed in a cell namely, mRNAs (including operons), tRNAs, ribosomal RNAs, cisacting antisense RNAs and trans-acting sRNAs can be mapped with 1-nucleotide resolution. These studies have shown that the transcriptional landscape of all organisms is much more complex than expected. This Review focuses on 5 and 3 UTRs, cis-acting antisense RNAs and trans-acting sRNAs, as well as on proteins that affect the expression of virulence genes and
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*Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Ume University, 90187 Ume, Sweden. Instituto de Agrobiotecnologa, Universidad Pblica de NavarraCSICGobierno de Navarra, 31006 Pamplona, Spain. Correspondence to J.J. email: jorgen.johansson@ molbiol.umu.se doi:10.1038/nrmicro2457

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DNA SD mRNA 5 RNase Trans-acting sRNA 70 5 UTR SD RNase 3 3 UTR 3 5 Cis-acting antisense RNA RNase Gene X

6S RNA

Figure 1 | control of mrNA activity and stability. The fate of an mRNA is controlled Nature coding | Microbiology by several factors. 5 untranslated regions (UTRs) lie upstream of theReviews sequences and include thermosensors, pH sensors and riboswitches. 3 UTRs lie at the other end of coding RNAs and, in many cases, determine transcription termination and stability. Cis-acting antisense RNAs are expressed from the opposite strand of the DNA. Trans-acting small non-coding RNAs (sRNAs) are expressed from a different location on the chromosome and most commonly bind to the ShineDalgarno (SD) sequence of a target mRNA in an antisense manner. Trans-acting sRNAs can also sequester target proteins; for example, 6S RNA sequesters the housekeeping RNA polymerase 70 (also known as RpoD). The fate of transcripts can also be controlled by RNases, which function either exonucleolytically (orange), by targeting transcripts from the 5 or 3 end (such as RNase J1 and RNase J2, which are found in some Gram-positive bacteria) or endonucleolytically (purple), by targeting sequences in the middle of the transcript.

translation of the ORF, which encodes the transcriptional activator listeriolysin regulatory protein (PrfA)8. Once present, PrfA, which is essential for L. mono cytogenes virulence, activates the expression of virulence genes encoding adhesins, phagosome-escaping factors and immune-modulating factors9,10. For instance, PrfA activates the expression of hly (encoding listeriolysin O, which is essential for bacterial escape from the phagosome) and actA (encoding actin assembly-inducing protein (ActA), which is essential for listerial intracellular movement). Thermosensors that function similarly to prfA have been identified in other pathogenic bacteria, such as Yersinia spp. and Salmonella spp.11,12. Yersinia pestis, the causative agent of plague, has a thermosensor that controls the expression of the transcriptional virulence regulator lcrF, ensuring its expression only at 37 c. lcrF is required for Y. pestis virulence13 because it activates the expression of Yope, which blocks phagocytosis14,15. lcrF and other thermosensors of the four-uracil family (but not prfA) are thought to achieve this control through their anti-SD sequence (made up of four uracils)16. Specifically, at low temperatures the anti-SD sequence sequesters the SD sequence, whereas at human body temperature the SD site dissociates from the antiSD sequence, allowing the ribosome to bind to the SD sequence and translation to progress. pH sensing. The alx gene in Escherichia coli encodes a putative transporter implicated in resistance against the antimicrobial agent tellurite17 and is expressed in highly alkaline conditions18. The 5 UTR in front of the alx mRNA has been shown to be a pH-responsive element 17. Under normal growth conditions (pH 7.0), transcription of the 5 UTR proceeds uninterrupted, leading to the formation of a translationally inactive structure. By contrast, under alkaline conditions an alternative structure that is translationally active is formed; in this case, the RNA polymerase pauses at two different sites in the 5 UTR, preventing the formation of the inactive structure. Instead, an open structure is formed, exposing the SD sequence and allowing binding of the ribosome and, hence, translation. It is possible that the basic regulatory mechanism controlling alx expression (that is, a difference in transcription speed generating alternative RNA structures) is used to regulate the expression of other genes as well. Metabolite sensing. One group of 5 UTRs is the riboswitches1922. These are metabolite-sensing regulatory RNA structures that function as sensors and regulators of various metabolic pathways in bacteria. each class of riboswitch binds a specific metabolite through its aptamer domain, and this interaction induces a structural change in the riboswitch regulatory domain that causes altered transcription or translation. For riboswitches acting at the transcriptional level, binding of the metabolite to the aptamer domain generally induces the formation of a terminator structure in the regulatory domain to prevent transcription of the downstream gene. For riboswitches acting at the translational level,
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therefore promote virulence. For scientific interest, one example of non-pathogenic RNA regulation (a lysine riboswitch) is also discussed.

Riboswitch
An mRNA control element that changes conformation in response to the binding of a metabolite (for example, glycine, lysine or coenzyme B12) and influences gene expression.

5 regulatory UTRs of mRNAs The region between the transcriptional start site and the start codon of an mRNA is known as the 5 UTR. This region can vary substantially in length, ranging from only a few to several hundred bases. Transcription can begin at various promoters, allowing the formation of many potential 5 UTRs and, hence, complex possibilities of post-transcriptional regulation3,4. 5 UTRs are used by pathogenic bacteria, among other organisms, to modify gene expression on the basis of changes in temperature, pH and the presence of metabolites.
Temperature control. Bacteria have developed thermosensor mechanisms that act at the protein, DNA and RNA levels to directly detect changes in temperature, although most act at the RNA level57. Such sensing mechanisms are especially important for pathogens, which need to fine-tune gene expression in response to host temperature. The food-borne human pathogen Listeria monocy togenes, which causes various brain and maternofetal infections, possesses such an RNA thermosensor. The 116-nucleotide 5 UTR upstream of the prfA mRNA coding sequence forms a secondary structure at lower temperatures, masking the shineDalgarno sequence (SD sequence) and thereby inhibiting translation. An alternative secondary structure is formed at human body temperature (37 c), exposing the SD region and allowing

ShineDalgarno sequence
A sequence that is located 5 of the AUG (start) codon on bacterial mRNAs and functions as the binding motif of the 30s subunit of the ribosome. The consensus sequence is AGGAGG.

Aptamer
An RNA domain, either engineered or natural, that forms a precise three-dimensional structure and selectively binds a target molecule.

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Box 1 | RNA sequencing
Isolated total RNA is enriched for mRNA (see the figure; green) through the removal of ribosomal RNA (rRNA) and tRNA (black) using several techniques. Capture of rRNA involves the use of probes that bind 16S and 23S rRNA. Processed RNA is removed by 5-to-3 exonucleases that recognize monophosphorylated 5 ends: 16S and 23S rRNAs are processed and thus carry a monophosphorylated 5 end, so these RNAs are degraded, whereas primary transcripts usually carry a triphosphorylated 5 end. Samples can also be enriched for mRNA though the addition of an artificial poly(A) tail to mRNAs followed by fishing with an oligo(dT). The mRNA is converted into cDNA (red) by reverse transcription. The lack of natural poly(A) tails enforces alternative priming approaches, such as random hexamer priming, addition of artificial poly(A) tails followed by priming with oligo(dT), or priming from RNA probes ligated to the mRNAs. The cDNA is amplified (normally when attached to beads) and sequenced by one of many different platforms (such as 454, Ilumina and Solid). Normally, each sequence read is 30400 bases, and this is mapped on the reference genome. The number of reads for each region gives a direct measurement of RNA expression and stability. The total number of sequence reads can reach 1 109.
RNA isolation

c-di-GmP drops in the intestine, and this is sensed by the riboswitch, thereby allowing the expression of GbpA and the consequent attachment of the bacteria to the intestine25.

Cyclic-di-GMP
A second messenger that is generated by diguanylate cyclases and hydrolysed by phosphodiesterase A.

Rho-independent transcriptional terminator

A strong secondary RNA structure followed by several uracils that destabilizes the RNADNA duplex so that the RNA polymerase falls off. Normally found after the coding sequence of an mRNA.

3 regulatory UTRs of mRNAs In eukaryotes, regulatory UTRs located at the 3 end of the transcript are known to control translation26,27. The 3 UTRs of bacterial mRNAs are thought to mainly harbour transcription termination structures, which might prevent access of exonucleases to the 3 end of the transcript but have no clear regulatory function. However, the recent advances in bacterial transcriptome analyses mRNA enrichment (BOXes 1,2) have indicated regulatory roles for 3 UTRs, albeit not specifically with regard to virulence. One such example is the lysine riboswitch of L. mono cytogenes 4 (FIG. 2), which lies in the 5 UTR, similarly to Reverse transcribe other bacterial riboswitches. The lysine riboswitch regufragments (cDNA) lates the expression of the downstream gene, lmo0798, which encodes a lysine transporter. In the presence of lysine, an intrinsic Rho-independent transcriptional terminator is formed and the downstream gene is not expressed. By Binding of cDNA to beads contrast, when lysine is absent, a structure that blocks termination (an anti-terminator structure) is formed and the downstream gene is expressed (FIG. 2). Interestingly, in addition to this well-known type of riboswitch regulation, the lysine riboswitch can also control the termiAmplify and sequence nation of the upstream gene, which encodes a putative blue-light receptor. The mRNA encoding the upstream gene harbours a long 3 UTR containing the lysine riboswitch but no other terminator. The presence of lysine terminates transcription, whereas the absence Analyse gene expression of lysine allows co-expression of the upstream and downstream gene on the same transcript (FIG. 2). Other RNA structures located in the 3 UTR with Nature Reviews | Microbiology binding of the metabolite to the aptamer domain putative regulatory functions have been identified in alters the interaction between the SD and an anti-SD Bacillus subtilis 28. Specifically, the transcripts of nine sequence, either preventing or allowing binding of the genes were found to have long 3 UTRs that have high ribosome to the SD sequence19. sequence similarity and form stable RNA secondary Recently, one riboswitch class was identified that structures. Because most of these genes encode proteins controls the expression of downstream genes includ- that associate with the membrane and are involved in ing genes important for DNA uptake, motility and viru- cell wall synthesis and transport, the 3 UTR structures lence in many pathogenic bacteria (Vibrio cholerae, might be involved in the translation and/or localizathe causative agent of cholera, Clostridium difficile, an tion of the proteins to specific compartments in the intestinal opportunistic pathogen, and Bacillus cereus, cell. Alternatively, the long 3 UTRs might protect from a major cause of food poisoning) by binding the sec- 3-to-5 exonucleolytic degradation through their second messenger cyclic-di-GMP23. Binding of c-di-GmP ondary structures. In addition to these structures, long to the riboswitch aptamer domain induces a structural putative 3 regulatory UTRs have been observed next alteration that can affect transcription termination or to transcripts encoding certain virulence factors in the translation initiation, depending on the bacterial spe- human pathogen Staphylococcus aureus 29. This bactecies. Interestingly, different riboswitches in the same rium has several long 3 UTRs that give rise to sRNAs30. bacterial species might respond differently to c-di-GmP, These examples suggest that large bacterial 3 UTRs either stimulating or repressing expression of the might have a role in mRNA stability, mRNA localizadownstream gene. tion, the generation of sRNAs or regulation by binding One riboswitch that is negatively regulated by trans-acting factors. c-di-GmP lies in front of gbpA, which encodes N-acetylglucosamine-binding protein A (GbpA), a pro- Cis-acting antisense RNAs tein important for V. cholerae intestinal attachment 24. Cis-acting antisense RNAs consist of two subtypes (FIG. 3): GbpA is expressed only under specific growth condi- bona fide antisense RNAs, which are present on the comtions, including when V. cholerae enters the intestine. plementary strand to one or several ORFs; or overlapping This is at least partly because the concentration of UTRs, which consist of a long 5 or 3 UTR of an mRNA
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Mycoplasma pneumoniae, Pseudomonas syringae, L. monocytogenes, B. subtilis and the cyanobacterium Each tiling array in bacteria Synechocystis sp. Pcc 6803 (ReFs 4,28,3638). It thereRNA isolation usually contains >105 fore seems that cis-acting antisense RNAs are extremely oligonucleotides. Total RNA abundant. Interestingly, the size of the antisense tran(black and green) is isolated and scripts found in a cell can sometimes range from a few converted into labelled cDNA bases to several kilobases, so one particular antisense RNA (red) before hybridization, and may overlap several genes, as shown in L. monocytogenes the array is then read under a and B. subtilis 4,28. fluorescence microscope. RNA Although the mechanisms involved in pervasive might also be spotted directly Conversion to cDNA onto the array and visualized antisense transcription are still unclear, some insights by antibodies recognizing have been obtained 33,34,36,37,3941 . For example, the DNARNA duplexes. The 1.2 kb antisense RNA AmgR was shown to regulate signal strength of each Salmonella enterica virulence. AmgR is complemenoligonucleotide reflects tary to the mgtC portion of the polycistronic mgtCBR the relative amount of that operon, which encodes mgtc, mgtB and mgtR. mgtc particular mRNA in each of is an inner-membrane protein that is present in several the tested samples. In newly Hybridize to microarray bacterial pathogens, in which it is required for survival designed tiling arrays, the in macrophages, virulence in mice and growth at low entire genome is covered by Tiling array mg 2+ concentrations. A variation in the expression of overlapping oligonucleotides, Genomic DNA 5 3 allowing non-coding regions AmgR promotes changes in mgtc protein levels, thereby of the chromosome (such as affecting virulence. Inactivation of the amgR promoter Partially overlapping intergenic regions and antisense derepresses the expression of mgtc, which makes this probes strands) to be read. S. enterica mutant more virulent than the wild-type strain; by contrast, overexpression of AmgR attenuates Analyse gene expression S. enterica virulence owing to a decrease in mgtc levels. Thus, S. enterica modulates its proliferation inside host tissues by regulating mgtc levels through the action of Nature Reviews | Microbiology that overlaps with the mRNA encoded by the other DNA the AmgR cis-acting antisense RNA42. Interestingly, the strand. For example, an overlapping antisense 5 UTR expression of both the antisense AmgR and the mgtCBR can be expressed from a promoter located on the oppo- operon is controlled by the same response regulator, PhoP, site strand of an ORF. A long 3 UTR, generated either by under low mg 2+ concentrations. However, PhoP binds the absence of a transcriptional terminator near the stop the amgR promoter with less affinity than it binds the codon or by termination read-through events, can lead mgtC promoter; this results in a regulatory loop in which to overlapping antisense 3 UTRs, as the transcription of low levels of active PhoP (caused by a slight increase in that gene would end in a position located inside or after mg 2+ concentrations) induce mgtC expression, whereas the gene encoded on the opposite strand2 (FIG. 3). high levels of active PhoP (caused by a high concentraUntil a few years ago, our understanding of cis- tion of mg 2+) induce AmgR expression, leading to mgtC acting antisense RNAs was limited to studies in bac- inactivation42. Thus, the antisense RNA might act as teriophages, plasmids and transposons. Although the a timing device, allowing a dynamic change in target number of identified cis-acting antisense RNAs in bac- mRNA levels in a PhoP-dependent manner. teria increased soon after, those identified were limited Similarly, in H. pylori several cis-acting antisense to sRNAs that acted on regions near the SD, owing to RNAs and their corresponding mRNA targets (encodthe biased nature of the methodologies used7,19. Recent ing proteins required for acid resistance) are induced technological advancements (BOXes 1,2) have shown that by the same signal (in this case, acid stress)33. This is antisense transcription occurs in all species, including in contrast to most trans-acting sRNAs, which are typibacteria. Indeed, thousands of transcripts that origi- cally controlled by a different regulator than their mRNA nate from antisense RNAs to genes or from intergenic target 19 (see below). regions that were previously thought to be silent have Another example of a cis-acting antisense RNA affectbeen identified in eukaryotes. This unanticipated level ing a crucial bacterial process is the L. monocytogenes of complexity is known as pervasive transcription, as 1.7 kb 5 UTR of the mRNA encoding mogR4, the negatranscripts are not restricted to well-defined features tive regulator of flagellum biosynthesis. mogR is tranResponse regulator such as ORFs31,32. scribed from two alternative promoters, P1 and P2, which A bacterial gene-regulatory Pervasive transcription has also been found in bac- are located at 1,697 and 45 nucleotides upstream of the protein that controls gene teria. For example, a study examining the primary tran- start codon, respectively. consequently, P1 generates a expression in response to scriptome of the gastric pathogen Helicobacter pylori long 5 UTR that overlaps the first three genes (lmo0675, external signals. Most response regulators consist of two found that around 46% of all ORFs were associated with lmo0676 and lmo0677) of the operon required for flagdomains: a regulatory domain, at least one antisense transcription start site33. Similarly, ellum biosynthesis, which is encoded on the opposite the activity of which is more than 1,000 antisense transcription start sites have strand. The transcription of this long overlapping 5 UTR modulated (indirectly) by been mapped in E. coli 34, supporting previous results35. depends on the stress-activated transcriptional regulator the external signal, and a DNA-binding domain. many antisense transcripts have also been found in RNA polymerase B, and it is therefore overexpressed
Box 2 | Tiling array
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DNA Imo0799 LysRS Imo0798

P mRNA + Lysine

Lys 5

Terminator

transcription has been shown to be a genome-wide phenomenon, further investigations are needed to determine whether the main function of cis-acting antisense RNAs is to control gene expression at the transcriptional level (by transcriptional interference) or at the posttranscriptional level (by mRNA processing or translational inhibition), or whether both regulatory mechanisms can coexist.

Lys 5 3

Lysine

Anti-terminator

Figure 2 | Untranslated region-mediated regulation. In the presence of lysine, an intrinsic Rho-independent terminator (T) is formed in the lysine riboswitch (LysRS) mRNA Nature Reviews | Microbiology sequence, preventing transcription of the downstream gene, lmo0798. In this case, a small non-coding transcript is generated that corresponds to LysRS. The LysRS terminator also functions as a terminator for the upstream gene, lmo0799, generating a transcript that consists of the lmo0799 sequence and LysRS. In the absence of lysine, the intrinsic terminator is not formed, allowing transcription to proceed into the downstream gene. Because there is no termination of lmo0799 expression in the absence of lysine, a long transcript comprising lmo0799, LysRS and lmo0798 is also generated from the lmo0799 promoter. P, promoter.

Transcriptional interference
The negative impact that one transcriptional activity can have on another transcriptional activity in cis.

Quorum sensing
The phenomenon in which the accumulation of signalling molecules enables a single cell to sense the number of bacteria that are present (the cell density); the purpose is to coordinate certain behaviours or actions between bacteria.

during the stationary growth phase. Absence of B increases bacterial motility, whereas overexpression of the P1-derived, B-dependent transcript decreases motility. Although the precise mechanism is not known, it is expected that bacteria modulate the expression of flagellum proteins by integrating environmental signals in a fast manner. Thus, the RNA levels of the flagellum operon can be controlled by post-transcriptional RNA processing that depends on the levels of antisense RNA, which are in turn controlled by B. In general, it is conceivable that only mRNA that is present at higher levels than its cis-acting antisense antagonist is translated, so translation of certain genes starts only when the mRNA concentration reaches a certain level2. Furthermore, it cannot be excluded that transcriptional regulation of mRNA might begin while the cis-acting antisense RNA is being transcribed and, consequently, transcriptional interference might occur between both elongation complexes43. Because antisense

Trans-acting sRNAs RNA molecules acting in trans on distant targets are commonly denoted as trans-acting sRNAs and are perhaps the best-studied form of regulatory RNA19. Transacting sRNAs, traditionally identified in intergenic regions, are encoded distally from their targets and function either by binding RNAs, leading to downregulation of target mRNA activity, typically through degradation, or by binding target proteins and affecting their activity. most trans-acting sRNAs are involved in responding to rapidly changing environmental conditions, and only a few have been shown to have roles in different infection models4,4451. One example of a trans-acting sRNA with a role in virulence has been identified in the Gram-positive opportunistic human pathogen S. aureus. S. aureus harbours the accessory gene regulator (agr) locus, which encodes an autoactivating quorum sensing system52 and is composed of two transcriptional units that are transcribed from the divergent P2 and P3 promoters. expression of the four genes encoding the quorum sensing two-component system (agrBDCA) is initiated at P2, whereas expression from P3 results in a transcript that can act as a trans-acting 514-nucleotide sRNA, known as RNAIII53, and can encode -haemolysin. RNAIII is structurally conserved among staphylococcal species and regulates several mRNAs involved in the pathogenicity of S. aureus. The expression of RNAIII peaks at late logarithmic and stationary phases and is upregulated by AgrA, the response regulator of the agr locus52. Because it can function as both an activator and a repressor, RNAIII can control the switch between the expression of secreted factors and the inhibition of surface proteins53. For example, RNAIII regulates the expression of -haemolysin (hla; also known as hly) mRNA54 by binding, through its 5 domain, to a region upstream of the hla coding sequence that normally sequesters the hla SD sequence and inhibits translation; the interaction releases the SD sequence of hla, and translation can begin55. Furthermore, RNAIII negatively regulates the earlyexpressed virulence factors immunoglobulin G-binding protein A and fibrinogen-binding protein56,57 and the transcriptional regulator Rot 58. In this case RNAIII binds the target RNA through its 3 UTR and central domain, thereby inhibiting translation and inducing target mRNA degradation. Finally, RNAIII has been found to regulate coa mRNA, which encodes staphylocoagulase, another early-expressed virulence factor that causes coagulation of human plasma and enables S. aureus to hide from the host immune system59. Two hairpins of RNAIII can directly interact with the coa
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Antisense RNAs RNA ORF1 Terminator Long antisense RNA Overlapping UTRs ORF6 Overlapping 5 UTRs Overlapping 3 UTRs ORF9 ORF10 ORF11 ORF12 ORF7 ORF8 ORF2 ORF3 Promoter Small antisense RNA ORF4

ORF5

Figure 3 | Cis-acting antisense rNAs. Schematic representation of the different types of antisense RNA molecules. These include bona fide antisense RNAs and overlapping Nature Reviews | Microbiology 5 and 3 untranslated regions (UTRs). The antisense RNA may be either a long antisense RNA covering more than one ORF (in the example, the long antisense RNA overlaps ORF1, ORF2 and ORF3) or a small antisense RNA that overlaps the ShineDalgarno sequence (which lies between the promoter and the start codon) and affects mRNA stability and/or protein translation (for example, the antisense RNA overlapping ORF4). Overlapping 5 UTRs are generated when the transcription of a certain gene (for example, ORF5) starts from a promoter located on the DNA strand opposite divergent genes (in this case, ORF6, ORF7 and ORF8). As a result, the ORF5 mRNA has a long 5 UTR that overlaps the ORF6, ORF7 and ORF8 mRNA. Overlapping 3 UTRs are generated when transcription of a certain gene ends in or after a gene located on the opposite DNA strand; for example, the transcription of the operon encoding ORF9, ORF10 and ORF11 ends after ORF12, so the long 3 UTR completely overlaps the ORF12 mRNA.

SD sequence and with parts of the coding sequence, thereby inhibiting translation initiation and promoting RNase III-dependent degradation of the coa transcript together with RNAIII60. In L. monocytogenes, approximately 50 trans-acting sRNAs have been discovered4,6164, but functional and mechanistic data are still limited. Two trans-acting sRNAs, RliB and Rli38 (which are absent in nonpathogenic strains of L. monocytogenes), were recently shown to have a role in L. monocytogenes pathogenicity4. RliB is 360 nucleotides long and contains five repeats of 29 nucleotides interspaced by 3536 nucleotides62. Rli38 is 369 nucleotides long and has homology to putative sRNAs in other pathogenic bacteria, such as S. aureus and Enterococcus faecalis. Interestingly, Rli38 is differentially expressed during various environmental stress conditions and is markedly induced when bacteria are exposed to human blood4. Infection experiments using rliB- or rli38-knockout bacteria showed that the rli38-knockout strain had a lower ability to colonize various mouse organs than the wildtype strain. Surprisingly, the rliB-knockout strain had a higher colonization ability than the wild-type strain; these findings highlight the complexity of the regulation exerted by these trans-acting sRNAs with regard to L. monocytogenes virulence.
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The complexity of RNA regulation was further increased by the recent finding that riboswitches can have dual regulatory functions in L. monocytogenes. A S-adenosylmethionine (SAm) riboswitch can function both as a classical cis-acting riboswitch, controlling the expression of its downstream gene by transcriptional termination, and as a trans-acting sRNA, directly binding to a target mRNA65. On binding to its metabolite (SAm), the riboswitch adopts a terminator structure, terminating its transcription and generating the transacting sRNA. This transcriptionally terminated SAm riboswitch, termed SreA (SAm riboswitch element A; 227 nucleotides), directly base-pairs with the 5 UTR of the prfA transcript (the distal part of the RNA thermosensor), thereby blocking its translation (FIG. 4a). This interaction occurs only at high temperatures (~37 c), when the thermosensor has a more open conformation, and in nutrient-rich conditions when the levels of SreA are high (that is, when SAm levels are high). Intriguingly, the expression of SreA RNA is also controlled by PrfA: when PrfA levels are high, PrfA switches off its own expression, thus downregulating SreA. In a sreA mutant background, the virulence gene hly (which is regulated by PrfA, as mentioned above) is upregulated because PrfA levels increase, suggesting that SreA antagonizes L. monocytogenes virulence and therefore inhibits pathogenesis. It therefore seems that SreA functions as a sensor of the metabolic state in bacteria, preventing the expression of PrfA if conditions are rich in the host cytoplasm (when SAm levels are high and SreA is terminated). The observation that terminated riboswitches can have a function by themselves markedly increases the number of putative trans-acting sRNAs. Six trans-acting sRNAs were recently discovered in the Gram-negative opportunistic pathogen Legionella pneumophila, and among these was a homologue of the well-studied E. coli 6S RNA50. In E. coli, 6S RNA associates with and sequesters the RNA polymerase holoenzyme containing 70 (also known as RpoD; the housekeeping sigma factor that facilitates the binding of RNA polymerases to specific promoter sequences) but not S (also known as 38 or RpoS; the stationary phase sigma factor). The 6S RNA70 interaction leads to increased transcription of S-dependent genes66. Because 6S RNA expression is increased at stationary phase, this allows the bacterium to switch from 70-dependent to S-dependent gene expression. Two sizes of 6S RNA have been identified in L. pneumophila, one of 182 nucleotides and another of 147 nucleotides. Interestingly, only the shorter, 3-processed 6S RNA could co-immunoprecipitate with 70. During infection of THP-1 macrophage-like cells, a strain with mutated 6S RNA (ssrS) was attenuated, indicating the importance of 6S RNA during L. pneu mophila replication inside human cells. The 6S RNA controls the expression of ~5% of L. pneumophila genes; among the positively regulated genes are vipA and legC5, which encode effector molecules exported by the type IvB secretion system50. Data suggest that 6S RNA in L. pneumophila could bind other, as-yet unknown, targets, the expression of which depends on 70 RNA polymerases50.
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a
Closed Open SreA 5

37 C SD 5 3 30 C 5 3 5 Ribosome + SreA

3 SreA 3 prfA 5 UTR 5 Translation inhibited 3

region of the ompT transcript, repressing its translation. The interaction between vrrA and ompT depends on Hfq, similarly to many other trans-acting RNAtarget mRNA interactions in E. coli and S. enterica71; interestingly, however, the vrrAompA interaction does not strictly depend on Hfq19,48.

prfA 5 UTR Translation inhibited

Accessory proteins Regulation by RNAs often requires an interaction with accessory proteins. Below, we discuss the interactions of these accessory proteins with the regulatory RNAs and the mechanisms by which they exert their effects.
Hfq. If the mRNA is controlled by a trans-acting sRNA, the RNA chaperone protein Hfq is often required to facilitate a stable trans-acting sRNAmRNA target interaction1,19,73. Hfq is especially important if the level of complementarity between the trans-acting sRNA and the target mRNA is low. Hfq displays structural homology to eukaryotic Sm proteins, which are involved in RNA degradation and splicing, and acts as a hexamer, forming a doughnut shape19. Recent data suggest that distinct parts of Hfq bind the target mRNA and the trans-acting sRNA74. However, the exact mechanism by which Hfq functions in currently unclear. Homologues of Hfq have been found in approximately half of the sequenced eubacteria examined to date. Intriguingly, Hfq has been shown to participate in virulence in many Gram-negative bacteria but is dispensable for virulence in Gram-positive species75. One exception is L. monocytogenes, in which a hfq strain displays a lower bacterial count in mice than the wildtype strain76. Hfq coordinates V. cholerae quorum sensing by facilitating the antisense interaction between the trans-acting sRNA Qrr (quorum regulatory RNA) and the hapR mRNA, which encodes a major transcriptional regulator, and this interaction allows virulence genes to be expressed at low cell density 77. Interestingly, in E. coli Hfq was recently shown to interact more frequently with the antisense strand of the protein-coding sequence than with the sense strand, indicating that it is involved in regulation mediated by both cis and trans antisense RNAs78. RNases. RNases are a class of enzymes that govern the maturation and degradation of target mRNAs and transacting sRNAs79. RNases are classified by their nature of cleavage, which can be endonucleolytic (within the transcript) or exonucleolytic (from the 5 or, most commonly, the 3 end of the transcript). RNases recognize either single-stranded or structured double-stranded RNA sequences. many recent reviews8084 have discussed RNases, so this Review describes only a few RNases involved in virulence. RNase e is a central endonuclease in Gram-negative bacteria that recognizes AU-rich segments as cleavage sites. It is also the major ribonuclease in the in vivo degradosome, which also contains enolase (a glycolytic enzyme), PNPase (polynucleotide phosphorylase) and RhlB (an RNA helicase)81,85. The degradosome components might vary depending on growth conditions,
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Translation initiated

b
ompA Translation 5 inhibited 3 VrrA 3 5 OmpA

Vesicle production Virulence

Figure 4 | Trans-acting rNAs. a | Expression of listeriolysin regulatory protein (PrfA) in Listeria monocytogenes is controlled by temperature and by the trans-acting riboswitch S-adenosylmethionine (SAM) riboswitch element A (SreA; 227 nucleotides). The prfA untranslated region (UTR) forms a closed stem loop structure at temperatures below Nature Reviews | Microbiology 30 C, masking the ShineDalgarno (SD) sequence. At 37 C, the closed stem loop structure is unstable, allowing the ribosome to bind to the SD and initiate translation. SreA can function in trans by binding to the 5 UTR of prfA. The SreAprfA UTR interaction leads to decreased expression of PrfA. b | The trans-acting small non-coding RNA VrrA (Vibrio regulatory RNA of OmpA) negatively regulates the expression of outer-membrane protein A (OmpA) in Vibrio cholerae. VrrA binds the ompA mRNA, thereby blocking ribosome access to the SD sequence. The reduction of OmpA protein levels leads to increased release of outer-membrane vesicles and decreased virulence in an infant-mouse infection model.

Degradosome
A complex of several proteins involved in the degradation and processing of various transcripts in Gram-negative bacteria.

So far, 36 trans-acting sRNAs have been identified in V. cholerae67, at least nine of which are involved in quorum sensing and biofilm formation6870. The recently discovered trans-acting sRNA vrrA (Vibrio regulatory RNA of OmpA) is 140 nucleotides long and is conserved among the Vibrio species48. expression of vrrA is regulated by e, an alternative sigma factor involved in the response to extracytoplasmic, high-temperature and oxidative stress conditions. vrrA acts as a repressor of outermembrane protein A (OmpA) synthesis. On ultraviolet irradiation, vrrA levels increase, repressing the expression of OmpA by directly base-pairing with the ompA mRNA and blocking its SD sequence. Indeed, an ompA mutant showed increased survival during ultraviolet irradiation compared with survival of the wild-type strain. This is because reduction of OmpA levels leads to increased production of outer-membrane vesicles (FIG. 4b), which have been proposed to physically protect the bacterium against ultraviolet light-induced damage. The absence of vrrA increases V. cholerae virulence in mice through an unknown mechanism, possibly owing to the increased expression of OmpA and/or the reduced production of outer-membrane vesicles. Another outer-membrane protein, OmpT, which is known to be involved in V. cholerae bile resistance, has recently been identified as the second target of vrrA71,72. vrrA directly base-pairs with the 5

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and the activity of the degradosome is modulated by regulator of ribosome activity A (RraA)86,87. RNase e is thought to preferentially bind monophosphorylated rather than triphosphorylated 5 ends, although this idea was recently challenged8892. In most cases, RNase e and Hfq act together to induce the degradation of trans-acting sRNAmRNA target complexes and have been shown to interact directly with trans-acting sRNAs in a complex that eventually degrades target mRNAs93. However, RNase e has been shown to compete with Hfq for some AU-rich single-stranded sequences, indicating that Hfq can sometimes protect target mRNAs and transacting sRNAs from endonucleolytic cleavage94. RNase e also controls the degradation of the AmgRmgtCBR cis-acting antisense RNAtarget mRNA complex in a mechanism that is independent of RNase III and Hfq42. RNase e-mediated processing is important for the proper stoichiometric expression of different ORFs in polycistronic mRNAs. One such example is observed with the expression of the pyelonephritis-associated pilus (composed of Pap proteins), which mediates binding of uropathogenic E. coli to the kidney, causing pyelonephritis95. The papBA bicistronic mRNA is processed by RNase e, which recognizes AU-rich sequences between the coding RNAs96. After processing, the transcript encoding the regulatory PapB protein is rapidly degraded, whereas papA, which encodes the major structural component of the pilus, is expressed, resulting in the formation of pili of the correct length. Another example of an RNase with a role in virulence is RNase III, which belongs to the family of double-stranded RNA-specific endoribonucleases. It cleaves phosphodiester bonds, creating 5-phosphate and 3-hydroxyl termini with overhangs of 2 nucleotides97. Trans-acting sRNA-mediated gene regulation has been shown to depend on RNase III activity in many cases. One such example is S. aureus RNAIII (see above), which requires RNase III to degrade the RNAIIItarget mRNA duplexes98. Although most bacterial exoribonucleases process RNA from the 3 to the 5 end, RNase J1 and RNase J2 in B. subtilis possess 5-to-3 exonuclease activity as well as endonucleolytic activity 99. RNase J1 and RNase J2 homologues are conserved in many Gram-positive bacteria and seem to function similarly to the RNase e of Gram-negative bacteria. As with RNase e, RNase J1 and RNase J2 are specific for AU-rich single-stranded RNA segments and show preference for monophosphorylated 5 ends100,101. Although RNase J1 and RNase J2 can work in a complex, only RNase J1 is essential for growth in B. subtilis 102; by contrast, both are essential for growth in Streptococcus pyogenes 103. Targets of RNase J1 and RNase J2 in S. pyogenes include the virulence factors hasA (which encodes hyaluronate synthase A, involved in capsule synthesis), sagA (which encodes streptolysin S) and streptodornase (sda; which encodes a DNase)103. Another endonucleolytic RNase in Gram-positive bacteria is RNase Y (encoded by ymdA), which targets, among other transcripts, SAm riboswitches in B. subti lis104. Interestingly, RNase Y of B. subtilis has been shown
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to associate with the RNA helicase cshA, enolase, PNPase and RNase J1, possibly forming a degradosome-like complex similar to the RNase e degradosome in E. coli 105,106. The RNase Y orthologue of S. pyogenes, cvfA, is induced in the absence of carbohydrates and downregulates genes involved in metabolism and transport. Strains of S. pyogenes and S. aureus lacking cvfA show attenuated virulence in mice and silkworms. Transcriptomics data revealed that cvfA controls the expression of the S. pyo genes virulence factors speB (encoding streptopain, a cysteine protease that is involved in the degradation of host cellular proteins) and sagA (which is important for rupturing host cells)107,108.

Concluding remarks The recent advances in detailed RNA expression studies carried out on a global scale have given us a vast amount of information about how RNA regulation is exerted in bacterial pathogens. what has become clear is that the activity and stability of an mRNA (or an operon) can be controlled by many factors. These regulatory factors might be part of the transcript itself (the 5 or 3 end) and can directly sense environmental cues such as temperature, pH and the concentration of specific metabolites. They might also function in an antisense manner, being true antisense transcripts (complementary to the coding RNA, like AmgR) or being expressed from locations that are distal to target mRNAs (like RNAIII and vrrA). These regulatory factors may interact with proteins such as Hfq to ultimately control the stability and processing of target transcripts. One advantage of regulation by RNA structures is the speed at which it can occur. As illustrated by the pH-mediated regulation of alx, the speed of transcription generating alternative RNA secondary structures probably applies for most 5 UTR-mediated regulation. Similarly, with the help of the chaperone Hfq, transacting sRNAs can be exposed to the correct target mRNA, allowing the interaction to occur; this can also be achieved by transcript maturation, which allows exposure of the correct sequence to the transacting sRNA. The speed of the interactions, along with the ability to react to environmental cues and for the mRNA to be degraded as soon as the reaction is complete, makes regulation by RNA structures ideal for pathogenic bacteria. These bacteria encounter many different, often hostile, environments (for example, the intestine, the blood and the phagosome) during the course of infection. This Review shows that we have started to grasp the role of RNAs during infection, although many questions need to be addressed. Are the regulatory RNAs expressed in the whole population at the same time or are they only expressed in some bacteria? This is particularly interesting for cis-acting antisense RNAs and their targets on the opposite strand because it might explain stochastic gene expression in a population (that is, expression of a particular gene in only a subset of a bacterial population). At what stages during infection are the regulatory RNAs active and expressed? can regulatory RNAs interact more directly with the host (can they be secreted and function as microRNAs, downregulating host RNAs)?
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It is also possible that the information we obtain on RNA regulation could be used to develop new methods of treatment. Different analogues of riboswitchbinding metabolites have already proved successful in reducing the expression of bacterial growth genes and decreasing pathogenesis in mice (for example, during S. aureus infection)109,110. Similar unbiased chemical biology experiments (blocking gene or protein function with libraries of chemical compounds) could be used to inhibit the function of certain 5 UTR elements, making them inactive and thereby preventing the expression of the downstream gene product.

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Acknowledgements

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J.G., S.N. and E.L. are supported by the J. C. Kempe foundation. A.T.-A. has a JAE-DOC research contract from Consejo Superior de Investigaciones Cientficas (CSIC; the Spanish National Research Council). J.J. is supported by Ume University, Sweden, by the Swedish Research Council grants 2008-58X-15144-05-3 and 621-2009-5677, and by European Research Council Starting Grant number 260764. We apologize to colleagues whose work could not be cited owing to space limitations.

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Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATION
Jrgen Johanssons homepage: http://www.molbiol.umu.se/ english/research/researchers/jorgen-johansson
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