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The role of RNAs in the regulation of virulence-gene expression
The role of RNAs in the regulation of virulence-gene expression Pascale Romby1, Franc¸ois Vandenesch2 and E Gerhart H Wagner3 Bacterial pathogens sense their environment, and in response, virulence genes are induced or repressed through spatial and temporal regulation. They are also subjected to stress conditions, which require appropriate responses. Recent research has revealed that RNAs are key regulators in pathogens. Small RNAs regulate the translation and/or stability of mRNAs that encode virulence proteins, or proteins with roles in adaptive responses, which are triggered by environmental cues and stresses. In most cases, these small RNAs act directly on target RNAs by an antisense mechanism. Other small RNAs act indirectly, by sequestration of regulatory proteins. Direct sensing of environmental signals can occur through induced structural changes in mRNAs.
pathogenic bacteria? The majority of E. coli sRNAs are present in pathogenic strains; this and the known involvement of sRNAs in stress-response regulation suggests they might be important for virulence. Recent genome searches have identified sRNAs in other pathogens (Table 1) such as Staphylococcus aureus [7], Pseudomonas aeruginosa [8] and Vibrio cholerae [9,10]. These sRNAs directly or indirectly regulate virulence genes, or affect adaptive stress-responses, which are important for bacterial survival in a host. This review focuses on recently discovered pathogenesis-related RNAs and their mechanisms of action.
Addresses 1 UPR 9002 CNRS, Institut de Biologie Mole´culaire et Cellulaire, 15 rue Rene´ Descartes, 67084 Strasbourg Cedex, France 2 Faculte´ de Me´decine Laennec, National Reference Center for Staphylococci, INSERM E0230, IFR62, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France 3 Institute of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, 751 24 Uppsala, Sweden
Regulatory RNAs and signaling pathways
Corresponding author: Romby, Pascale ([email protected])
Current Opinion in Microbiology 2006, 9:229–236 This review comes from a themed issue on Cell regulation Edited by Werner Goebel and Stephen Lory Available online 10th March 2006 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.02.005
Introduction Pathogenic bacteria inhabit numerous ecological niches and have to adapt to rapidly changing conditions. They have evolved a plethora of sensory systems that activate or repress the expression of virulence genes in response to both environmental and host signals. Much of this regulation is carried out by proteins at a transcriptional level; lately however, RNAs have emerged as major regulators of adaptive responses. It has been established that antisense RNAs regulate essential functions of plasmids in bacteria [1]. Genome-wide searches have subsequently uncovered >70 small non-coding RNAs (sRNAs), encoded by the chromosome of the enterobacterium Escherichia coli [2,3,4]. Thus, it is not surprising that RNA-mediated regulation plays key roles in virulence [5,6]. What is known about RNA-based regulators in www.sciencedirect.com
The transcription of many pathogenesis-related RNAs is dependent on growth phase. Promoters are tightly regulated, frequently as part of well-understood regulons, responding to specific signals. In several pathogens, the secretion of virulence factors is regulated by cell-density sensing (quorum sensing), a process that involves communication through secreted signaling molecules [11]. Several regulatory RNAs are the main effectors of quorum-sensing systems [9,10,12]. In V. cholerae, the sensory signals converge on a response regulatory protein, LuxO (Figure 1). At low cell density, when the autoinducer is absent, phosphorylated LuxO activates the transcription of four redundant Qrr RNAs (quorum regulatory RNAs) that regulate the mRNA of the downstream target gene hapR [10]. In S. aureus, the effector of quorum sensing is encoded by the agr system, which is composed of two divergent transcription units. The first operon combines a density-sensing cassette (agrD and agrB) and a two-component sensory transduction system (agrA and agrC), which is required for its autocatalytic activation as well as for the activation of transcription of RNAIII, the intracellular effector of the agr regulon (Figure 1) [12]. In Streptococcus pyogenes, the pathogenesis-related RNA encoded by the pel locus is part of a signal transduction cascade [13]. Transcription of pel is dependent on growth phase; the addition of conditioned media to early logarithmic cells triggers pel transcription. The CsrA-binding RNAs are also positively regulated by two-component signal transduction systems (for examples, see [9,14–16]). Direct binding of the response regulator to the csrB promoter was demonstrated in S. typhymurium [17]. The elusive signal to which the sensor kinase responds must be dependent on growth phase, because the concentration of these RNAs reaches a peak as the cells enter stationary phase. Current Opinion in Microbiology 2006, 9:229–236
230 Cell regulation
Table 1 Several trans-acting RNAs as mediators of virulence sRNA
Function
Regulatory mechanism
References
ompA ompC* ompF*
Porines/outer membrane proteins
Inhibition of translation and mRNA degradation
[27,28] [30] [29]
OmrA-OmrB
ompT, cirA, fecA, fepA
Outer membrane proteins
Inhibition of translation and mRNA degradation
[31]
RyhB
Iron metabolism
Inhibition of translation and mRNA degradation
[23,24]
IstR
sodB*, acnA, sdhD, fumA, bfr, ftn tisAB*
Antitoxin/toxin system
Inhibition of translation and target cleavage
[46]
SgrS
ptsG*
Glucose transport
[35,36]
gadX Hc1
Acid response Nucleoid structure
Inhibition of translation and mRNA degradation mRNA stabilization mRNA translation ?
[38] [47]
MicA MicC MicF
GadY LhtA
Bacteria E. coli
Chlamydia trachomatis
Known targets *
RatA
Bacillus subtilis
txpA
Antitoxin/toxin system
mRNA degradation
[37]
PrrF1 or PrrF2
P. aeruginosa
sodB sdhD bfr
Iron metabolism
Inhibition of translation and mRNA degradation
[8]
Qrr1–Qrr4 sRNA
V. cholerae
hapR
Virulence
Inhibition of translation and mRNA degradation
[10]
Vibrio harveyi
luxR
Bioluminescence
S. aureus
hla
Hemolysin synthesis
Activation of translation
[12,32]
Host–pathogen interaction
Inhibition of translation and mRNA degradation
[34]
sa1000y
Virulence
Inhibition of translation and mRNA degradation
–
?
Virulence?
?
[7]
RNAIII
spa
SprA-SprG FasX
S. pyogenes
Pel VR RNA
Clostridium perfringens
VirX
*
?
Virulence (secreted factors)
?
[48]
?
Virulence (protease, M protein, streptokinase)
?
[13]
?
Virulence (secreted toxins)
?
[49]
?
Virulence (secreted toxins)
?
[50]
CsrB/CsrC
E. coli
CsrA protein
Glycogen biosynthesis, biofilm formation, hostbacteria interaction
Protein sequestration
[15,51]
CsrBz
Salmonella typhymurium
CsrA protein
Virulence
Protein sequestration
[52]
RsmZ/RsmY/RsmX
Pseudomonas fluorescens
RsmA protein
Exoenzymes, secondary metabolites
Protein sequestration
[14,53]
RsmZ/RsmB
P. aeruginosa
RsmA protein
Elastase, pyocyanin, secondary metabolites
Protein sequestration
[16,26]
CsrB/CsrC/CsrD
V. cholerae
CsrA protein
Virulence
Protein sequestration
[9]
The interaction between mRNA and sRNA has been shown experimentally. ?, Unknown mechanism or target. y SA1000, an adhesin-like factor has been recently demonstrated as a new target of RNAIII (Romby, Vandenesch et al., unpublished). z RNAs with a similar function to CsrB were also predicted in genomes of other pathogenic bacteria such as Yersinia pestis, and S. flexneri [54].
*
Current Opinion in Microbiology 2006, 9:229–236
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The role of RNAs in the regulation of virulence-gene expression Romby, Vandenesch and Wagner 231
Figure 1
sRNAs in regulatory cascades and connections between signals and global regulators. (a) The global transcriptional regulators Fur and SgrR sense signals such as intracellular iron concentration and the accumulation of glucose phosphate [2], respectively, and regulate the transcription of many target genes, in part through controlling transcription of sRNAs. Iron limitation causes inactivation of Fur protein, and consequently derepression of RyhB synthesis. (b) Schematic models of S. aureus and V. cholerae quorum-sensing circuits, adapted from [12] and [9], respectively. Activation is denoted by arrows and repression by lines. Abbreviation: AIP, autoinducer peptide.
Diversity in structure and mechanism of action Work on E. coli and other bacteria has shown that in these bacteria, sRNAs are present in abundance and, according to functional analysis, are genuine regulators (Table 1). These sRNAs are highly divergent in structure and size (Figure 2). The determinants required for regulation cannot usually be predicted from structure alone. Most sRNAs studied to date act using antisense mechanisms, which involve base-pairing to target RNAs in order to affect post-transcriptional upregulation or downregulation of gene expression (Table 1, Figure 2a). Mechanistically, binding to a target often results in translational inhibition and/or facilitated mRNA decay; however, sometimes antisense RNAs promote the conversion of a translationally inert mRNA conformation to an active conformation [3,12]. A second mechanism of control involves sequestration of regulatory proteins such as E. coli CsrA and its homologs which are present in other pathogenic bacteria (Table 1, Figure 2c). In this mechanism, out-titration of CsrA by the CsrB RNA modifies the expression of genes that are regulated by this translational repressor. Structural elements in mRNAs are used as sensors and regulatory switches. Sensing could be www.sciencedirect.com
dependent on temperature, for example, which might induce the formation of a translation-competent mRNA structure (Figure 2b). Metabolites could also bind to nascent mRNAs, thereby inducing formation of either a terminator or a translationally inactive state — these cases are known as riboswitches [18,19]. Thus, riboregulators are related in general function but display a great diversity in size, structure and mechanism of action.
Regulating the regulators Regulatory RNAs can affect many downstream genes; they can initiate regulatory cascades to modulate the expression of multiple genes involved in or required for pathogenesis. This can be demonstrated in several scenarios. The first scenario is in Listeria monocytogenes, which utilizes mRNA thermo-sensors to detect its presence in a warm-blooded host and turn on virulence functions (Figure 2b). The transcriptional regulator PrfA (the activator of several virulence genes), is thermo-regulated at the translational level [20]. Temperature change is detected by a structural switch in the 50 leader of the prfA mRNA. At low temperature ribosomes cannot initiate translation as a result of a ‘closed’ ribosomal binding site (RBS) structure and degradation of the translation-incompetent mRNA ensues. Current Opinion in Microbiology 2006, 9:229–236
232 Cell regulation
Figure 2
RNA-mediated mechanisms used in virulence gene regulation. (a) sRNAs that directly target mRNAs. (Left) The mechanism of action of S. aureus RNAIII on spa expression. The region complementary to the RBS of spa mRNA is shown by the black boxes [34]. The initial contacts involve a loop–loop interaction. (Right) The predicted secondary structure of V. cholerae Qrr1 RNA [10], with nucleotides complementary to the RBS of hapR mRNA framed. Repression of hapR mRNA is Hfq-dependent and involves mRNA degradation, probably mediated by RNase E. (b) Cis-acting elements in mRNAs. A riboswitch can adopt alternative structures, depending on changes in temperature or metabolite concentration. Conformational switches modulate the accessibility of the RBS to either repress or activate translation. (c) An example of an sRNA that targets a regulatory protein. The predicted secondary structure of V. cholerae CsrB [9]. Postulated CsrA binding motifs are shown in bold. Abbreviation; SD, Shine and Dalgarno sequence.
Increasing the temperature destabilizes the structure of the leader, permitting translation initiation (Figure 2b). Downstream gene expression, thus, relies on the thermo-control of one regulator gene. In the second scenario, because chromosomally encoded antisense sRNAs usually form only a limited number of basepairs with target RNAs, one single sRNA is able to bind several different targets (Table 1). An example of stress-response regulation that should affect virulence is the regulation of iron acquisition and storage (Figure 1). In E. coli, the sRNA RyhB [21] — which has homologs in S. typhymurium, Klebsiella pneumoniae, Yersinia pestis, V. cholerae [21] and Shigella flexnerii [22] — and two functionally related sRNAs from P. aeruginosa [8], PrrF1 and PrrF2, are controlled by the transcriptional repressor Fur (ferric uptake regulator). During iron limitation, de-repression of E. coli RyhB affects the expression of at least 18 operons, mainly encoding iron-binding proteins, thus achieving Current Opinion in Microbiology 2006, 9:229–236
coordinated regulation of this class of target genes [23]. RyhB is an antisense RNA that binds to the RBSs of several mRNAs, such as to sodB [21], facilitated by the Hfq (host factor for phage Qb) protein [24]. This blocks translation of sodB and triggers rapid RNase E-dependent degradation of both RyhB and sodB mRNA [21]. This coupled degradation might result in rapid depletion of the sRNA pool when de novo synthesis of the sRNA is shut off. This ‘limited complementarity’ scenario not only permits single sRNAs to act on multiple targets but also permits several sRNAs — induced under different conditions — to act on a single target. This allows integration of different environmental signals and is exemplified by DsrA, RprA and OxyS, three differentially induced stress-response sRNAs the regulatory activities of which converge on rpoS (stress/stationary phase Sigma factor) in E. coli and in S. typhymurium [3]. The apparent redundancy of some regulatory RNAs (Table 1) might be essential for the finetuning of regulatory switches [10]. www.sciencedirect.com
The role of RNAs in the regulation of virulence-gene expression Romby, Vandenesch and Wagner 233
The third scenario involves RNAs that indirectly regulate virulence gene expression by targeting regulatory proteins (Table 1). Here, the sRNA structurally mimics natural substrates of the regulatory protein [25]. V. cholerae uses a quorum-sensing cascade that involves at least three parallel signal transduction pathways and seven sRNAs, four of which are Qrr RNAs and three of which are Csr RNAs (Table 1). Three of these sRNAs, CsrB, CsrC and CsrD, act on the global regulatory protein CsrA [9]. As in E. coli, CsrA binds to these RNAs through multiple AGGA motifs (Figure 2c). As a result of sequestration, the normal target mRNAs of CsrA are upregulated. This also indirectly upregulates Qrr1–Qrr4 RNAs, which then bind to and destabilize the hapR mRNA (Figure 1). Absence of this transcription factor results in downregulation of several proteases, upregulation of several virulence factors and in formation of biofilms [9]. Orthologs of CrsA are present in other human pathogens (Table 1); in P. aeruginosa, two similar RNAs trap the regulatory protein RsmA, resulting in increased synthesis of virulence factors [16,26].
sRNA and modulation of bacterial surface properties Several regulatory RNAs target mRNAs that encode membrane proteins (Table 1). Recent studies have shown that regulation of ompA (outer membrane protein A), previously solely attributed to Hfq–mRNA binding, is mediated by the sRNA MicA [27,28]. Interestingly, three major outer membrane protein genes, ompA, ompC and ompF, appear to be regulated by sRNAs that are induced under different stress conditions (Table 1). MicA, MicC and MicF bind to functionally equivalent regions in their respective target mRNAs in order to repress translation and to induce rapid RNase E-dependent mRNA degradation [27,28,29,30]. A recent paper reported that two additional E. coli sRNAs, OmrA and OmrB, promote the decay of several outer membrane protein mRNAs [31]. Proposed targets for the binding of these sRNAs include the protease OmpT and TonBdependent receptors that are involved in iron transport and act as phage and colicin receptors. Thus, at least five sRNAs could modulate and fine-tune the cell-surface composition and membrane properties in E. coli and related pathogens [27,31]. Similarly, S. aureus RNAIII controls the switch between the expression of surface proteins and excreted toxins — a dichotomy between colonization and pathogenicity [12]. RNAIII is a fascinating example of a multiple-target regulator of virulence. This RNA uses different structural domains to act in three roles: as an mRNA encoding for hemolysin @; as an antisense RNA for hla (hemolysin a) mRNA translational activation [32]; and as an antisense RNA for the repression of protein A synthesis (Table 1, Figure 2a). In addition, RNAIII potentially regulates the expression of >100 S. aureus genes [33]. It is not yet known whether this occurs directly through effects on www.sciencedirect.com
transcription, or indirectly through post-transcriptional regulation of transcription factors. RNAIII inhibits the synthesis of the main surface adhesin, protein A, using an antisense-RNA mechanism [34]. Binding of RNAIII sequesters the RBS of spa mRNA and induces rapid degradation by the double-strand-specific RNase III (Figure 2a). Additional surface proteins appear to be repressed by RNAIII using the same antisense-RNA mechanism (Table 1). Several sRNAs target membrane-located transporter proteins (Table 1). During sugar stress, SgrS regulates glucose transport in E. coli [35,36], and SprA in S. aureus is predicted to regulate an ABC (ATP binding cassette) transporter [7]. Thus, modulation of membrane and surface properties in bacterial pathogens by sRNAs might contribute to virulence by ensuring the entry of essential nutrients into the cell and by creating routes of escape from host-defense mechanisms, for example by changing the range of antigenic determinants presented to cells of the immune system.
Antisense mechanisms and the involvement of helper proteins sRNAs often base-pair within the 50 leaders of target mRNAs, usually sequestering RBSs; however, a few sRNAs might bind to target mRNAs near their 30 ends [7,37,38]. Often, rapid decay of the repressed mRNA is observed, but it is still debatable whether degradation is a secondary effect of inhibited translation. The halflife of bacterial mRNA is strongly affected by its association with ribosomes [39]. Thus, sRNA-mediated translational inhibition might accelerate RNase Emediated degradation of target mRNAs. Many sRNAs that form only a limited number of base pairs with target mRNAs require the hexameric Sm-like protein Hfq [40]. Hfq could affect regulation by stabilizing sRNAs, by promoting antisense–target RNA pairing or by acting in an unfolding or chaperone role. Hfq might also act as an adaptor between sRNAs (e.g. SgrS and RhyB) and RNase E to target mRNAs for rapid degradation [41]. Mutations in hfq decrease virulence in several pathogens, for example, L. monocytogenes [42], P. aeruginosa [43] and V. cholerae [44]. In Legionella pneumophila, Hfq induces slight defects in a macrophage infection model [45]. S. aureus RNAIII is recognized by Hfq in vivo and in vitro, suggesting that Hfq might also be required for full virulence [34]. Whether an Hfq requirement for virulence can be explained by direct regulatory activity of this protein, or by the Hfq-bound sRNAs, is a matter for extensive investigation. By contrast, some sRNAs that form more extensive base-pairing with targets do not require Hfq. In several of these cases, RNase III initiates the degradation of the sRNA–target duplex (Figure 2a) [34,46]. Even in this case, the relative contribution of ribosome occlusion or mRNA degradation to the efficiency of control is not well known. However, whereas these RNA duplexes are Current Opinion in Microbiology 2006, 9:229–236
234 Cell regulation
probably reversible, degradation of target mRNAs irreversibly prevents translation. Thus, the action of sRNAs at the post-transcriptional level results in instantaneous downregulation of mRNAs.
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Conclusions RNAs are essential intracellular effectors of virulence traits and are part of many regulatory networks in pathogenic bacteria. This field is still in its infancy and many questions remain to be addressed. How many riboregulators are there? Can we find rules for structure–function relationships? What is the importance of RNA-binding proteins in assisting these RNAs? Although much methodological progress has been made to identify regulatory RNAs [4], the identification of their direct targets still lags behind, as does an understanding of the roles of these RNAs in signal-transduction pathways, stress responses and in the general physiology of pathogens. To date, the known regulatory RNAs provide links among metabolism, defense mechanisms and virulence (Figure 1). Additional connections between virulence and housekeeping networks should be revealed as more is learnt about these networks. A particular challenge concerns the roles played by RNAs in response to the host environment and during the infection process. There might be many surprises waiting for us yet.
Acknowledgements We thank the laboratory members of our research groups for their discussions. PR is grateful for financial support from the Centre National de la Recherche Scientifique (CNRS), FV for support from the Institut de Recherche de la Sante´ et de la Recherche for Me´dicale (INSERM), PR and FV for grants from the Ministe`re de la Recherche (ANR), GW for grants from the Swedish Natural Science Research Council (VR), and GW and PR for support from the European Community (FOSRAK, EC005120).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Wagner EGH, Vogel J: Approaches to identify novel nonmessenger RNAs in bacteria and to investigate their biological functions: functional analysis of identified non-mRNAs. In Handbook of RNA Biochemistry. Edited by Hartmann RK, Bindereif A, Scho¨n A, Westhof E. Wiley-VCH; 2005:614-654. This is an overview of the different experimental approaches used to identify novel sRNAs in E. coli and to investigate their biological functions. This review outlines the advantages and limitations for each method, and lists useful protocols. 5.
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Current Opinion in Microbiology 2006, 9:229–236
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Lenz DH, Miller MB, Zhu J, Kulkarni RV, Bassler BL: CsrA and three redundant small RNAs regulate quorum sensing in Vibrio cholerae. Mol Microbiol 2005, 58:1186-1202. See annotation to [10].
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The role of RNAs in the regulation of virulence-gene expression Romby, Vandenesch and Wagner 235
This study shows that several sRNAs are rapidly degraded with their target mRNA after pairing. Therefore, this coupled degradation causes a rapid depletion of the sRNA pool when the synthesis of the sRNA has stopped. This, thus, allows instantaneous re-adaptation of the target mRNAs to the absence of the sRNA. 22. Oglesby AG, Murphy ER, Iyer VR, Payne SM: Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP. Mol Microbiol 2005, 58:1354-1367. 23. Masse E, Vanderpool CK, Gottesman S: Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 2005, 187:6962-6971. 24. Geissmann TA, Touati D: Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. EMBO J 2004, 23:396-405. 25. Dubey AK, Baker CS, Romeo T, Babitzke P: RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction. RNA 2005, 11:1579-1587. 26. Heurlier K, Williams F, Heeb S, Dormond C, Pessi G, Singer D, Camara M, Williams P, Haas D: Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1. J Bacteriol 2004, 186:2936-2945. 27. Udekwu KI, Darfeuille F, Vogel J, Reimega˚rd J, Holmqvist E, Wagner EGH: Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA. Genes Dev 2005, 19:2355-2366. See annotation to [28] 28. Rasmussen AA, Eriksen M, Gilany K, Udesen C, Thomas F, Petersen C, Valentin-Hansen P: Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase-dependent control. Mol Microbiol 2005, 58:1421-1429. These two studies [27,28] solved the long-standing mystery of how translation of E. coli ompA mRNA is regulated. An antisense RNA, MicA, base-pairs with the ribosome binding site of ompA mRNA, blocking ribosome binding and facilitating RNase E cleavage and subsequent mRNA decay. MicA requires Hfq for full activity. 29. Andersen J, Forst SA, Zhao K, Inouye M, Delihas N: The function of micF RNA. micF RNA is a major factor in the thermal regulation of OmpF protein in Escherichia coli. J Biol Chem 1989, 264:17961-17970. 30. Chen S, Zhang A, Blyn LB, Storz G: MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli. J Bacteriol 2004, 186:6689-6697. 31. Guillier M, Gottesman S: Remodeling of the Escherichia coli outer membrane by two small regulatory RNAs. Mol Microbiol 2006, 59:231-247. Genetic analysis, combined with the use of microarrays, revealed that the two E. coli sRNAs OmrA and OmrB repress the expression of several mRNAs encoding outer-membrane proteins. The regulatory mechanisms still remain to be solved, but the data suggest that these sRNAs induce rapid degradation of target mRNAs. Thus, like MicA, MicC, and MicF, these sRNAs modulate the cell surface composition of the bacteria upon environmental change. 32. Morfeldt E, Taylor D, von Gabain A, Arvidson S: Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J 1995, 14:4569-4577. 33. Dunman PM, Murphy E, Haney S, Palacios D, Tucker-Kellogg G, Wu S, Brown EL, Zagursky RJ, Shlaes D, Projan SJ: Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J Bacteriol 2001, 183:7341-7353. 34. Huntzinger E, Boisset S, Saveanu C, Benito Y, Geissmann T, Namane A, Lina G, Etienne J, Ehresmann B, Ehresmann C et al.: Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J 2005, 24:824-835. In this study it was shown that S. aureus RNAIII represses the synthesis of one of the major virulence factors, protein A, by an antisense mechanism. RNAIII blocks translation by base pairs with the ribosome binding site of spa mRNA and facilitates double-strand specific cleavage by RNase III, www.sciencedirect.com
thus initiating mRNA decay. RNAIII upregulates or downregulates several virulence-related mRNAs using independent regulatory domains. 35. Kawamoto H, Morita T, Shimizu A, Inada T, Aiba H: Implication of membrane localization of target mRNA in the action of a small RNA: mechanism of post-transcriptional regulation of glucose transporter in Escherichia coli. Genes Dev 2005, 19:328-338. This study shows that efficient translational repression of ptsG mRNA, encoding the major glucose transporter in E. coli, by the sRNA SgrS requires inner-membrane localization of the mRNA, promoted by anchoring through the nascent peptide. It is proposed that localization-dependent repression decreases competition between SgrS and the ribosome, possibly by affecting second rounds of translation. 36. Vanderpool CK, Gottesman S: Involvement of a novel transcriptional activator and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate phosphotransferase system. Mol Microbiol 2004, 54:1076-1089. 37. Silvaggi JM, Perkins JB, Losick R: Small untranslated RNA antitoxin in Bacillus subtilis. J Bacteriol 2005, 187:6641-6650. 38. Opdyke JA, Kang JG, Storz G: GadY, a small-RNA regulator of acid response genes in Escherichia coli. J Bacteriol 2004, 186:6698-6705. 39. Deana A, Belasco JG: Lost in translation: the influence of ribosomes on bacterial mRNA decay. Genes Dev 2005, 19:2526-2533. 40. Valentin-Hansen P, Eriksen M, Udesen C: The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol 2004, 51:1525-1533. This is a review that describes the multiple functions of the Sm-like Hfq protein addresses several unresolved questions concerning the mechanism of action of Hfq, and that discusses the molecular mechanism by which Hfq recognizes sRNAs and/or mRNAs. 41. Morita T, Maki K, Aiba H: RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev 2005, 19:2176-2186. This work shows that RNase E, through its C-terminal domain, forms ribonucleoprotein complexes with Hfq and several (antisense) E. coli sRNAs. This study provides, for the first time, mechanistic clues on how mRNA destabilization can be mediated by sRNAs, and suggests that all Hfq-dependent antisense sRNAs might work through this pathway. 42. Christiansen JK, Larsen MH, Ingmer H, Sogaard-Andersen L, Kallipolitis BH: The RNA-binding protein Hfq of Listeria monocytogenes: role in stress tolerance and virulence. J Bacteriol 2004, 186:3355-3362. 43. Sonnleitner E, Hagens S, Rosenau F, Wilhelm S, Habel A, Jager KE, Blasi U: Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb Pathog 2003, 35:217-228. 44. Ding Y, Davis BM, Waldor MK: Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression. Mol Microbiol 2004, 53:345-354. 45. McNealy TL, Forsbach-Birk V, Shi C, Marre R: The Hfq homolog in Legionella pneumophila demonstrates regulation by LetA and RpoS and interacts with the global regulator CsrA. J Bacteriol 2005, 187:1527-1532. 46. Vogel J, Argaman L, Wagner EGH, Altuvia S: The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide. Curr Biol 2004, 14:2271-2276. 47. Grieshaber NA, Grieshaber SS, Fischer ER, Hackstadt T: A small RNA inhibits translation of the histone-like protein Hc1 in Chlamydia trachomatis. Mol Microbiol 59: 541–550. 48. Kreikemeyer B, Boyle MD, Buttaro BA, Heinemann M, Podbielski A: Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule. Mol Microbiol 2001, 39:392-406. 49. Shimizu T, Yaguchi H, Ohtani K, Banu S, Hayashi H: Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Mol Microbiol 2002, 43:257-265. Current Opinion in Microbiology 2006, 9:229–236
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50. Ohtani K, Bhowmik SK, Hayashi H, Shimizu T: Identification of a novel locus that regulates expression of toxin genes in Clostridium perfringens. FEMS Microbiol Lett 2002, 209:113-118. 51. Liu Y, Cui Y, Mukherjee A, Chatterjee AK: Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites. Mol Microbiol 1998, 29:219-234. 52. Altier C, Suyemoto M, Ruiz AI, Burnham KD, Maurer R: Characterization of two novel regulatory genes affecting
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Salmonella invasion gene expression. Mol Microbiol 2000, 35:635-646. 53. Kay E, Dubuis C, Haas D: Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHAO. Proc Natl Acad Sci USA 2005, 102:17136-17141. 54. Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy SR: Rfam: an RNA family database. Nucleic Acids Res 2003, 31:439-441.
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pathogenic bacteria? The majority of E. coli sRNAs are present in pathogenic strains; this and the known involvement of sRNAs in stress-response regulation suggests they might be important for virulence. Recent genome searches have identified sRNAs in other pathogens (Table 1) such as Staphylococcus aureus [7], Pseudomonas aeruginosa [8] and Vibrio cholerae [9,10]. These sRNAs directly or indirectly regulate virulence genes, or affect adaptive stress-responses, which are important for bacterial survival in a host. This review focuses on recently discovered pathogenesis-related RNAs and their mechanisms of action.
Addresses 1 UPR 9002 CNRS, Institut de Biologie Mole´culaire et Cellulaire, 15 rue Rene´ Descartes, 67084 Strasbourg Cedex, France 2 Faculte´ de Me´decine Laennec, National Reference Center for Staphylococci, INSERM E0230, IFR62, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France 3 Institute of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, 751 24 Uppsala, Sweden
Regulatory RNAs and signaling pathways
Corresponding author: Romby, Pascale ([email protected])
Current Opinion in Microbiology 2006, 9:229–236 This review comes from a themed issue on Cell regulation Edited by Werner Goebel and Stephen Lory Available online 10th March 2006 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.02.005
Introduction Pathogenic bacteria inhabit numerous ecological niches and have to adapt to rapidly changing conditions. They have evolved a plethora of sensory systems that activate or repress the expression of virulence genes in response to both environmental and host signals. Much of this regulation is carried out by proteins at a transcriptional level; lately however, RNAs have emerged as major regulators of adaptive responses. It has been established that antisense RNAs regulate essential functions of plasmids in bacteria [1]. Genome-wide searches have subsequently uncovered >70 small non-coding RNAs (sRNAs), encoded by the chromosome of the enterobacterium Escherichia coli [2,3,4]. Thus, it is not surprising that RNA-mediated regulation plays key roles in virulence [5,6]. What is known about RNA-based regulators in www.sciencedirect.com
The transcription of many pathogenesis-related RNAs is dependent on growth phase. Promoters are tightly regulated, frequently as part of well-understood regulons, responding to specific signals. In several pathogens, the secretion of virulence factors is regulated by cell-density sensing (quorum sensing), a process that involves communication through secreted signaling molecules [11]. Several regulatory RNAs are the main effectors of quorum-sensing systems [9,10,12]. In V. cholerae, the sensory signals converge on a response regulatory protein, LuxO (Figure 1). At low cell density, when the autoinducer is absent, phosphorylated LuxO activates the transcription of four redundant Qrr RNAs (quorum regulatory RNAs) that regulate the mRNA of the downstream target gene hapR [10]. In S. aureus, the effector of quorum sensing is encoded by the agr system, which is composed of two divergent transcription units. The first operon combines a density-sensing cassette (agrD and agrB) and a two-component sensory transduction system (agrA and agrC), which is required for its autocatalytic activation as well as for the activation of transcription of RNAIII, the intracellular effector of the agr regulon (Figure 1) [12]. In Streptococcus pyogenes, the pathogenesis-related RNA encoded by the pel locus is part of a signal transduction cascade [13]. Transcription of pel is dependent on growth phase; the addition of conditioned media to early logarithmic cells triggers pel transcription. The CsrA-binding RNAs are also positively regulated by two-component signal transduction systems (for examples, see [9,14–16]). Direct binding of the response regulator to the csrB promoter was demonstrated in S. typhymurium [17]. The elusive signal to which the sensor kinase responds must be dependent on growth phase, because the concentration of these RNAs reaches a peak as the cells enter stationary phase. Current Opinion in Microbiology 2006, 9:229–236
230 Cell regulation
Table 1 Several trans-acting RNAs as mediators of virulence sRNA
Function
Regulatory mechanism
References
ompA ompC* ompF*
Porines/outer membrane proteins
Inhibition of translation and mRNA degradation
[27,28] [30] [29]
OmrA-OmrB
ompT, cirA, fecA, fepA
Outer membrane proteins
Inhibition of translation and mRNA degradation
[31]
RyhB
Iron metabolism
Inhibition of translation and mRNA degradation
[23,24]
IstR
sodB*, acnA, sdhD, fumA, bfr, ftn tisAB*
Antitoxin/toxin system
Inhibition of translation and target cleavage
[46]
SgrS
ptsG*
Glucose transport
[35,36]
gadX Hc1
Acid response Nucleoid structure
Inhibition of translation and mRNA degradation mRNA stabilization mRNA translation ?
[38] [47]
MicA MicC MicF
GadY LhtA
Bacteria E. coli
Chlamydia trachomatis
Known targets *
RatA
Bacillus subtilis
txpA
Antitoxin/toxin system
mRNA degradation
[37]
PrrF1 or PrrF2
P. aeruginosa
sodB sdhD bfr
Iron metabolism
Inhibition of translation and mRNA degradation
[8]
Qrr1–Qrr4 sRNA
V. cholerae
hapR
Virulence
Inhibition of translation and mRNA degradation
[10]
Vibrio harveyi
luxR
Bioluminescence
S. aureus
hla
Hemolysin synthesis
Activation of translation
[12,32]
Host–pathogen interaction
Inhibition of translation and mRNA degradation
[34]
sa1000y
Virulence
Inhibition of translation and mRNA degradation
–
?
Virulence?
?
[7]
RNAIII
spa
SprA-SprG FasX
S. pyogenes
Pel VR RNA
Clostridium perfringens
VirX
*
?
Virulence (secreted factors)
?
[48]
?
Virulence (protease, M protein, streptokinase)
?
[13]
?
Virulence (secreted toxins)
?
[49]
?
Virulence (secreted toxins)
?
[50]
CsrB/CsrC
E. coli
CsrA protein
Glycogen biosynthesis, biofilm formation, hostbacteria interaction
Protein sequestration
[15,51]
CsrBz
Salmonella typhymurium
CsrA protein
Virulence
Protein sequestration
[52]
RsmZ/RsmY/RsmX
Pseudomonas fluorescens
RsmA protein
Exoenzymes, secondary metabolites
Protein sequestration
[14,53]
RsmZ/RsmB
P. aeruginosa
RsmA protein
Elastase, pyocyanin, secondary metabolites
Protein sequestration
[16,26]
CsrB/CsrC/CsrD
V. cholerae
CsrA protein
Virulence
Protein sequestration
[9]
The interaction between mRNA and sRNA has been shown experimentally. ?, Unknown mechanism or target. y SA1000, an adhesin-like factor has been recently demonstrated as a new target of RNAIII (Romby, Vandenesch et al., unpublished). z RNAs with a similar function to CsrB were also predicted in genomes of other pathogenic bacteria such as Yersinia pestis, and S. flexneri [54].
*
Current Opinion in Microbiology 2006, 9:229–236
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The role of RNAs in the regulation of virulence-gene expression Romby, Vandenesch and Wagner 231
Figure 1
sRNAs in regulatory cascades and connections between signals and global regulators. (a) The global transcriptional regulators Fur and SgrR sense signals such as intracellular iron concentration and the accumulation of glucose phosphate [2], respectively, and regulate the transcription of many target genes, in part through controlling transcription of sRNAs. Iron limitation causes inactivation of Fur protein, and consequently derepression of RyhB synthesis. (b) Schematic models of S. aureus and V. cholerae quorum-sensing circuits, adapted from [12] and [9], respectively. Activation is denoted by arrows and repression by lines. Abbreviation: AIP, autoinducer peptide.
Diversity in structure and mechanism of action Work on E. coli and other bacteria has shown that in these bacteria, sRNAs are present in abundance and, according to functional analysis, are genuine regulators (Table 1). These sRNAs are highly divergent in structure and size (Figure 2). The determinants required for regulation cannot usually be predicted from structure alone. Most sRNAs studied to date act using antisense mechanisms, which involve base-pairing to target RNAs in order to affect post-transcriptional upregulation or downregulation of gene expression (Table 1, Figure 2a). Mechanistically, binding to a target often results in translational inhibition and/or facilitated mRNA decay; however, sometimes antisense RNAs promote the conversion of a translationally inert mRNA conformation to an active conformation [3,12]. A second mechanism of control involves sequestration of regulatory proteins such as E. coli CsrA and its homologs which are present in other pathogenic bacteria (Table 1, Figure 2c). In this mechanism, out-titration of CsrA by the CsrB RNA modifies the expression of genes that are regulated by this translational repressor. Structural elements in mRNAs are used as sensors and regulatory switches. Sensing could be www.sciencedirect.com
dependent on temperature, for example, which might induce the formation of a translation-competent mRNA structure (Figure 2b). Metabolites could also bind to nascent mRNAs, thereby inducing formation of either a terminator or a translationally inactive state — these cases are known as riboswitches [18,19]. Thus, riboregulators are related in general function but display a great diversity in size, structure and mechanism of action.
Regulating the regulators Regulatory RNAs can affect many downstream genes; they can initiate regulatory cascades to modulate the expression of multiple genes involved in or required for pathogenesis. This can be demonstrated in several scenarios. The first scenario is in Listeria monocytogenes, which utilizes mRNA thermo-sensors to detect its presence in a warm-blooded host and turn on virulence functions (Figure 2b). The transcriptional regulator PrfA (the activator of several virulence genes), is thermo-regulated at the translational level [20]. Temperature change is detected by a structural switch in the 50 leader of the prfA mRNA. At low temperature ribosomes cannot initiate translation as a result of a ‘closed’ ribosomal binding site (RBS) structure and degradation of the translation-incompetent mRNA ensues. Current Opinion in Microbiology 2006, 9:229–236
232 Cell regulation
Figure 2
RNA-mediated mechanisms used in virulence gene regulation. (a) sRNAs that directly target mRNAs. (Left) The mechanism of action of S. aureus RNAIII on spa expression. The region complementary to the RBS of spa mRNA is shown by the black boxes [34]. The initial contacts involve a loop–loop interaction. (Right) The predicted secondary structure of V. cholerae Qrr1 RNA [10], with nucleotides complementary to the RBS of hapR mRNA framed. Repression of hapR mRNA is Hfq-dependent and involves mRNA degradation, probably mediated by RNase E. (b) Cis-acting elements in mRNAs. A riboswitch can adopt alternative structures, depending on changes in temperature or metabolite concentration. Conformational switches modulate the accessibility of the RBS to either repress or activate translation. (c) An example of an sRNA that targets a regulatory protein. The predicted secondary structure of V. cholerae CsrB [9]. Postulated CsrA binding motifs are shown in bold. Abbreviation; SD, Shine and Dalgarno sequence.
Increasing the temperature destabilizes the structure of the leader, permitting translation initiation (Figure 2b). Downstream gene expression, thus, relies on the thermo-control of one regulator gene. In the second scenario, because chromosomally encoded antisense sRNAs usually form only a limited number of basepairs with target RNAs, one single sRNA is able to bind several different targets (Table 1). An example of stress-response regulation that should affect virulence is the regulation of iron acquisition and storage (Figure 1). In E. coli, the sRNA RyhB [21] — which has homologs in S. typhymurium, Klebsiella pneumoniae, Yersinia pestis, V. cholerae [21] and Shigella flexnerii [22] — and two functionally related sRNAs from P. aeruginosa [8], PrrF1 and PrrF2, are controlled by the transcriptional repressor Fur (ferric uptake regulator). During iron limitation, de-repression of E. coli RyhB affects the expression of at least 18 operons, mainly encoding iron-binding proteins, thus achieving Current Opinion in Microbiology 2006, 9:229–236
coordinated regulation of this class of target genes [23]. RyhB is an antisense RNA that binds to the RBSs of several mRNAs, such as to sodB [21], facilitated by the Hfq (host factor for phage Qb) protein [24]. This blocks translation of sodB and triggers rapid RNase E-dependent degradation of both RyhB and sodB mRNA [21]. This coupled degradation might result in rapid depletion of the sRNA pool when de novo synthesis of the sRNA is shut off. This ‘limited complementarity’ scenario not only permits single sRNAs to act on multiple targets but also permits several sRNAs — induced under different conditions — to act on a single target. This allows integration of different environmental signals and is exemplified by DsrA, RprA and OxyS, three differentially induced stress-response sRNAs the regulatory activities of which converge on rpoS (stress/stationary phase Sigma factor) in E. coli and in S. typhymurium [3]. The apparent redundancy of some regulatory RNAs (Table 1) might be essential for the finetuning of regulatory switches [10]. www.sciencedirect.com
The role of RNAs in the regulation of virulence-gene expression Romby, Vandenesch and Wagner 233
The third scenario involves RNAs that indirectly regulate virulence gene expression by targeting regulatory proteins (Table 1). Here, the sRNA structurally mimics natural substrates of the regulatory protein [25]. V. cholerae uses a quorum-sensing cascade that involves at least three parallel signal transduction pathways and seven sRNAs, four of which are Qrr RNAs and three of which are Csr RNAs (Table 1). Three of these sRNAs, CsrB, CsrC and CsrD, act on the global regulatory protein CsrA [9]. As in E. coli, CsrA binds to these RNAs through multiple AGGA motifs (Figure 2c). As a result of sequestration, the normal target mRNAs of CsrA are upregulated. This also indirectly upregulates Qrr1–Qrr4 RNAs, which then bind to and destabilize the hapR mRNA (Figure 1). Absence of this transcription factor results in downregulation of several proteases, upregulation of several virulence factors and in formation of biofilms [9]. Orthologs of CrsA are present in other human pathogens (Table 1); in P. aeruginosa, two similar RNAs trap the regulatory protein RsmA, resulting in increased synthesis of virulence factors [16,26].
sRNA and modulation of bacterial surface properties Several regulatory RNAs target mRNAs that encode membrane proteins (Table 1). Recent studies have shown that regulation of ompA (outer membrane protein A), previously solely attributed to Hfq–mRNA binding, is mediated by the sRNA MicA [27,28]. Interestingly, three major outer membrane protein genes, ompA, ompC and ompF, appear to be regulated by sRNAs that are induced under different stress conditions (Table 1). MicA, MicC and MicF bind to functionally equivalent regions in their respective target mRNAs in order to repress translation and to induce rapid RNase E-dependent mRNA degradation [27,28,29,30]. A recent paper reported that two additional E. coli sRNAs, OmrA and OmrB, promote the decay of several outer membrane protein mRNAs [31]. Proposed targets for the binding of these sRNAs include the protease OmpT and TonBdependent receptors that are involved in iron transport and act as phage and colicin receptors. Thus, at least five sRNAs could modulate and fine-tune the cell-surface composition and membrane properties in E. coli and related pathogens [27,31]. Similarly, S. aureus RNAIII controls the switch between the expression of surface proteins and excreted toxins — a dichotomy between colonization and pathogenicity [12]. RNAIII is a fascinating example of a multiple-target regulator of virulence. This RNA uses different structural domains to act in three roles: as an mRNA encoding for hemolysin @; as an antisense RNA for hla (hemolysin a) mRNA translational activation [32]; and as an antisense RNA for the repression of protein A synthesis (Table 1, Figure 2a). In addition, RNAIII potentially regulates the expression of >100 S. aureus genes [33]. It is not yet known whether this occurs directly through effects on www.sciencedirect.com
transcription, or indirectly through post-transcriptional regulation of transcription factors. RNAIII inhibits the synthesis of the main surface adhesin, protein A, using an antisense-RNA mechanism [34]. Binding of RNAIII sequesters the RBS of spa mRNA and induces rapid degradation by the double-strand-specific RNase III (Figure 2a). Additional surface proteins appear to be repressed by RNAIII using the same antisense-RNA mechanism (Table 1). Several sRNAs target membrane-located transporter proteins (Table 1). During sugar stress, SgrS regulates glucose transport in E. coli [35,36], and SprA in S. aureus is predicted to regulate an ABC (ATP binding cassette) transporter [7]. Thus, modulation of membrane and surface properties in bacterial pathogens by sRNAs might contribute to virulence by ensuring the entry of essential nutrients into the cell and by creating routes of escape from host-defense mechanisms, for example by changing the range of antigenic determinants presented to cells of the immune system.
Antisense mechanisms and the involvement of helper proteins sRNAs often base-pair within the 50 leaders of target mRNAs, usually sequestering RBSs; however, a few sRNAs might bind to target mRNAs near their 30 ends [7,37,38]. Often, rapid decay of the repressed mRNA is observed, but it is still debatable whether degradation is a secondary effect of inhibited translation. The halflife of bacterial mRNA is strongly affected by its association with ribosomes [39]. Thus, sRNA-mediated translational inhibition might accelerate RNase Emediated degradation of target mRNAs. Many sRNAs that form only a limited number of base pairs with target mRNAs require the hexameric Sm-like protein Hfq [40]. Hfq could affect regulation by stabilizing sRNAs, by promoting antisense–target RNA pairing or by acting in an unfolding or chaperone role. Hfq might also act as an adaptor between sRNAs (e.g. SgrS and RhyB) and RNase E to target mRNAs for rapid degradation [41]. Mutations in hfq decrease virulence in several pathogens, for example, L. monocytogenes [42], P. aeruginosa [43] and V. cholerae [44]. In Legionella pneumophila, Hfq induces slight defects in a macrophage infection model [45]. S. aureus RNAIII is recognized by Hfq in vivo and in vitro, suggesting that Hfq might also be required for full virulence [34]. Whether an Hfq requirement for virulence can be explained by direct regulatory activity of this protein, or by the Hfq-bound sRNAs, is a matter for extensive investigation. By contrast, some sRNAs that form more extensive base-pairing with targets do not require Hfq. In several of these cases, RNase III initiates the degradation of the sRNA–target duplex (Figure 2a) [34,46]. Even in this case, the relative contribution of ribosome occlusion or mRNA degradation to the efficiency of control is not well known. However, whereas these RNA duplexes are Current Opinion in Microbiology 2006, 9:229–236
234 Cell regulation
probably reversible, degradation of target mRNAs irreversibly prevents translation. Thus, the action of sRNAs at the post-transcriptional level results in instantaneous downregulation of mRNAs.
6.
Geissmann T, Possedko M, Huntzinger E, Fechter P, Ehresmann C, Romby P: Regulatory RNAs as mediators of virulence gene expression in bacteria. In Handbook of Experimental Pharmacology: RNA towards medicine. Edited by Erdmann V, Brosius J, Barciszewski J. Springer-Verlag; 2006:9-43.
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Wilderman PJ, Sowa NA, FitzGerald DJ, FitzGerald PC, Gottesman S, Ochsner UA, Vasil ML: Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc Natl Acad Sci USA 2004, 101:9792-9797.
Conclusions RNAs are essential intracellular effectors of virulence traits and are part of many regulatory networks in pathogenic bacteria. This field is still in its infancy and many questions remain to be addressed. How many riboregulators are there? Can we find rules for structure–function relationships? What is the importance of RNA-binding proteins in assisting these RNAs? Although much methodological progress has been made to identify regulatory RNAs [4], the identification of their direct targets still lags behind, as does an understanding of the roles of these RNAs in signal-transduction pathways, stress responses and in the general physiology of pathogens. To date, the known regulatory RNAs provide links among metabolism, defense mechanisms and virulence (Figure 1). Additional connections between virulence and housekeeping networks should be revealed as more is learnt about these networks. A particular challenge concerns the roles played by RNAs in response to the host environment and during the infection process. There might be many surprises waiting for us yet.
Acknowledgements We thank the laboratory members of our research groups for their discussions. PR is grateful for financial support from the Centre National de la Recherche Scientifique (CNRS), FV for support from the Institut de Recherche de la Sante´ et de la Recherche for Me´dicale (INSERM), PR and FV for grants from the Ministe`re de la Recherche (ANR), GW for grants from the Swedish Natural Science Research Council (VR), and GW and PR for support from the European Community (FOSRAK, EC005120).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Wagner EGH, Altuvia S, Romby P: Antisense RNAs in bacteria and their genetic elements. Adv Genet 2002, 46:361-398.
2. Gottesman S: Micros for microbes: non-coding regulatory RNAs in bacteria. Trends Genet 2005, 21:399-404. See annotation to [3]. 3. Storz G, Altuvia S, Wassarman KM: An abundance of RNA regulators. Annu Rev Biochem 2005, 74:199-217. These [2,3] are recent comprehensive reviews on the physiological roles of newly discovered sRNAs in E. coli and their mechanisms of action. 4.
Wagner EGH, Vogel J: Approaches to identify novel nonmessenger RNAs in bacteria and to investigate their biological functions: functional analysis of identified non-mRNAs. In Handbook of RNA Biochemistry. Edited by Hartmann RK, Bindereif A, Scho¨n A, Westhof E. Wiley-VCH; 2005:614-654. This is an overview of the different experimental approaches used to identify novel sRNAs in E. coli and to investigate their biological functions. This review outlines the advantages and limitations for each method, and lists useful protocols. 5.
Johansson J, Cossart P: RNA-mediated control of virulence gene expression in bacterial pathogens. Trends Microbiol 2003, 11:280-285.
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