Small noncoding RNAs controlling pathogenesis Alejandro Toledo-Arana, Francis Repoila and Pascale Cossart

Infectious diseases are a leading cause of mortality worldwide. biological processes, affecting all steps of gene expression A major challenge in achieving their eradication is a better [7]. In bacteria, sRNAs mostly function as coordinators understanding of bacterial pathogenesis processes. The recent of adaptation processes in response to environmental discovery of small noncoding RNAs (sRNAs) as modulators of changes, integrating environmental signals and control- gene expression in response to environmental cues has ling target gene expression [10–12]. Usually, sRNAs brought a new insight into bacterial regulation. sRNAs regulate gene expression either by pairing to mRNAs coordinate complex networks of stress adaptation and and affecting their stability and/or translation or by bind- virulence gene expression. sRNAs generally ensure such a ing to proteins and modifying their activity [7]. Here, we regulation by pairing to mRNAs of effector and/or regulatory focus on noncoding RNAs encoded by bacterial genomes genes, or by binding to proteins. An updated view on bacterial and demonstrated to regulate pathogenesis (Table 1)[1]. models responsible for important infections illustrates the key Other regulatory RNA elements, such as untranslated role of sRNAs in the control of pathogenesis mRNA regions or plasmidic sRNAs, have been reviewed Addresses elsewhere [3,5 ,7 ,13–15]. Institut Pasteur, Unite´ des Interactions Bacte´ ries-Cellules; INSERM, U604; and INRA, USC2020, Paris, F-75015, France RNAIII of Staphylococcus aureus, a paradigm for an sRNA controlling Corresponding author: Cossart, Pascale ([email protected]) expression of virulence factors Staphylococcus aureus is one of the most common causes of Current Opinion in Microbiology 2007, 10:182–188 nosocomial infections and toxin-mediated diseases. It expresses a large number of virulence factors, including This review comes from a themed issue on Cell regulation toxins, exoenzymes and extracellular matrix-binding sur- Edited by Gisela Storz and Dieter Haas face proteins. Numerous regulatory systems control the expression of these virulence factors [16]. Among these, Available online 23rd March 2007 the agr system seems to be the master regulator of S. aureus 1369-5274/$ – see front matter virulence. agr consists of two divergent transcription units, # 2006 Elsevier Ltd. All rights reserved. RNAII and RNAIII. RNAII encodes a typical two- component system (AgrA, the response regulator, and DOI 10.1016/j.mib.2007.03.004 AgrC, the sensor kinase), a propeptide AgrD and a pepti- dase AgrB. AgrB processes AgrD into a small autoinducing peptide, which binds to and activates AgrC. AgrA, AgrB, Introduction AgrC and AgrD constitute a quorum-sensing system During infection, pathogenic bacteria must be able to (QSS), with the higher concentration of the autoinducing express their virulence genes properly, and to survive peptide inducing the transcription of the divergent unit — in the environmental conditions imposed by their hosts. that is, RNAIII, the effector molecule of the agr system In general, the coordinated expression of genes involved in (Figure 1)[16]. RNAIII was the first regulatory sRNA virulence and adaptation to environmental cues is under discovered to be involved in bacterial pathogenesis [17]. the control of common regulatory cascades. Numerous RNAIII is a 514 nucleotide (nt) transcript folded into 14 proteins are involved in these regulatory pathways [1]. stem–loop structures, with a dual function: it encodes a 26 RNAs are also emerging as regulators, enabling the amino acid peptide, d-hemolysin (hld), and also acts as a pathogen to adapt its metabolic needs during infection regulatory sRNA controlling virulence. RNAIII is able to and to express its virulence genes when required. Among pair with at least three mRNA targets, for example hla, spa the RNA-based regulatory elements controlling pathogen- and rot mRNAs (Figure 2a,b). The 50-leader of hla mRNA, esis are riboswitches, 50-untranslated regions of mRNAs encoding a-hemolysin, can form a secondary structure that and small noncoding RNAs (sRNAs) [2,3,4,5,6]. occludes the ribosome-binding site (RBS). The 50-end of RNAIII, when binding to this folded hla mRNA structure, sRNAs — other than ribosomal RNAs (rRNAs) or transfer desequesters the RBS, thereby activating the translation of RNAs (tRNAs) — have been found in all organisms in the a-hemolysin mRNA (Figure 2a) [18]. The two other which they have been searched for (i.e. bacteria, archaea targets, spa and rot mRNA, which encode protein A and the and eukaryotes). In recent years, sRNAs have been transcription factor Rot, respectively, are negatively identified by different methods, and their number is affected by RNAIII, which pairs with and sequesters their constantly growing [7,8,9]. In addition, sRNAs have RBS [19,20]. The hybrid formed by RNAIII and spa been recognized as regulators involved in many important mRNA was shown to be a substrate for RNase III and a

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Table 1

sRNAs involved in bacterial pathogenesis.

Bacteria Diseases sRNA (nt) Target Refs Gram-positive bacteria Staphylococcus aureus Skin infections, bacteremia, endocarditis, osteomyelitis, toxin-mediated RNAIII (514) hla mRNA [18] diseases, abscesses, nosocomial infections spa mRNA [19] rot mRNA [20] SA1000 mRNA [21] Streptococcus pyogenes Pharyngitis, skin infections, acute rheumatic diseases, scarlet fever, FasX (300) ? [55] necrotizing fasciitis, glomerulonephritis Pel (459) ? [56] Clostridium perfringens Food poisoning, wound infections, gas gangrene VR RNA (400) ? [57] VirX (400) ? [58] Gram-negative bacteria Pseudomonas aeruginosa Burn and wound infections, endocarditis, cystitis, pneumonia in cystic RsmY (120) RsmA [27–29,34] fibrosis patients, septicaemia in immunocompromised patients RsmZ (119) Vibrio cholerae Cholera CsrB (417) CsrA [41] CsrC (366) CsrD (351) Qrr1 (96) hapR mRNA [40] Qrr2 (108) Qrr3 (107) Qrr4 (107) Salmonella typhimurium Gastroenteritis, enterocolitis, septicaemia in immunocompromised CsrB (350) CsrA [59] patients tmRNA (363) [60] Chlamydia trachomatis Sexually transmitted genital infections, trachoma IhtA (120) hctA mRNA? [44] target for degradation [19]. A similar situation has been present in the genome of the S. aureus N315 strain. sprD, proposed for the RNAIII–rot mRNA hybrid but further sprE, sprF and sprG sRNAs are encoded by the bacterio- investigation is required to establish fully the mode of phage fN315, also present in the N315 strain genome. action of RNAIII in this case [20]. Other mRNA targets of Interestingly, sprA, sprF and sprG are present in multiple RNAIII have been identified, including SA1000-mRNA, copies localized in other places of the genome and differ- which encodes an adhesin-like factor involved in adher- ent from SaPIn3 and fN315 [25]. sprA sRNA was shown ence and invasion of epithelial cells [21–23] (T Geissmann to pair with mRNAs encoding an ABC transporter operon, and P Romby, personal communication). including two ORFs (open reading frames; SA2216– SA2217) and a putative a-acetolactate decarboxylase Thus, RNAIII seems to be a paradigm in RNA-mediated (SA2007) [25], suggesting a possible modulation of the virulence gene expression. The agr-dependent expres- bacterial metabolism by a sRNA present in either a sion of virulence factors is subject to temporal control – pathogenicity island or a phage. However, it is not yet that is, during growth, adhesins (e.g. protein A) are known if some of the sprA–G sRNAs are involved in the produced before hemolysins (e.g. a-hemolysin and d- virulence of S. aureus. hemolysin), proteases and other degradative enzymes [16]. This sequence of events depends on various signals, RsmY and RsmZ sRNAs and the RsmA including cell density: when the cell number increases, translational repressor protein: a network the abundance of RNAIII also increases, resulting in controlling Pseudomonas aeruginosa decreased expression of adhesins (e.g. spa mRNA) and pathogenesis activation of translation of hemolysins (e.g. a-hemolysin Pseudomonas aeruginosa is a ubiquitous saprophyte and an and d-hemolysin). At the same time, through its trans- opportunistic human pathogen that causes severe infec- lation control of the transcription regulator Rot, RNAIII tions in immunocompromised patients. It forms biofilms also modulates the transcription of other genes involved on a variety of surfaces, including the lungs of cystic in virulence and metabolic adaptation [20,24]. There- fibrosis patients [26]. P. aeruginosa pathogenesis relies fore, the integration of the cell density signal by RNAIII mainly on the tightly regulated expression of a type III is a key process to regulate the expression of virulence secretion system, on biofilm (adherence) properties and traits in S. aureus in a time-dependent manner. on a large number of N-acyl-homoserine lactone- regulated extracellular toxins and secondary metabolites. Besides RNAIII, eleven other sRNAs have been ident- This control is based on numerous interlinked systems, ified in S. aureus. Among those, sprA, sprB and sprC are including the GacS-GacA–RsmY–Z-RsmA pathways localized in the pathogenicity island, SaPIn3, which is [27–30]. The GacS-GacA–RsmY–Z-RsmA network is www.sciencedirect.com Current Opinion in Microbiology 2007, 10:182–188 184 Cell regulation

Figure 1

Regulatory pathways involving sRNAs controlling virulence in bacteria responsible for prevalent worldwide infectious diseases. The sRNAs in red act by base-pairing mechanisms; the sRNAs in blue sequester regulatory proteins. Dashed lines correspond to possible pathways (see text). The question mark (?) signifies an unknown environmental signal (for references, see text). Abbreviation: TTSS, type III secretion system. homologous to the Escherichia coli BarA-UvrY–CsrB–C- these sRNAs is to inhibit the activity of their target CsrA CsrA system, which is conserved in many bacterial protein (Figure 2c). CsrA — the carbon storage regulator species. E. coli CsrB–C are sRNAs belonging to the — is a translational regulator of diverse bacterial processes carbon storage regulatory system. The main function of including carbon metabolism and biofilm formation

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Figure 2

Mechanisms of action of sRNAs controlling virulence. (a) Translation activation by sRNA–mRNA hybrid formation. Stem–loop structure blocks the translation of the hla mRNA by occluding the RBS. RNAIII hybridizes to the anti-RBS region of the mRNA, desequestering the RBS [18]. (b) Translation repression by sRNA–mRNA hybrid formation. The sRNA hybridizes to the RBS region, repressing translation by occluding the RBS and promoting mRNA degradation [19,20] (c) Translation modulation by protein sequestration. CsrA (or RsmA) family proteins are translation regulators binding to the RBS region of mRNAs. Csr (or Rsm) sRNAs have several stem–loop structures mimicking the RBS regions recognized by CsrA (or RsmA) proteins. Csr (or Rsm) sRNAs sequester and antagonize CsrA (or RsmA) proteins [31].

[5,31–33]. In P. aeruginosa, the GacS-GacA two- tration (Figure 2c). RsmA is a translation regulator affect- component system positively controls the expression of ing positively or negatively the expression of a variety of two sRNAs, RsmY and RsmZ, which are homologous to genes important for virulence and survival, such as those CsrB–C sRNAs [28,34]. RsmZ is also under control of required for biofilm formation, carbon metabolism, LadS and RetS, two sensor proteins possibly acting posi- quorum sensing and type III secretion (Figure 1) tively and negatively on GacA, respectively (Figure 1) [27,34,35]. [29]. The RsmY and RsmZ sRNAs bind to RsmA (repres- sor of secondary metabolites), a homologue of the RNA- Using a bioinformatics search, 19 other sRNAs have been binding protein CsrA, and prevent its activity by seques- described in P. aeruginosa [9,36]. Among them, the two Fur- www.sciencedirect.com Current Opinion in Microbiology 2007, 10:182–188 186 Cell regulation

repressed sRNAs, PrrF1 and PrrF2, homologous to the The IhtA sRNA of Chlamydia, RyhB sRNA of E. coli, were shown to be involved in iron a developmental cycle switcher homeostasis and oxidative stress resistance, suggesting a Chlamydia trachomatis, is an obligate intracellular possible role for these sRNAs during infection [36,37]. pathogen, and the leading cause of blindness and sexually transmitted urogenital infections [42]. The chlamydial Vibrio cholerae Qrr1–4 and CsrBCD sRNAs: life cycle is controlled by two histone-like proteins, Hc1 integrators of quorum-sensing regulated and Hc2, expressed only during the late stages of the networks cycle. Their expression is concomitant with the differen- Vibrio cholerae inhabits different aquatic niches, in sym- tiation of reticulate bodies (RBs) into elementary bodies biotic and/or commensal association with phytoplankton (EBs), the infectious but transcriptionally and translation- and zooplankton, respectively. V. cholerae is also able to ally inactive form of the bacteria. Hc1 and Hc2 expression colonize the human small intestine, following ingestion and activity are controlled at different levels [43,44]. of contaminated food or water, and produces cholera Using a heterologous system, the sRNA IhtA was found to toxin responsible for cholera, a diarrhoeal disease that repress the translation of hctA mRNA without affecting its cankillanadultwithin24hours[38]. To adapt to stability. When C. trachomatis undergoes EB to RB differ- different environmental cues and survive, V. cholerae entiation, after bacterial entry into cells, the abundance of has multiple QSSs and at least seven sRNAs controlling IhtA sRNA increases, Hc1 synthesis stops and the global biofilm formation and virulence (Figure 1)[39,40,41]. Hc1 level decreases, probably owing to degradation [44]. Two QSSs, composed of the sensor proteins CqsS and When RB differentiates into EB, IhtA transcription LuxP–Q, responding, respectively, to the autoinducers decreases and Hc1 synthesis takes place. Therefore, IhtA CAI-1 and AI-2, transmit the cell density signal to the acts indirectly as a global transcription activator by imped- response regulator LuxO through the common relay ing the synthesis of the histone Hc1, and thereby avoiding protein LuxU. Subsequently, the activated form of chromatin condensation during the replicative stage of C. LuxO, together with the alternative sigma factor s54, trachomatis (Figure 1). How IhtA expression itself is triggers the transcription of four sRNAs, Qrr1, Qrr2, Qrr3 regulated during the RB–EB switch remains a challen- and Qrr4. Using the RNA chaperone protein Hfq, the ging issue. Qrr1–4 sRNAs bind to the RBS of hapR mRNA, targeting the messenger for degradation and leading to a decreased Is Hfq always involved in pathogenesis? levelofHapR,themasterregulatorofbiofilmandviru- Hfq is a hexameric RNA-binding protein highly con- lence genes (Figure 1)[40]. The apparent redundancy of served in many bacterial genomes, homologous to the four sRNAs (Qrr1–4) acting on the same regulator eukaryotic Sm-like protein involved in RNA splicing [45]. (HapR) is assumed to reflect the integration of different Hfq acts as an RNA chaperone modulating translation of environmental cues [40]. many mRNAs and the stability of sRNA–mRNA hybrids in E. coli [12,46,47]. It has also been shown that Hfq Recently, VarS-VarA (a third phosphorelay system), interacts with the RNA polymerase (RNAP) in the pre- initially identified for its role in V. cholerae pathogenesis, sence of the ribosomal protein S1 [48]. Although, Hfq is was shown also to control LuxO through another sRNA- reported to have, within this complex, an ATPase activity dependent mechanism [41]. VarS-VarA is homologous [48], more studies are necessary to understand the to and functions in the same fashion as the GacS-GacA implications of this activity in the Hfq function. The and BarA-UvrY systems from Pseudomonas species and E. presence of Hfq, in both the sRNA–mRNA complex and coli, respectively (see above). VarS-VarA activates the RNAP–S1 complex, suggests that Hfq might couple transcription of the sRNAs CsrB, CsrC and CsrD, which transcription and sRNA-dependent translation, or have bind to and sequester the CsrA protein, resulting in the another sRNA-independent function within the RNAP– indirect activation of luxO expression (Figure 1, 2c) S1 complex. Several hfq mutants in pathogenic bacteria, [41,33]. including P. aeruginosa, V. cholerae, Brucella abortus, Lis- teria monocytogenes, Legionella pneumophila, are affected in Finally, additional evidence exists for the presence of a virulence [49–53]. In V. cholerae, this phenotype is due, at fourth QSS, also acting through LuxU (Figure 1) [41]. least in part, to the involvement of Hfq in the pairing of How this regulatory cascade is integrated into the Qrr1–4 sRNAs with hapR mRNA (see earlier) [40]. In L. complex regulation of V. cholerae pathogenesis is still monocytogenes, 15 sRNAs have been identified and three of unknown. these coimmunoprecipitate with Hfq [22,54]. Whether these sRNAs are associated with the hfq defective viru- The fact that V. cholerae involves at least seven sRNAs lence phenotype is unknown. It therefore seems that Hfq controlling the master regulator HapR is a clear example might have several functions that remain to be clarified. It of the important role of sRNAs in the integration of cannot be ruled out that Hfq in pathogenic species could environmental signals to regulate adaptation processes affect virulence independently of an interaction with and pathogenesis. sRNAs.

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Conclusions 7. Storz G, Altuvia S, Wassarman KM: An abundance of RNA regulators. Annu Rev Biochem 2005, 74:199-217. sRNAs are key components of regulatory cascades, coor- This review provides an insight into the modes of action of sRNAs in dinating the expression of virulence genes in response to bacteria. It also abundantly illustrates that these are similar to eukaryotic sRNA-dependent mechanisms. environmental or other changes. These sRNAs function either directly on virulence genes (i.e. RNAIII and spa 8. Vogel J, Sharma CM: How to find small non-coding RNAs in bacteria. Biol Chem 2005, 386:1219-1238. and hla mRNAs) and/or on regulators of virulence genes This is an in-depth review outlining the advantages and limitations of (i.e. Qrr and hapR mRNA). They are able to adapt the different methods to identify sRNAs. expression of virulence genes to stress and metabolic 9. 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Mol Microbiol 2003, 48:855-861. affect virulence and/or the metabolic adaptations 12. Gottesman S: The small RNA regulators of Escherichia coli: required during the infection? Which regulatory cascades roles and mechanisms. Annu Rev Microbiol 2004, 58:303-328. are they involved in, and which signals trigger them? Are 13. Wagner EG, Altuvia S, Romby P: Antisense RNAs in bacteria and sRNAs redundant in some cases? Answers to these ques- their genetic elements. Adv Genet 2002, 46:361-398. tions will probably be found soon. Such answers could 14. Brantl S: Regulatory mechanisms employed by cis-encoded introduce new ways of conceiving and generating drugs antisense RNAs. Curr Opin Microbiol 2007, 10:102-109. that target bacterial signaling or essential metabolic path- 15. Weaver K: Emerging plasmid-encoded antisense RNA regulated systems. Curr Opin Microbiol 2007, 10:110-116. ways during infection. 16. 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