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Intercellular communication in bacteria L. Caetano, M. Antunes, and Rosana B. R. Ferreira Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

Abstract Bacteria have been long considered primitive organisms, with a lifestyle focused on the survival and propagation of single cells. However, in the past few decades it became obvious that bacteria can display sophisticated group behaviors. For instance, bacteria can communicate amongst themselves and with their hosts, by producing, sensing, and responding to chemical signals. By doing so, they can sense their surroundings and adapt as to increase their chances of survival and propagation. Here, we review the discovery of ­bacterial intercellular communication, some of the signaling molecules identified to date, the role of intercellular signaling in symbiotic and pathogenic relationships between bacteria and their hosts and its implications for the development of new therapeutic strategies against human disease. Keywords: 

The discovery of bacterial intercellular communication The realization that bacteria can utilize chemical signals to communicate dates back to the 1960s and 1970s. The pivot of such discovery was the research on the regulation of competence by Streptococcus pneumoniae and bioluminescence by Vibrio fischeri. In both cases, scientists noticed that these bacterial traits were expressed only after a particular cell density had been achieved during the growth of a culture. This phenomenon was attributed to the production of signaling molecules that allowed cells within a population to communicate with each other (Tomasz et al. 1964; Tomasz 1965; Kempner & Hanson et al. 1968; Nealson, Platt & Hastings et al. 1970). This discovery has led to a hunt for the molecular details of these processes.

A linear peptide controls genetic competence in S. pneumoniae The phenomenon of bacterial competence, the capacity of bacterial cells to take up extracellular DNA and integrate it as part of their genetic material, has been studied in S. pneumoniae for several decades. In the 1960s, it was

known that the development of competence occurred at particular physiological states of a bacterial population (Hotchkiss 1954). Early studies on the subject revealed that growing cultures of S. pneumoniae showed a lag in competence during the initial stages of growth. Once the lag was overcome, a striking increase in the competence of the culture was observed (Tomasz et al. 1964; Tomasz 1965). At this time, the increase in competence occurred at a rate that was much faster than the rate of growth. Additionally, the addition of cell-free supernatants of competent S. pneumoniae cultures to incompetent cultures caused them to bypass the lag in the expression of competence (Tomasz et  al. 1964). These observations led to the conclusion that S. pneumoniae cells produced an extracellular macromolecule that could activate the development of competence and that the production of such molecule occurred at a particular point during growth. Thirty years later, the molecule responsible for this phenomenon was identified as a linear heptadecapeptide, the competence-stimulating peptide, or CSP (Figure 1) (Havarstein, Coomaraswamy & Morrison et al. 1995). The gene encoding a precursor of the peptide signal was also identified and named comC (Havarstein, Coomaraswamy & Morrison et  al. 1995). The peptide signal is processed and exported to the extracellular environment by the transport system encoded by comAB

Address for Correspondence:  L. C. M. Antunes, The University of British Columbia, Michael Smith Laboratories, 367 - 2185 East Mall, Vancouver, BC, Canada V6T 1Z4. E-mail: [email protected] (Received 00 00 0000; accepted 00 00 0000) ISSN 1040-841X print/ISSN 1549-7828 online © 2009 Informa UK Ltd DOI: 10.1080/10408410902733946

http://www.informapharmascience.com/mcb

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O

O

O

O

O N OH

H 3-oxohexanoyl-homoserine lactone

3-hydroxytridecan-4-one

H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg-Lys-Lys-OH HO

B

O

O

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N

HO

H

HO

O

O

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p-coumaroyl-homoserine lactone

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(2S, 4S)-2-methyl 2,3,3,4-tetrahydroxytetrahydrofuran borate

Figure 1.  Examples of intercellular signaling molecules made by bacteria.

PG CM

ComD

ComAB

Pre-CSP

ComE comC ComW

ComX ?

CSP

ComE

Competence genes

Figure 2.  Control of competence of S. pneumoniae by the c­ ompetence-stimulating peptide (CSP). CSP is encoded by comC. The peptide signal is processed and exported to the extracellular environment by comAB and sensed by the two-component regulatory system comDE. ComD is the signal receptor histidine kinase and ComE is the cognate response regulator. Binding of the peptide signal to ComD induces a phosphorylation cascade that activates ComE. ComE can then induce expression of ComX and ComW, which ultimately activate multiple genes involved in the competence phenotype, such as cilABCDE, coi, cinA, recA and cfl. PG, peptidoglycan; CM, cytoplasmic membrane.

(Hui & Morrison et al. 1991; Hui, Zhou & Morrison et al. 1995) and sensed by the two-­component regulatory system comDE, where comD encodes the signal receptor histidine kinase and comE encodes its cognate response regulator (Pestova, Havarstein & Morrison et  al. 1996). Presumably, binding of the peptide signal to ComD induces a phosphorylation cascade that results in the activation of ComE. ComE can then induce expression

of ComX, an alternative sigma factor that ultimately activates multiple genes involved in the competence phenotype (Lee & Morrison et  al. 1999; Luo, Li & Morrison et  al. 2003). The result is that S. pneumoniae cultures are competent only after the peptide signal has accumulated and has been sensed by the cells in a population. Although ComX is required for competence (Lee & Morrison et  al. 1999), it is not sufficient for the development of competence in the absence of the inducing peptide (Luo, Li & Morrison et al. 2003). Instead, an additional ComE-controlled gene is required (Luo, Li & Morrison et al. 2004). The gene, comW, is required for a high level of competence and is regulated by the signal peptide, but its function is unknown. ComX and ComW are predicted to act in concert to activate the genes involved in the competence phenotype, such as cilABCDE, coi, cinA, recA, and cfl (Figure 2) (Lee & Morrison et  al. 1999). A recent investigation of genes controlled by the S. pneumoniae competence-inducing peptide revealed that this signal molecule exerts much broader effects on the physiology of S. pneumoniae than first anticipated (Peterson et  al. 2004). Dozens of genes are differentially expressed when S. pneumoniae is grown in the presence of the signal peptide and many of the genes affected have no obvious relation to the competence phenotype (Peterson et al. 2004).

An acyl-homoserine lactone controls light production in V. fischeri After the initial observation of bacterial signaling was made in S. pneumoniae, it was not long until a similar discovery was made in a Gram-negative bacterium.

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In the late 1960s, it was well known that certain marine bacteria have the capacity to emit light, and this phenomenon had attracted the attention of scientists for several decades. Among the most studied bioluminescent bacterial species was V. fischeri. During that time, scientists were intrigued by the observation that during the growth of V. fischeri in the laboratory, luminescence levels remained low throughout the initial stages of growth but skyrocketed as soon as the culture achieved a certain optical density (Kempner & Kemper et al. 1968; Nealson et  al. 1970). Analogously to the conclusions drawn from studies of S. pneumoniae, the fact that the rate of light emission was higher than the rate of growth after the threshold optical density had been achieved suggested that, at a critical point in the growth curve, an activator of light production acted to induce luminescence. Curiously, addition of supernatants from stationary-phase cultures eliminated the lag in light emission (Kempner & Hanson et al. 1968). This phenomenon was then attributed to the production of molecules that, upon accumulation during growth, activated the production of light. These molecules were named autoinducers and the phenomenon, autoinduction. In 1981, the structure of the V. fischeri autoinducer was solved and the molecule proved to be 3-oxohexanoyl-homoserine lactone (3OC6-HSL; Figure 1) (Eberhard et al. 1981). Only a few years later, the entire genetic locus involved in the production and regulation of light production by V. fischeri was identified and characterized (Engebrecht, Nealson & Silverman et  al. 1983; Engebrecht & Silverman et  al. 1984). The genes are organized in two adjacent, divergently-transcribed loci. The leftward locus contains a single gene, luxR, whereas the rightward locus contains the luxICDABEG operon. Genetic and biochemical studies implicated LuxI as the 3OC6-HSL synthase and LuxR as the receptor of this signal molecule and the transcriptional activator of the rightward locus (Engebrecht, Nealson & Silverman et al. 1983; Engebrecht & Silverman et al. 1984; Schaefer et al. 1996; Urbanowski, Lostroh & Greenberg et  al. 2004). The luxAB genes were shown to encode the  and  subunits of the enzyme responsible for the light-emitting reaction, luciferase, respectively (Engebrecht, Nealson & Silverman et  al. 1983; Engebrecht & Silverman et al. 1984). The luxCDEG genes were shown to be involved in the production of substrates for luciferase (Engebrecht, Nealson & Silverman et  al. 1983; Engebrecht & Silverman et  al. 1984; Zenno & Saigo et  al. 1994). Vibrio fischeri produces a second acyl-homoserine lactone, octanoyl-homoserine lactone (C8-HSL), which also controls lux expression (Gilson, Kuo & Dunlap et  al. 1995). While 3OC6-HSL activates lux transcription through direct interactions with LuxR (Urbanowski, Lostroh & Greenberg et al. 2004), based on homologous systems of other Vibrio species, C8-HSL is predicted to function through a complex phosphorelay

cascade that activates light production indirectly (Visick 2005) (see section on the control of light production by V. harveyi and Figure 3). C8-HSL is also able to bind LuxR directly but with lower affinity than 3OC6-HSL (Schaefer et al. 1996). Although the regulation of luminescence by V.  fischeri has been the prototype of studies of quorum sensing in bacteria, quorum sensing does more than just activate lux in this organism. A transcriptome analysis of the C8-HSL regulon has been performed and the results of such study revealed that another important bacterial trait controlled by this signal molecule is motility (Lupp & Ruby et  al. 2005). Additionally, several non-lux genes that are controlled by 3OC6-HSL have been identified through proteomics and transcriptomics approaches (Callahan & Dunlap et al. 2000; Antunes et al. 2007).

Bacterial intercellular communication is a widespread phenomenon After the initial discovery that V. fischeri and S. pneumoniae cells can communicate through the production and detection of extracellular signaling molecules, several published studies indicated that bacterial communication was more widespread than initially thought (Fuqua, Parsek & Greenburg et al. 2001; Lyon Novick et al. 2004; Bassler & Losick et  al. 2006; Smith et  al. 2006). In several Gram-negative bacteria, the signaling molecule was

OM IM

AinR LuxU

LuxO

C8-HSL 3OC6-HSL

LitR

LuxR

AinS

LuxI LIGHT

luxR

luxlCDABEG

Figure 3.  Acyl-HSL control of light production by V. fischeri. Vibrio fischeri produces two acyl-HSLs, 3OC6-HSL and C8-HSL, both of which control lux expression. 3OC6-HSL activates lux transcription through direct interactions with LuxR. Based on homologous systems of other Vibrio species, C8-HSL is predicted to function through a complex phosphorelay cascade that activates luxR transcription. Under low cell-density conditions, neither C8-HSL nor 3OC6-HSL accumulates to inducing levels. When the cell density increases, C8-HSL accumulates and activates the regulatory cascade that results in transcription of luxR. C8-HSL can then bind LuxR and induce low levels of light production. Since C8-HSL has a lower affinity for LuxR than does 3OC6-HSL, under higher cell density conditions, 3OC6HSL accumulates and overcomes the interaction of C8-HSL with LuxR, fully activating light production. IM, inner membrane; OM, outer membrane.

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also an acyl-homoserine lactone and the genes involved in their production and detection were homologues of luxI and luxR, respectively (Fuqua, Winans & Greenburg et  al. 1994). Most remarkably, it has been shown that the human opportunistic pathogen Pseudomonas aeruginosa utilizes acyl-homoserine lactones to control virulence factors such as extracellular proteases and secondary metabolites, among other traits (Schuster et  al. 2003). Conversely, additional examples of Grampositive bacteria that utilize peptide signals to communicate emerged (Kleerebezem et al. 1997). For instance, the human pathogen Staphylococcus aureus also utilizes peptide signaling to control the expression of extracellular degradative enzymes and virulence (Recsei et  al. 1986; Balaban & Novick et al. 1995). These findings led to the concretization of a new field of research in bacteriology. Because the signaling molecules can only accumulate to inducing concentrations after a threshold population density has been achieved, this phenomenon was named quorum sensing (Fuqua, Winans & Greenberg et al. 1994). Many species of bacteria are now known to either produce or respond to acyl-homoserine lactones or peptides (Fuqua, Winans & Greenberg et al. 1996; Kleerebezem et al. 1997; de Kievit & Iglewski et al. 2000; Fuqua, Parsek & Greenberg et  al. 2001; Fuqua & Greenberg et al. 2002; Lyon & Novick et al. 2004; Bassler & Losick et  al. 2006; Smith et  al. 2006; Williams et  al. 2007). Some of the bacterial features controlled by quorum sensing are the production of extracellular enzymes, exopolysaccharides and antibiotics, among others.

Bacterial intercellular signal molecules: An expanding list Although signaling through acyl-homoserine lactones and peptides was the cornerstone of the study of bacterial communication for several years, it was not long until the scrutiny of molecular pathways used by bacteria to communicate revealed novel molecules. Most of the early developments in this field can be attributed to the study of quorum sensing in V. harveyi and V. cholerae and the discoveries that emerged from such studies are described in more detail below.

A furanosyl borate diester controls gene expression in V. harveyi The observation that V. fischeri controls light production using a diffusible signal molecule prompted the investigation of the production of such molecules by other marine luminescent bacteria. Amongst those, the best studied is the shrimp pathogen Vibrio harveyi. Early studies on the regulation of light production by V.

harveyi have identified a complex regulatory cascade. Like V. fischeri, V. harveyi uses an acyl-homoserine lactone to control the expression of genes involved in light production (Cao & Meighen et  al. 1989). The autoinducer of V. harveyi is 3-hydroxybutanoyl-homoserine lactone (3OHC4-HSL) and it is produced by the protein encoded by luxLM, which represents a second family of acyl-homoserine lactone synthases that are not homologous to V. fischeri luxI (Bassler et al. 1993). The receptor for 3OHC4-HSL is not a LuxR-type protein either. Rather, 3OHC4-HSL is sensed by the sensor kinase LuxN (Bassler et al. 1993; Timmen, Bassler & Jung et al. 2006). In the absence of the signal, LuxN autophosphorylates and transfers the phosphate to the phosphotransferase LuxU (Timmen, Bassler & Jung et al. 2006). LuxU, in turn, transfers it to the response regulator LuxO (Freeman & Bassler et  al. 1999). LuxO is a 54-dependent transcriptional activator (Lilley & Bassler et  al. 2000). In V. harveyi, LuxO activates the transcription of at least one small non-coding RNA (sRNA) (Lenz et  al. 2004). The sRNA, qrr1, acts in concert with the RNA chaperone Hfq to destabilize the messenger RNA of its target gene, luxR (not related to V. fischeri luxR) (Lenz et al. 2004). LuxR is the direct activator of light production and other traits in V. harveyi (Swartzman, Silverman & Meighen et  al. 1992; Pompeani et al. Aug 4th 2008). Therefore, LuxO is an indirect repressor which modulates light production through regulation of mRNA stability of luxR (Bassler, Wright & Silverman 1994b et al. 1994; Lenz et al. 2004). In the presence of 3OHC4-HSL this cascade is reversed, resulting in inactivation of LuxO and derepression of light production through an increase in luxR mRNA levels (Lenz et al. 2004). Vibrio harveyi possesses a second quorum sensing system (Bassler, Wright & Silverman 1994a et al. 1994). The identity of the second V. harveyi autoinducer remained elusive for a long time although it has been known that the gene luxS is involved in the synthesis of the molecule, termed autoinducer 2 (AI-2) (Surette, Miller & Bassler et al. 1999). A two-component regulatory system is responsible for detecting AI-2. LuxP, a periplasmic protein, together with the sensor kinase LuxQ, compose the receptor complex of AI-2 (Bassler, Wright & Silverman 1994b et  al. 1994). In the absence of the signal, LuxQ autophosphorylates and transfers the phosphate to LuxU, which, in turn, transfers it to LuxO (Bassler, Wright & Silverman 1994b et  al. 1994; Freeman & Bassler et al. 1999; Neiditch et al. 2005). As previously mentioned, phospho-LuxO acts to indirectly repress the production of light by V. harveyi through the modulation of luxR mRNA levels (Lenz et  al. 2004). In the presence of AI-2, this cascade is reversed, resulting in inactivation of LuxO, and derepression of light production (Lenz et  al. 2004). In 2002, AI-2 was identified as the furanosyl borate diester (2S,4S)-2-methyl 2,3,3,4tetrahydroxytetrahydrofuran borate (Figure 1) (Chen

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et  al. 2002). The luxS/AI-2 signaling system is widespread among Gram-negative and Gram-positive bacteria. Many bacterial species have been shown to either produce AI-2-like molecules and/or contain orthologs of luxS (Xavier & Bassler et al. 2003; Vendeville et al. 2005). Because acyl-homoserine lactones and peptides are usually species-specific and AI-2 production and sensing is more promiscuous, it has been proposed that AI-2 may be a signal used for interspecies communication (Bassler, Greenburg & Stevens et al. 1997). Although the role of AI-2 as a signaling molecule is well established in V. harveyi, evidence that it represents a bona fide signal in other bacterial species is scarce. AI-2 is produced from S-adenosylmethionine (SAM), the major donor of methyl groups in bacteria (Schauder et al. 2001). During methyl-transfer reactions involving SAM, the toxic intermediate S-adenosylhomocysteine (SAH) is formed (Schauder et al. 2001). Conversion of SAH to non-toxic molecules is accomplished by the enzymes Pfs and LuxS (Schauder et al. 2001). The fact that LuxS is involved in such an important metabolic pathway has raised skepticism about its role as a bacterial communication molecule. In fact, a recent survey of sequenced bacterial genomes has showed that most bacterial species that possess luxS orthologs lack known receptors for AI-2, suggesting that in most cases LuxS may play a primarily metabolic role (Rezzonico & Duffy et al. 2008).

-hydroxyketones: Yet another family of bacterial intercellular signaling molecules Like V. harveyi, V. cholerae also produces and responds to AI-2 (Bassler, Greenburg & Stevens et  al. 1997; Zhu et al. 2002). Additionally, V. cholerae has another autoinducer and the molecule has been named CAI-1 (Miller et  al. 2002). CAI-1 has been recently identified as the -hydroxyketone 3-hydroxytridecan-4-one (Figure 1)) (Higgins et al. 2007). The systems used by V. cholerae to sense its autoinducers are analogous to those used by V. harveyi. CAI-1 is synthesized by CqsA and sensed by CqsS (Miller et al. 2002). Similarly to the other quorum sensing systems of V. harveyi and V. cholerae, CAI-1 invokes a dephosphorylation cascade through LuxU and LuxO that results in the inactivation of the latter (Miller et al. 2002). However, the quorum sensing systems of V. cholerae work backwards. In the absence of CAI-1 and AI-2, virulence factors are activated and biofilm formation is induced (Zhu et  al. 2002). Conversely, when CAI-1 and/or AI-2 accumulate, these traits are repressed (Zhu et al. 2002). It has been proposed that this singular mechanism by which V. cholerae uses quorum sensing to control virulence gene expression has implications for the ecology of the organism. Namely, it has been hypothesized that V. cholerae represses gene expression in high

cell-densities to promote its dissemination by inducing shedding from the host and release into the environment (Zhu et  al. 2002). Recently, another -hydroxyketone has been added to the growing list of bacterial signaling molecules. Tiaden and colleagues have shown that Legionella pneumophila possesses orthologs of V. cholerae cqsAS (Tiaden et al. 2007). In L. pneumophila, LqsA synthesizes the signal LAI-1, 3-hydroxypentadecan-4one (Spirig et al. 2008). LqsS is predicted to be the receptor for the signal molecule (Tiaden et  al. 2007; Spirig et  al. 2008). The observation that an -hydroxyketone signaling system has been selected for since the evolutionary divergence of L. pneumophila and V. cholerae suggest that other bacterial species may utilize similar systems to control gene expression. In fact, it has been shown that multiple Vibrio species possess ortologues of cqsAS and/or activate the CAI-1 signaling system of V. cholerae (Henke & Bassler et al. 2004). However, the role of such signaling systems in the control of gene expression in these organisms remain to be determined.

Other bacterial signaling molecules The explosion of research in the field of bacterial intercellular signaling has led to the dissection of the minute molecular details of how bacteria produce, sense, and respond to signaling molecules. This is particularly true for the canonical bacterial quorum sensing systems that utilize acyl-homoserine lactones, peptides, and AI-2. However, investigation of this field has also led to the realization that bacterial signaling is not limited to such systems that we now understand in detail. In the past years, many extracellular molecules that can be produced and sensed by bacteria have been unveiled. Perhaps one of the most interesting examples of this is the finding that the phototrophic soil bacterium Rhodopseudomonas palustris and a few other bacterial species utilize an ortholog of V. fischeri luxI (rpaI) to synthesize a compound structurally different from acyl-homoserine lactones (Schaefer et  al. 2008). The signal molecule in this case is p-coumaroyl-homoserine lactone (pC-HSL; Figure 1) and this finding suggests that multiple molecules can be synthesized using the same bacterial signal synthases (Schaefer et  al. 2008). Besides pC-HSL, many other bacterial intercellular signals have been isolated and characterized. For example, P. aeruginosa produces the molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal, or PQS) and this signal has been shown to control the production of a major virulence factor (Pesci et  al. 1999). Additionally, the plant pathogen Ralstonia solanacearum utilizes the signal molecule 3-hydroxypalmitic acid methyl ester (3OH-PAME) to control virulence (Flavier et  al. 1997). In Escherichia

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coli, a peptide termed ‘extracellular death factor’ has been identified and implicated in quorum-sensingdependent controlled cell death (Kolodkin-Gal et  al. 2007). Recently, formate has been implicated as a signaling molecule produced and sensed by Salmonella enterica (Huang et al. 2008). Although the role of some of these molecules in bacterial communication per se might be controversial, they nevertheless demonstrate the complex biology of bacterial chemical signaling.

Bacterial eavesdropping The search for new signal molecules and receptors involved in bacterial communication that took place in the past few years has revealed some unique cases that escape the dogma of how signal production and sensing occur. For instance, a few bacterial species have been shown to be able to respond to signal molecules without producing them (Michael et al. 2001; Pereira et al. 2008). This has led to the speculation that such systems might be involved in sensing signals produced by other species in complex microbial ecosystems. Perhaps one of the most convincing and best-studied cases of bacterial sensing of non-self-produced molecules is represented by S. enterica. A luxR ortholog has been identified in S. enterica (Ahmer et  al. 1998). However, attempts to detect acyl-homoserine lactones produced by S. enterica cultures have failed (Michael et al. 2001). With the sequencing of the S. enterica genome (http:// cmr.jcvi.org/), it became obvious that the reason for this is that luxI or luxLM orthologs are not present in the genome and, consequently, S. enterica does not synthesize acyl-homoserine lactones. Nevertheless, the S. enterica luxR ortholog, sdiA, encodes and active protein that can detect and respond to exogenously added signals (Michael et al. 2001; Janssens et al. 2007). Recently, it has been shown that SdiA is active in vivo during the transit of S. enterica through the gastrointestinal tract of hosts (Smith et  al. 2008). Interestingly, SdiA was not active during the transit of S. enterica through the intestines of mammals or birds. However, transit of S. enterica through the intestines of turtles resulted in SdiA activation (Smith et al. 2008). The authors were able to isolate an acyl-homoserine lactone producer from the animals used and identified it as Aeromonas hydrophila. As expected, coculturing A. hydrophila and S. enterica in vitro resulted in SdiA activation (Smith et al. 2008). These findings suggest that signals produced by the intestinal microbiota of turtles can activate the quorum sensing system of S. enterica and that the differences in activation by different hosts can likely be explained by differences in the composition of their resident bacterial communities. Nevertheless, the impact of such phenomenon on host interactions of S. enterica remains to be determined.

More recently, another example of bacterial eavesdropping has been described. Pereira et al. (2008) have shown that the plant symbiont Sinorhizobium meliloti, although not capable of producing AI-2, can respond to exogenously added signal (Pereira et  al. 2008). Co-culturing S. meliloti with the AI-2-producing, plant pathogen, Erwinia carotovora results in the removal of AI-2 from the external environment by S. meliloti cells (Pereira et al. 2008). The authors show that this phenomenon is not due to signal degradation but active internalization of AI-2. Although S. meliloti and E. carotovora coexist in the environment, in the rhizosphere of several plants, the question of whether or not S. meliloti can actively sense E. carotovora-produced AI-2 in nature remains to be addressed.

Bacterial communication and host interactions A common theme that has emerged from the study of bacterial signaling is that it often controls bacterial traits involved in interactions with their hosts (Parsek & Greenberg et al. 2000). This is true for both beneficial and detrimental bacterium-host relationships. Some of the most studied examples of how bacterial signaling can control bacterial symbiosis and pathogenesis are described in detail below.

Intercellular signaling controls the symbiosis between V. fischeri and its host The regulatory control of bioluminescence by V. fischeri has important consequences for its biology. Vibrio fischeri forms symbiotic associations with species of fish and squid (Ruby & Nealson et al. 1976; Boettcher & Ruby et al. 1990). Among those, the best studied is the symbiosis with the Hawaiian bobtail squid Euprymna scolopes (Visick & Ruby et al. 2006). The squid acquires V. fischeri cells from the surrounding sea water upon hatching. The cells are housed in a specialized organ termed the light organ, where they grow to high numbers and emit light. The squid has a sophisticated apparatus of lens and reflector tissues that can control the intensity and direction of the light emitted (McFall-Ngai & Montgomery et  al. 1990). Euprymna scolopes lives in shallow waters and has a nocturnal behavior. By controlling the emission of light by V. fischeri cells present in the light organ to match the intensity of the moonlight, it is believed that the squid can mask its shadow in the sea floor and, therefore, avoid predators (McFall-Ngai 1990). Vibrio fischeri also benefits from this relationship by utilizing growth substrates provided by the squid (Graf & Ruby et  al. 1998). In the context of this symbiosis, the

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complex control of light production by quorum sensing is crucial. Bioluminescence is energetically expensive and, therefore, must be tightly controlled. When luminescence is fully induced, it is estimated that 10% of all cellular energy is used for light production and that luciferase constitutes up to 5% of all cellular protein (Karl & Nealson et al. 1980). By controlling the expression of lux through quorum sensing, V. fischeri ensures that maximum light production occurs only where it is useful, in the host environment. As a consequence, bioluminescence and host colonization are intrinsically linked and mutations in both structural and regulatory bioluminescence genes impair host colonization by V. fischeri (Visick et al. 2000).

The human opportunistic pathogen P. aeruginosa uses intercellular signaling to control virulence Several studies have shown that not only symbiotic but also pathogenic bacteria produce acyl-homoserine lactones. In many cases it has been demonstrated that pathogenic bacteria utilize quorum sensing to control features that are likely to be involved in virulence (de Kievit et al. 2000; Kong, Vuong & Otto et al. 2006; Kendall & Sperandio et al. 2007; White & Winans et al. 2007). This has caused an explosion in the interest on the subject. One of the most studied bacterial species that utilize quorum sensing to control virulence is Pseudomonas aeruginosa. P. aeruginosa is a human opportunistic pathogen that can colonize the lungs of patients with the genetic disorder cystic fibrosis, eventually leading to their death (Murray, Egan & Kazmierczak et al. 2007). Pseudomonas aeruginosa possesses multiple quorum sensing systems that work in a complex cascade to regulate the production of virulence factors and many other genes. Two luxI homologues and three luxR homologues are involved in this process (Schuster & Greenberg et al. 2006). The las quorum sensing system of P. aeruginosa is composed of the lasI and lasR genes, homologues of V. fischeri luxI and luxR, respectively. LasI is responsible for the production of the quorum sensing signal 3-­oxododecanoyl-homoserine lactone (3OC12-HSL) and LasR is the transcriptional regulator that responds to this signal (Passador et  al. 1993; Pearson et al. 1994; Schuster, Urbanowski & Greenberg et al. 2004). In the presence of activating concentrations of 3OC12-HSL, LasR controls the expression of virulence factors such as genes involved in hydrogen cyanide production and extracellular degradative enzymes (Schuster et al. 2003; Wagner et al. 2003). Besides those, a second quorum sensing system, the rhl system, is controlled by the las quorum sensing system. RhlI synthesizes the quorum sensing molecule butanoyl-

homoserine lactone (C4-HSL) and RhlR binds and responds to it (Ochsner et  al. 1994; Ochsner & Reiser et al. 1995; Pearson et al. 1995). Among the traits controlled by C4-HSL is the production of rhamnolipid, a surfactant important for biofilm formation and virulence (Ochsner & Reiser et al. 1995; Davey, Caiazza & O’Toole et al. 2003; Zulianello et al. 2006). Besides LasR and RhlR, P. aeruginosa possesses a third LuxR-type signal receptor, QscR (Chugani et al. 2001). No cognate signal synthase exists for QscR, which has been shown to detect and respond to 3OC12-HSL to regulate a set of genes that partially overlaps the LasR and RhlR regulons (Lee, Lequette & Greenburg et al. 2006; Lequette et al. 2006). Using 3OC12-HSL and C4-HSL, P. aeruginosa can control the expression of more than 300 genes (Schuster et  al. 2003; Wagner et  al. 2003; Lequette et  al. 2006). This represents about 6% of the genome, showing that quorum sensing plays an immense role in the physiology of this human pathogen. The quorum sensing regulatory circuit of P. aeruginosa is complex and intertwines with several other regulatory pathways (Schuster & Greenberg et  al. 2006). Additionally, the timing of expression of quorum-controlled genes is highly variable indicating that other regulatory events must be acting upon genes controlled by quorum sensing (Schuster & Greenberg et al. 2006).

Inter-kingdom communication A recent area of study on bacterial signaling has revealed an ongoing communication between microorganisms and their hosts (Rumbaugh 2007; Hughes & Sperandio et  al. 2008). Several host-derived signals are sensed by bacteria. Some of these compounds include interferon gamma (Wu et  al. 2005), tumor necrosis factor alpha (Luo et al. 1993), interleukin-1 (Porat et al. 1991), adenosine (Kohler et al. 2005), epinephrine (Sperandio et al. 2003), and antimicrobial peptides (Bader et  al. 2005). Conversely, hosts have also been shown to sense and respond to bacterial signals, such as acyl-homoserine lactones (Kravchenko et  al. 2008). Below we discuss a few examples of bacteria-host communication. Recent studies have suggested that bacteria can sense the metabolic stress of the host to take advantage of a weakened state. In enterohemorrhagic E. coli, the mammalian hormones epinephrine and norepinephrine, which are released by the host during stress, are sensed by the QseC receptor to regulate bacterial virulence genes (Sperandio et al. 2003; Clarke et al. 2006). A qseC mutant is attenuated for virulence, which underscores the importance of this inter-kingdom communication to the development of disease (Clarke et  al. 2006). In a more recent report, it was observed that P. aeruginosa can sense host-produced dynorphin and

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respond through increased virulence (Zaborina et  al. 2007). Like epinephrine and norepinephrine, dynorphin is released by the host during stress. Dynorphin was shown to activate the expression of the pqsABCDE operon resulting in increased production of the quorum sensing signal PQS, among others (Zaborina et al. 2007). Bacteria have also evolved to sense the immune system of hosts by detecting and responding to small innate immune molecules. For example, P. aeruginosa can sense a component of the immune system to mount an effective countermeasure to immune activation by its host. Host-derived interferon gamma, which has the main function of bacterial clearance, can bind to the outer membrane protein OprF to activate quorum sensing-dependent virulence factors, such as PA-I lectin and pyocyanin (Wu et  al. 2005). Additionally, interferon gamma increases transcription of rhlI, the C4-HSL signal synthase (Wu et al. 2005). In S. enterica, host-derived antimicrobial peptides directly activate the sensor kinase PhoQ, promoting the expression of virulence genes through a phosphorelay cascade (Bader et al. 2005). Together these studies begin to shed light on how pathogens can sense stress signals and immune response factors to adapt to the host environment and establish disease. On the other side of the communication, the ability of host cells to sense bacterial signaling molecules, such as acyl-homoserine lactones, has also been shown. The effects of these molecules are diverse and often deleterious for the host, leading to increased bacterial pathogenesis. A host receptor for these molecules remains to be identified, but their activity has been shown to modulate host immune responses (Telford et al. 1998; Ritchie et al. 2003; Ritchie et al. 2005), promote apoptosis (Tateda et  al. 2003; Shiner et  al. 2006) and elicit proinflammatory effects (Smith et  al. 2001; Smith et al. 2002; Smith et al. 2002; Zimmermann et al. 2006). For instance, it has been recently shown that the P. aeruginosa quorum sensing signal 3OC12-HSL can dampen host immune responses by disrupting signaling through nuclear factor kappa B, the major eukaryotic immunomodulatory regulator (Kravchenko et  al. 2008). Administration of 3OC12-HSL impaired LPSinduced inflammation in a murine model, suggesting that P. aeruginosa might benefit from 3OC12-HSL production by preventing host immune activation during infection (Kravchenko et al. 2008). Research on signaling between prokaryotes and eukaryotes has made significant progress in the recent past and it is now obvious that such phenomenon has important implications for bacteria-host interactions. Future studies will not only reveal novel layers of interactions between bacteria and eukaryotes but also help further understand the molecular mechanisms involved in bacteria-host communication.

Bacterial signaling as a target for therapeutic intervention The discovery that bacterial pathogens often utilize intercellular signaling systems to control virulence has opened a new avenue for the development of new therapeutic strategies against bacterial infections. Bacterial signaling could be targeted by therapeutics in several different ways. Drugs that interfere with the biochemical pathways for synthesis of signaling molecules may prove useful to attenuate bacterial virulence. For instance, it has been shown that multiple chemicals can inhibit C4-HSL production by P. aeruginosa RhlI (Parsek et  al. 1999). Among the inhibitors are end-products of the reaction catalyzed by RhlI, such as 59-methylthioadenosine as well as analogues of SAM, such as S-adenosylhomocysteine. Another potential approach for targeting bacterial intercellular signaling for therapeutics would be to inhibit signal binding to cognate receptors. This could be achieved by chemically engineering synthetic analogs of the signal molecules that retain the ability to specifically bind the receptors without activating them and significant progress has been made in this area (Rasmussen & Givskov et  al. 2006; Geske, O’Neill & Blackwell et  al. 2008). For example, the identification of one such analog was recently described (Rasko et al. 2008). By screening a library of small organic molecules, Rasko et al. (2008) identified an inhibitor of the bacterial adrenergic receptor QseC. The analog, N-phenyl-4-{[(phenylamino) thioxomethyl]amino}-benzenesulfonamide, inhibited binding of QseC to its cognate signal, norepinephrine. Additionally, it inhibited virulence gene expression and decreased morbidity and mortality in models of infection with enterohemorragic E. coli, S. entericaenterica, and Francisella tularensis (Rasko et  al. 2008). Besides interfering with signals and signal receptors, another potential strategy for using bacterial communication as a target for therapeutic intervention would be to manipulate downstream events that are activated after signal binding to the receptor. Some species of bacteria utilize complex regulatory cascades to link the detection of a signal molecule to the regulation of virulence gene expression. In theory, every step along such cascades is a potential target and efforts towards the development of therapeutic strategies targeting signaling systems should not be limited to the signals per se. For example, a small molecule inhibitor of virulence that acts on a bacterial transcriptional regulator has been described (Hung et  al. 2005). Through screening of a library of small molecules, Hung et al. (2008) identified 4-[N-(1,8naphthalimide)]-n-butyric acid (virstatin) as an inhibitor of virulence gene expression and host colonization in V. cholerae (Hung et  al. 2005). Virstatin was shown to inhibit the production of both cholera toxin and the

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toxin coregulated pilus, two major virulence factors of V. cholerae. This inhibition occurred through a post-transcriptional effect on ToxT, a transcriptional regulator of both virulence factors (Hung et al. 2005). Later on, the mechanism of action of virstatin was dissected and the drug was shown to affect ToxT activity by inhibiting its dimerization (Shakhnovich et al. 2007). Alternatively to the inactivation of bacterial intercellular signaling systems, another possibility for therapeutic use is the early activation of such systems. It has been hypothesized that bacterial signaling systems are used by pathogens to hide their virulence factors until a sufficient number of cells is present to overcome host defenses (de Kievit et al. 2000). By delaying the production of extracellular molecules, bacterial pathogens may avoid detection by the host immune system. Therefore, the use of signal analogues that could activate the expression of such genes early during infection might be beneficial by alerting the host immune system that an infection is occurring before bacteria can build up and cause significant damage. Nevertheless, apart from the identification of chemical molecules that act as agonists of bacterial signaling molecules (Smith, Bu & Suga et al. 2003; Lowery et  al. 2005), very little development has been made in their use as potential therapeutic agents. Additionally, this particular application of bacterial signal molecules as therapeutics might be harder than the inhibition of chemical signaling because it relies on very early detection of disease. Limitations notwithstanding, the study of bacterial signaling may provide us with a plethora of potential therapeutic targets. In the current face of widespread bacterial antibiotic resistance, the discovery on new drugs with mechanism of action considerably different from the ones currently in use is of major urgency. Bacterial signaling systems are a very attractive target because they are not required for bacterial survival. Because bacterial cells targeted will not be killed by the drugs, the selection of resistant variants may occur more slowly, which would represent a major advantage over the antibacterial drugs currently in use.

Concluding remarks Over forty years have passed since the initial observation that bacteria can communicate. Still, the field of research that has bloomed over this observation continues to produce exciting discoveries. We now understand how bacteria produce several intercellular signals, how they sense them, how they respond to them and how they affect the bacterial population in general. Still, new studies suggest that we have just started touching the diversity of signaling molecules and their effects on bacterial cells. This is remarkably true when one considers poorly

explored environments where microbial diversity is extremely rich such as soil and the gastrointestinal tract of animals. Exploring bacterial signaling in such environments is likely to provide us with an astonishing number of molecules involved in bacterial communication as well as intricate species interactions dependent upon them. By itself, this may open several new avenues for manipulating microbes in ways beneficial to humans.

Acknowledgements Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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