When being alone is enough: noncanonical functions of canonical bacterial quorum-sensing systems

Abstract

A number of bacterial pathogens are capable of detecting the presence of other bacteria located within their surrounding niche through a process of bacterial signaling and cell-to-cell communication commonly referred to as quorum sensing (QS). QS systems are commonly now described in the context of collective behaviors exhibited by groups of bacteria coordinating diverse arrays of physiological functions to enhance survival of the community. However, QS systems have also been implicated in a variety of processes distinct from the measure of bacterial cell density. This review will highlight noncanonical adaptations of canonical QS systems that have evolved to enable bacteria to detect nonself individuals within a population or to detect occupation of confined spaces.

Bacterial quorum sensing (QS), or ‘knowing thy neighbor’, is a form of bacterial cell-to-cell communication that enables bacteria to sense the presence of other bacteria within their surrounding environment and to rapidly respond to changes in population density [1–4]. It has become widely accepted that, in most cases, bacteria do not simply exist as single isolated microorganisms within an environment but have instead evolved a variety of communication systems that enable them to detect and essentially count other members within a population, a phenomenon more formally known as cell density-dependent QS. QS enables bacteria to coordinate complex activities with bacterial neighbors, including biofilm formation and DNA transfer from one cell to another. QS can essentially serve to ensure a successful party by preventing bacteria from starting the festivities until enough guests have arrived.

The phenomenon of QS was initially recognized as a mechanism contributing to the regulation of bioluminescence via the LuxI/LuxR QS system in the Gram-negative bacteria Vibrio fischeri and Vibrio harveyi [1,4–6]. Briefly, LuxI is a synthase that catalyzes a reaction between S-adenosyl methionine and an acyl carrier protein to produce an acyl-homoserine lactone (AHL) that freely diffuses out of the bacterial cell [7]. At high concentrations, the AHL will diffuse back into the cell and bind to its cognate cytoplasmic receptor LuxR and induce the transcription of the luciferase operon, forming an autoregulatory feedback loop (Figure 1) [8–12]. Since this first description, numerous LuxI/LuxR homologous systems have been identified in more than a 100 Gram-negative bacteria and have been shown to regulate a variety of genes involved in a plethora of functions including virulence gene expression, motility, biosynthesis of bacteriocins and biofilm formation [1,3,13].

Figure 1. . Canonical quorum-sensing systems.

The signaling molecules (AI for Gram-negatives or AIP for Gram-positive bacteria) either freely diffuse out of the cell (AI) or are processed and secreted at low levels (AIP). Once the cell density increases, the molecules accumulate to a certain threshold level and either diffuse back into the cell (for Gram-negatives), are actively imported back into the bacterial cell by an oligopeptide transporter or bind to a histidine kinase on the cell surface to activate a two-component signaling system. Molecules transported back into the bacterial cell usually bind transcriptional regulators to influence gene expression, whereas activating a two-component system will induce a phosphorylation cascade of a response regulator to control gene expression. Most of these systems are also autoregulating, forming a positive feedback loop on expression of the signaling molecule.

AI: Auto-inducer; AIP: Auto-inducing peptide; HK: Histidine kinase; IM: Inner membrane; OM: Outer membrane; QS: Quorum sensing; RR: Response regulator; TR: Transcriptional regulator.

A single bacterium can possess a number of different QS systems based on distinct small molecules [1–3,6,14]. In Gram-negative bacteria, the signaling molecules that induce QS are typically homoserine lactone derivatives, whereas small peptides serve as the signaling molecules in Gram-positive bacteria. These small peptides can either be encoded by small distinct open reading frames within the bacterial chromosome, or can be derived from the proteolytic processing of secretion signal sequences (SS) [2,4,14,15]. In each case, the initial peptide requires further proteolytic processing to generate the active peptide pheromone. The signaling peptides of Gram-positive bacteria may be short hydrophobic linear peptides, cyclic peptides or additionally modified, resulting in a structurally diverse signaling group. In both Gram-negatives and -positives, signaling molecules (called as auto-inducers [AIs] or auto-inducing peptides [AIPs] in Gram-positives) are generally secreted at low levels. Once the cell density increases, the signaling molecules accumulate until a certain threshold level is reached. Signaling molecules can either diffuse into cells (for Gram-negatives), be actively imported back into the bacterial cell by an oligopeptide transporter or bind to a histidine kinase (HK) receptor on the cell surface to activate a two-component signaling system (Figure 1). Signaling molecules transported into the bacterial cell usually bind transcriptional regulators (TR) to modulate gene expression, whereas activation of a two-component system induces a phosphorylation cascade to control gene expression (Figure 1). Most of these systems are autoregulating and form a positive feedback loop to amplify gene expression.

Since the initial discovery of QS, many types of QS systems have been identified in both Gram-negative and -positive bacteria, and as mentioned above, these systems have been implicated in a diverse range of physiological functions such as the development of competence for uptake of DNA by Streptococcus pneumoniae and Bacillus subtilis, and the induction of virulence factors and/or biofilm formation by Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, and Streptococcus pyogenes. There have been a number of excellent reviews written about various aspects of classical or canonical QS to which the reader can refer [1–4,6,14–17]. This review will focus instead on an alternative aspect of bacterial signaling that depends on QS systems independent of bacterial cell density. These noncanonical uses of QS system components have evolved to either detect distinct individuals within a population that differ from one's self, or to inform a single bacterial cell that it is located within a spatially confined environment. These variations on canonical QS are discussed in the following sections.

Finding the perfect mate: use of QS systems to find individuals who are different from oneself

Enterococcus faecalis is a Gram-positive bacterium that possesses a number of different QS systems that function to enhance its pathogenesis within the host and also to facilitate plasmid transfer from donor cells to recipient cells [2,18,19]. Enterococcus faecalis is a facultative anaerobic commensal bacterium that makes up part of the normal intestinal microbiota. However, it is also an opportunistic pathogen capable of causing of variety of infections including endocarditis, sepsis, surgical wound infections, and both catheter-associated and noncatheter associated urinary tract infections [20–22]. The bacterium represents a healthcare concern because it is highly tolerant to heat, aseptic treatments, and is one of the most prevalent multidrug-resistant pathogens isolated in hospital settings [23–26]. Enterococcus faecalis makes use of a canonical QS system, known as Fsr, which is similar to the well-characterized Agr system of S. aureus (described below in the ‘Staphylococcus aureus & host-cell vacuoles’ section) [27]. This system controls expression of two extracellular proteases: a gelatinase (GelE) and a serine protease (SpreE), which have been shown to be important for biofilm formation, development of endocarditis, infections associated with indwelling catheters, and in translocation across human colon cells, which may be a mechanism used by this opportunistic pathogen to disseminate to other locations [27–30]. Enterococcus faecalis also produces a cytolysin that is subject to regulation by another canonical QS system [31–35]. This cytolysin is a major virulence factor and has both bacteriocin and hemolytic activity, making it toxic to both other Gram-positive bacteria and also to eukaryotic cells [34,36].

In addition to these canonical QS systems, E. faecalis uses QS signaling components and small hepta- or octa-hydrophobic linear peptides (termed sex pheromones) in a noncanonical way to coordinate the conjugal transfer of virulence plasmids between donor cells and plasmid-free recipient cells within a population [2,18,33,37]. Via this adaptation, the plasmid-free recipient cells secrete the sex peptide pheromones while the plasmid-containing donor cells respond to the pheromones and stimulate a mating response (Figure 2). Five sex pheromones have been characterized (cAD1, cPD1, cCF10, cAM373 and cOB1). Interestingly, these peptide pheromones are all encoded within the cleaved N-terminal SS of much larger lipoproteins of unknown function. The SS directs these proteins for secretion through the general secretory pathway and once the signal sequence is cleaved by signal peptidase II (SPII), the released SS peptide is further processed by the activity of one or more proteases to produce the fully mature active peptide. The mature peptide pheromone secreted by a plasmid-free recipient cell binds to an oligopeptide-binding protein present on the surface of the plasmid-containing donor cell (known as TraC/PrgZ) and the peptide is then imported by chromosomally encoded oligopeptide transport machinery. Once inside the donor cell, the pheromone binds to a negative regulator (TraA/PrgX) with the result that the repression of gene expression of plasmid encoded genes is relieved, resulting in the expression of positive regulators and the subsequent expression of gene products required for the mating response and plasmid transfer machinery. One initial product that contributes to the process of plasmid transfer is a large surface protein, referred to as an aggregation substance (Asa1/Asa10) that is expressed on the surface of the donor cell and helps to initiate close contact with nearby recipient cells. Additional induced machinery provides for the complete transfer of a copy of the plasmid (along with any virulence factors and antibiotic resistance genes encoded on the plasmid itself) to the recipient cell (Figure 2). Once the recipient acquires the plasmid, masking of the cognate pheromone occurs to prevent self-induction through two plasmid-encoded products: a plasmid-encoded inhibitor peptide that competes for binding to TraC/PrgZ and a membrane-bound protein (TraB/PrgY) that somehow sequesters, modifies and/or degrades cognate peptide that escapes from the bacterium. This noncanonical adaptation of a canonical QS system thus appears to have evolved to differentiate like-cells (plasmid containing) from unlike-cells (plasmid free), thereby distinguishing individuals within the population instead of responding to changes in bacterial cell numbers.

Figure 2. . Sensing of distinct individuals by Enterococcus faecalis.

One is the loneliest number: bacterial QS-like cell signaling within the spacial confines of a host cell

• Listeria monocytogenes & bacterial escape from host-cell vacuoles

The concept of adapting QS systems so as to respond to situations distinct from changes in population density has been recognized in association with a thus far limited number of other bacterial pathogens [38–42]. Given that a basic feature of canonical QS is the detection of an accumulating signal within a population, it can be readily appreciated that situations other than an overall increase in bacterial numbers can lead to signal accumulation and detection. As an example, the secretion of a peptide pheromone by a single bacterium within a confined space where diffusion is limited could lead to local increases in pheromone concentration and the stimulation of pheromone-responsive pathways. One example of a spatially confined environment experienced by single bacterial cells occurs during the course of host-cell infection for the facultative intracellular bacterium Listeria monocytogenes [38,40].

Listeria monocytogenes is a ubiquitous environmental bacterium capable of surviving in many diverse environments both outside and inside the mammalian host cell [41–47]. Listeria monocytogenes’ highly adaptable nature has resulted in widespread problems in the food industry as it can easily contaminate and proliferate in food supplies despite commonly used preservation methods to prevent growth of micro-organisms [48–52]. Consumption of L. monocytogenes-contaminated food products normally leads to mild gastroenteritis in healthy individuals, but can result in more serious diseases and even death in immunocompromised persons, such as the elderly and fetuses of pregnant women [44,46,53]. Transitioning from life as a saprophyte to an intracellular pathogen within the host requires the ability to sense environmental changes and induce the upregulation of gene products to facilitate intracellular replication and spread of the bacterium to adjacent cells [54–57]. Listeria monocytogenes expresses a variety of virulence factors that enable the bacterium to invade mammalian cells, escape from host vacuoles into the cytosol, replicate within the cytosol and spread to neighboring cells using an actin-based motility mechanism.

The majority of known gene products required for L. monocytogenes intracellular growth are controlled by the master transcriptional regulator known as PrfA, a 27 kD protein which belongs to the cAMP receptor protein (Crp)-Fnr family of transcriptional regulators [47,56,58]. Members of this family are known to bind small-molecule cofactors that induce a conformational change in the protein resulting in protein activation and induction of target gene expression [59–61]. Activation of PrfA, presumably by the small molecule glutathione obtained from the host cell, serves as a critical switch that enables L. monocytogenes to survive within the host [62]. Outside of host cells, PrfA normally exists in a low activity form with full activation occurring once L. monocytogenes escapes from the vacuole into the glutathione-rich cytosol following bacterial entry of host cells. Thus, PrfA is a critical regulator of L. monocytogenes pathogenesis that responds to host-derived environmental cues.

Our lab has recently identified a PrfA-inducible secreted PplA (peptide pheromone-encoding lipoprotein A) that shares significant homology with the E. faecalis Cad lipoprotein encoding cAD1 peptide pheromone [19]. The synthesis of PplA is increased following PrfA activation and, similar to the cAD1 pheromone of E. faecalis, the pPplA peptide is processed from the released PplA lipoprotein N-terminal SS peptide following secretion of the lipoprotein through the general secretory pathway [40]. In contrast to E. faecalis where the cAD1 pheromone stimulates a mating response between plasmid-containing and plasmid-free cells, the pPplA peptide pheromone has functionally evolved to enhance vacuolar escape of L. monocytogenes in nonprofessional phagocytic cells (Figure 3A). Studies of mutants lacking both the PplA lipoprotein and its SS-encoded peptide pheromone versus the lipoprotein alone have demonstrated that the pPplA peptide pheromone is a critical virulence factor that contributes to both bacterial aggregation in broth culture and survival in mouse models of infection, whereas the PplA lipoprotein has no apparent role.

Figure 3. . Listeria monocytogenes peptide signaling within host-cell vacuoles.

(A) In nonprofessional phagocytic cells where pplA is expressed, a basal level of PplA is secreted and processed across the bacterial cellular membrane by the general secretory pathway, resulting in the release of the mature pPplA peptide into the extracellular space and the lipidation and anchoring of the remaining PplA to the bacterial membrane. Once L. monocytogenes enters a host cell and is contained within a vacuole, the pPplA peptide accumulates in the restricted space of the vacuolar compartment and is transported back into the bacterial cell through the CtaP oligopeptide-transport system. The transported peptide may bind to a transcriptional regulator and initiate a signaling cascade that induces the expression of gene products that contribute to complete vacuolar escape and also the steps leading up to PrfA activation. (B) In professional phagocytic cells, where the expression of pplA is decreased and/or inhibited, an unknown peptide stimulates the expression of the late com genes through the activity of the regulator ComK to enhance escape of L. monocytogenes from the vacuole. Expression of the late com genes results in the assembly of a pseudopilus or DNA translocation channel that is predicted to exert a mechanical force on the vacuolar membrane, thereby enhancing complete escape of the bacterium.

PrfA: Positive regulatory factor A.

While the sequence and nature of the pPplA peptide remains to be defined, experimental evidence supports the existence of an active secreted peptide based on: an increased rate of bacterial aggregation occurs when logarithmically growing cells are exposed to bacterial supernatants derived from dense culture of bacteria that encode the pPplA peptide versus those that do not; bacterial aggregation does not occur if supernatants are treated with proteinase K; and amino acid substitutions within the PplA SS can abolish aggregation without interfering with SS processing and lipoprotein secretion [19]. The addition of synthetic peptide (based on homology to cAD1) had no measurable effect on bacterial aggregation, suggesting that the pPplA active peptide is different from the peptide sequence predicted based on E. faecalis peptide or that the pPplA peptide is post-translationally modified.

With respect to L. monocytogenes infection of mammalian cells, mutants lacking the pPplA peptide exhibit delays in escape from the vacuoles of nonprofessional phagocytic cells but escape with normal kinetics from the phagosomes of macrophage-like cell lines or in bone marrow-derived macrophages. The vacuolar escape defect is correlated with reduced bacterial polymerization of host cell actin, an activity that occurs within the cytosol, and also with bacterial co-localization with the late endosomal marker Rab7. Interestingly, loss of the pPplA pheromone did not impair perforation of the vacuole, only bacterial escape into the cytosol [19]. These data strongly suggest that complete vacuole escape requires an additional mechanism beyond initial pore formation mediated by the activity of the hemolysin listeriolysin O (LLO). The pPplA peptide was also found to contribute to maintenance of surface-associated and secreted proteins, and it is possible that some of these gene products may contribute to either the stabilization of LLO-induced membrane pores and/or the physical disruption of the vacuole membrane.

Proteins identified based on increased abundance following accumulation of the pPplA pheromone include stress-induced chaperones and gene products involved in anaerobic respiration and oxidation reduction, suggesting a potential link between peptide-signaling pathway and the redox state of the vacuole. All bacterial virulence defects associated with the loss of the pPplA peptide could be completely compensated by the introduction of a constitutively active form of the PrfA virulence regulator (prfA* allele), suggesting a possible connection between the pPplA signaling pathway and PrfA activation. Taken together, these studies suggest a model in which L. monocytogenes senses the confines of the host vacuoles as a result of the accumulation and import of the pPplA peptide, leading to PrfA activation and enhanced bacterial escape into the cytosol (Figure 3A). Thus, in contrast to E. faecalis, which uses a peptide-signaling system to differentiate between two distinct cell types, the L. monocytogenes pPplA peptide appears to have evolved to enable the bacterium to sense the confined environment of the vacuole so as to induce the expression of gene products required for full membrane disruption and entry of the bacterium into the cytosol.

In addition to the pPplA peptide-signaling pathway, there is another potentially peptide-based signaling system related to competence development that has been implicated in enhancing escape of L. monocytogenes from host-cell vacuoles, specifically the phagosomes of macrophages [38]. Peptide signaling has been shown to regulate competence, or the uptake of DNA from the environment, for both B. subtilis and S. pneumoniae through the use of canonical QS systems [63–65]. Competence is a brief physiological state during which the bacterial cells collectively become primed to transport extracellular DNA across the cell wall and bacterial membrane, at times resulting in integration of the newly acquired DNA into the bacterial genome [63,64]. Several gene products, including those encoded by com genes, are required for peptide pheromone signaling and DNA uptake. Although it has not been demonstrated to exhibit natural competence, L. monocytogenes possesses a number of com genes that share homology with those involved in competence development in other bacteria, namely B. subtilis; however, not all the genes required for this system are present [66,67]. Listeria monocytogenes appears to lack comX, which encodes the inducing peptide, the transmembrane protein comQ and the two component signaling system encoded by comP/comA. While it remains true that a peptide has not been identified and no experimental evidence exists thus far to support the presence of a peptide, the competence systems described to date for other bacteria all share the property of peptide-induced stimulation of com gene expression. Most but not all of the regulatory components and late genes involved in assembly of the Com apparatus required for DNA transport are present in the Listeria genome [38]. Because of these missing components, it is unclear if the competence system is functional in L. monocytogenes and as mentioned above, natural competence has not been demonstrated under laboratory conditions.

One competence regulator that is encoded by L. monocytogenes is ComK, a master transcriptional regulator that induces the expression of late com genes involved in assembly of the Com apparatus for DNA uptake, and it is this system which has recently been reported to be important for L. monocytogenes in vacuole escape of professional phagocytic cells [38,67]. The late com genes are organized into three separate operons: the comG operon, comE operon and the comF operon. The comG operon encodes several prepilin proteins that assemble a pseudopilus that crosses the cell wall and two additional accessory proteins for its synthesis. The comE operon encodes a protein that functions to bind extracellular DNA and also a protein of unknown function. The comF operon encodes a DNA helicase protein for DNA transport and also proteins of unknown function. Interestingly, the comK gene in some strains of L. monocytogenes is inactivated by the presence of an A118-like prophage that integrates at a specific attachment site within the comK gene [68]. However, Rabinovich et al. have found that the late competence genes regulated by ComK are highly expressed during intracellular growth as a result of prophage excision [38]. In addition, mutants missing the specific components of the pseudopilus or the DNA translocation channel were impaired for vacuole escape in macrophages and were attenuated for bacterial virulence in mice, whereas the DNA-binding components were dispensable for these processes. A strain cured of the prophage and containing an intact comK gene grew similarly to wild-type L. monocytogenes in macrophages, and many naturally occurring strains of L. monocytogenes lack this prophage and are capable of growth within macrophages. For strains that do contain prophage, their data suggest that prophage integration/excision serves to regulate virulence gene expression and that the ComK system is somehow involved in sensing L. monocytogenes’ presence within host-cell vacuoles in professional-phagocytic cells (Figure 3B). Thus, while no peptide pheromone has yet been identified to stimulate the induction of the L. monocytogenes com genes within the phagosome, it is tempting to speculate that the bacterium has adapted components of the canonical com QS system for special sensing, similar to pPplA.

Given the requirement for selected Com gene products and the pPplA peptide for vacuole escape in different cell types, it is tempting to speculate that a pseudopilus may help stabilize the pores initially formed by LLO or that maybe the pseudopilus exerts physical pressure on the phagosomal membrane to aid in membrane disruption and bacterial escape into the cytosol. It is possible that the pPplA and ComK systems are somehow interconnected, and that fundamental differences in the vacuole membranes of professional phagocytic cells versus nonprofessional phagocytic cells necessitate the requirement for differentially regulated components. Additional characterization of the gene products regulated by pPplA and com gene products should help to clarify how these components enhance bacterial entry into the cytosol.

• Staphylococcus aureus & host-cell vacuoles

Staphylococcus aureus is a Gram-positive bacterium commonly found among normal human skin flora and is also a leading cause of nosocomial and community-acquired bacterial infections [15,69]. It is the etiological agent of numerous infections in both humans and animals that can range from minor wound infections to more serious life-threatening diseases such as endocarditis, osteomyelitis and toxic shock syndrome. The expression of many of the virulence factors involved in establishing these diseases is under the control of what is considered to be a canonical QS system known as the accessory gene regulator (Agr) system [15]. The agr system in S. aureus is comprised of four genes, agrACDB, where agrA and agrC encode a two-component detection system, agrD encodes a secreted hydrophobic peptide and agrB encodes a membrane bound protein that secretes, processes and modifies the released peptide into its mature cyclic form. The role and regulation of this system as part of the classical definition of QS has been described in detail in numerous reviews elsewhere [15,69]. In brief, it is believed that when S. aureus is present at low cell density early in infection, the bacteria express proteins (such as protein A, fibronectin-binding proteins and clumping factors) required for attachment and colonization. However, as the cell density increases, the AIP produced by Agr system autoregulates and activates transcription of RNAIII, a regulatory RNA that downregulates expression of these surface proteins and induces expression of numerous toxins and other factors that enhance nutrient acquisition, survival and dissemination.

Interestingly, other studies have demonstrated that the Agr system is not only important for monitoring cell density but can also function in a bacterial cell density-independent manner, similar to what has been observed for L. monocytogenes, to facilitate the escape of single bacteria trapped within the confined endosomal compartment of mammalian host cells (Figure 4) [39,41,42]. While S. aureus is not classically considered an intracellular pathogen, a number of studies have shown it has the ability to invade host cells, escape the endosome and replicate within the host cytosol. The Agr system appears to be required cytosolic bacterial replication as an agr mutant is defective for intracellular growth as a result of defects associated with vacuole escape. Shompole et al. demonstrated that biphasic expression of agr occurs during intracellular growth [39]. The first wave of expression occurs while S. aureus is still contained within the endosomal compartment, which coincides with peak transcription of hla (α-toxin) and hlb (β-toxin) as well as with escape from the endosome, while the second wave of agr expression correlated with damage to the cytoplasmic membrane of host cells. It was speculated by the authors that the Agr-regulated toxins may aid in lysis of the endosomal membrane in this confined environment, a term they referred to as ‘diffusion sensing’ rather than QS (Figure 4). Indeed, the term diffusion sensing (versus QS) has been used to describe the ability of a cell to detect whether secreted molecules move away from the cell in a microenvironment, as isolated cells in confined spaces may produce enough of the signaling molecule to induce self-induction [70]. These studies thus indicate a cell density-independent role for the Agr system in S. aereus that appears similar to that of pPplA and the com gene products for L. monocytogenes.

Figure 4. . Staphylococcus aureus accessory gene regulator signaling within the endosome.

Staphylococcus aureus displays biphasic expression of agr during intracellular growth. Expression of this peptide signaling system is first observed during containment within the endosomal compartment of a host cell and coincides with peak transcription of hla (α-toxin) and hlb (β-toxin), two Agr-regulated toxins may aid in lysis of the endosomal membrane in this confined environment, a term referred to as ‘diffusion sensing’ rather than quorum sensing.

Agr: Accessory gene regulator.

It is getting crowded in here: bacterial single-cell QS in manufactured confined spaces

The above studies describe adaptations of classical bacterial QS systems within the host to promote bacterial replication and survival. Mathematical modeling and simulation tools have also been developed to demonstrate QS in small confined spaces; however, these simulations have been generally designed to address QS-based behaviors for groups of bacteria in confined spaces rather than for a single confined bacterium [71,72]. In vitro techniques have been described to artificially confine bacteria in small spaces to monitor the expression of canonical QS systems [73–75]. Carnes et al. developed a low-volume nanostructured lipid–silica matrix droplet, mimicking an endosomal compartment, to confine a single S. aureus cell to specifically determine whether confinement alone could induce QS by monitoring the expression of the agr system (∼1 cell per 2 × 10³ μm³, which is equivalent to 0.5 × 10⁹ cells/ml, similar to the reported QS threshold of 10⁷–10⁹ cells/ml) [74]. The authors developed a dihexanoylphosphatidylcholine lipid vesicle encased in an ordered silicon dioxide nanostructure. The lipid vesicle is maintained at a pH of 5.5 (to mimic the pH of early endosome) and the surrounding silicon droplet serves as a reservoir for added buffer and media, all to construct a structure to mimic an endosome of a host cell. The pore forming α-toxin encoded by the hla gene was fused to gfp as a reporter to monitor auto-induced QS stimulated by the Agr system, where it was discovered that the system was indeed expressed by a single bacterium. The QS signal AIP accumulated over time in the confined space (∼10 h) resulting in Agr regulon expression demonstrating that autoinduction of the system within the spatial constraint had occurred. This study was the first to experimentally demonstrate confinement-induced QS for a single bacterium, hence the refinement of the term QS. These results clearly show that QS can function independently of cell density in an artificial setting.

Another experimental technique to confine bacteria to small spaces and examine the relationship between confinement and QS has been demonstrated with the Gram-negative bacterium P. aeruginosa [75]. P. aeruginosa is a bacterium ubiquitously found in the environment and is considered an opportunistic pathogen that has the ability to cause both acute and chronic infections in humans [1,4]. P. aeruginosa infections can lead to skin complications but this bacterium is more notorious for being associated with the lung disease known as cystic fibrosis [76,77]. In order to establish a disease, P. aeruginosa contains canonical QS systems to control the expression of a plethora of virulence factors [1,4,78]. This bacterium contains three QS systems, with the main one being LasI/LasR, which is a homologue of the LuxI/LuxR system described in the Vibrio species. The LasI AI is synthesized at a low level, and at high cell density it accumulates and diffuses back into the cell and is detected by the cytoplasmic LasR protein, which activates the transcription of target genes including those encoding virulence factors such as elastase, proteases and exotoxin A. The LasI/LasR complex can further activate lasI to form an auto-feedback loop and also target another luxI-encoded homolog called RhiI. When the AI produced by RhiI reaches a high enough concentration, it can bind to RhlR, a second LuxR homolog, which can further activate additional virulence genes encoding elastase, proteases, pyocyanin and siderophores. P. aeruginosa has not been reported to be contained within host cell endosomes or phagosomes. However, similar to what has been shown for S. aureus, it has been demonstrated that P. aeruginosa confined in vitro to a small microfluidic chamber can initiate QS within a single bacterial cell [75]. Boedicker et al. optimized a microfluidic technique to create droplets made from a biocompatible resin SU-8 containing approximately 100 fL volume and a range of a small number of bacterial cells. QS was detected in P. aeruginosa by constructing a reporter-fusing gfp to lasB, which encodes an elastase and is controlled by the LasI/LasR QS system. The authors effectively demonstrated that one bacterial cell could initiate QS over a period of 10 h. Interestingly, they also discovered that QS is heterogenic and not all bacterial cells underwent QS in a single well containing either one or more bacteria. These artificial confinement experiments lack host molecules and proteases that might be anticipated to further influence the induction of QS within the infected host. However, this study provides an additional example that confinement alone, without the influence of host factors or large cell densities, could nonetheless induce QS systems within a single bacterial cell.

Conclusion

It has become increasingly evident that bacterial communication systems have evolved to perform a variety of roles extending beyond conversations between individuals within a population. These adapted functions include the ability of a single bacterium to detect non-self-like individuals for the purposes of passing on virulence factors as seen with E. faecalis conjugation, and the enhancement of vacuolar escape through the detection of confined quarters as demonstrated for L. monocytogenes and S. aureus. Communication systems enable bacteria to multitask and manipulate regulatory systems so as to adapt to a variety of environments, thus promoting continued survival. Given that so many bacteria harbor cell-to-cell communication systems and that communication can take place either within populations or within manufactured confined spaces containing single bacteria, it will interesting to determine what other adaptations of QS systems exist for perhaps other distinct and as yet undefined purposes. The recognition that a bacterium can talk to itself as well as communicate with others means we need to listen more carefully to these potentially important conversations.

Future perspective

With an expanded view of the functional roles of bacterial QS systems, it is clear that bacteria can not only talk to each other but also find circumstances where they appear completely at ease talking only to themselves. The concept of diffusion sensing through small molecules has actually existed for some time, and some have proposed that it is this self-serving function that has been selected for over evolutionary time versus community communication through QS [70]. Overall, whether the concept is called diffusion sensing, confinement-induced QS or spatial sensing, small molecule signaling has been adapted for a diversity of roles, apparently enabling the ability of bacteria to accurately sense one's physical environment in the absence of other bacteria as well as having been adapted and perhaps exploited for the control of coordinated behaviors in populations. As we understand more regarding the varied functional roles of small molecule signaling, we can better eavesdrop on both these bacterial conversations and the monologues, and potentially influence the behavioral outcome by targeting these signal transduction systems. Sometimes, it may be as simple as just knowing when and where to listen.

EXECUTIVE SUMMARY.

Canonical quorum-sensing systems can perform noncanonical functions

• Both Gram-negative and -positive bacteria harbor systems that allow individual bacterial cells to sense communities and communicate with one another within those communities. Traditionally, quorum sensing (QS)-dependent cell signaling responds to increasing bacterial cell densities with alterations in patterns of bacterial gene expression and coordination of complex group behaviors. However, it is becoming increasingly appreciated that QS can function at the level of individual cells, thus demonstrating the functional flexibility of QS systems.

QS systems can function to differentiate oneself from another community member

• Enterococcus faecalis contains a number of canonical QS systems that respond to increases in cell density; however, this bacterium also secretes a peptide pheromone that serves to stimulate the conjugal transfer of virulence plasmids between two distinct individual cells.

QS systems can function at the single cell level to sense the spatial constraints of mammalian host-cell vacuoles

• Listeria monocytogenes and Staphylococcus aureus utilize canonical QS systems to sense their presence in the confined space of the host cell vacuole so as to enhance vacuolar escape. This serves as an example of a single bacterium using a QS-like system in the absence of other bacteria for self-signaling.

QS systems can function in manufactured small spaces

• Techniques have been developed for demonstrating QS-like signaling in both S. aureus and Pseudomonas aeruginosa by physically isolating single bacteria in an artificially engineered confined area, whereupon QS-dependent changes in gene expression can be observed in the absence of any additional factors.