The las and rhl quorum sensing systems

These key discoveries in P. aeruginosa QS systems inspired further researches on their functions, regulons and the molecular mechanisms with which the las and rhl circuits activate the expression of QS-responsive genes. The results showed that upon binding with the respective autoinducers OdDHL and BHL, the receptor proteins LasR and RhlR get activated and form complexes. The LasR-OdDHL and RhlR-BHL complexes bind to the conserved las-rhl boxes residing in the promoters of target genes, thereby activating their transcriptional expression (Schuster and Greenberg, 2007; Whiteley and Greenberg, 2001; Whiteley et al., 1999). Transcriptomic studies based on lasI and rhlI mutants revealed that the regulons are on a continuum, with some genes that respond dramatically well to OdDHL (e.g. lasA), some with BHL specificities (e.g. rhlAB), and some equally well to both signals (Schuster and Greenberg, 2006; Schuster et al., 2003). These genes constitute nearly 10% of P. aeruginosa genome, and therefore accounts for a majority of the physiological processes and virulence phenotypes (Schuster and Greenberg, 2006). Some of these key virulence genes are listed for the convenience of discussion (Table 1).

LasR also induces the expression of RsaL, a transcriptional repressor of lasI. Binding of RsaL to the bidirectional rsaL-lasI promoter inhibits the expression of both genes, which generates a negative feedback loop that counteracts the positive signal feedback loop mentioned earlier, thereby balancing the levels of OdDHL (Rampioni et al., 2007). Whilst LasR/OdDHL and RsaL do not compete for the same binding site on the lasI promoter region, the repression by RsaL is stronger than the activation by LasR (Rampioni et al., 2007). RsaL also inhibits the expression of some QS target genes such as biosynthetic genes of pyocyanin and cyanide (Rampioni et al., 2007). A range of positive and negative regulatory proteins were subsequently identified and they control the las and rhl systems in a variety of ways. Noteworthy are the regulatory effects of QscR and VqsR, which are homologues of LuxR. QscR forms heterodimers with LasR/OdDHL and RhlR/BHL and prevents their binding with the promoter DNA of downstream responsive genes, therein dampening the las and rhl QS signalling effects (Ledgham et al., 2003a). QscR also binds to OdDHL and utilize it for activating its own regulon (Chugani et al., 2001; Fuqua, 2006; Schuster and Greenberg, 2006). VqsR is a positive regulator of the las QS system and is itself regulated by the LasR/OdDHL complex (Li et al., 2007). More recently, an anti-activator QslA was identified, which binds to LasR via protein-protein interaction and prevents the interaction of the latter with promoter DNA of the las responsive genes. The inhibitory effect of QslA on LasR is irrespective of OdDHL concentrations. By disrupting the ability of LasR to trigger the expression of downstream genes and cause a QS response, QslA controls the overall QS activation threshold (Seet and Zhang, 2011). There are quite a few other super-regulators of the AHL-based QS systems which are summarized in the table below (Table 2). In addition, quorum quenching enzymes, which degrade AHL signals, the AHL-acylases PvdQ and QuiP, are also involved in balancing the level of AHL signals in P. aeruginosa (Huang et al., 2006; Sio et al., 2006).

Quinolone-based intercellular signaling

The third QS signal, PQS, was purified and characterized in 1999 by Pesci and co-workers when they observed that spent culture media from wild type PAO1 causes a dramatic induction of lasB expression in a lasR mutant of P. aeruginosa, which could not be mimicked by OdDHL or BHL (Pesci et al., 1999). PQS is structurally identified as 2-heptyl-3-hydroxy-4-quinolone, and it is chemically unique from the AHL signals of the las and rhl systems. Originally studied as an antibacterial molecule (Cornforth and James, 1956; Lightbown and Jackson, 1956), this is the first instance that a 4-quinolone compound was reported as a signalling molecule in bacteria. The PQS synthesis cluster has been identified to consist of pqsABCD, phnAB and pqsH (Gallagher et al., 2002). Shortly after the identification of PQS signal, the receptor PqsR (then known as MvfR) has been implicated in the regulation of PQS production (Cao et al., 2001). PqsA is an anthranilate-coenzyme A ligase (Coleman et al., 2008; Gallagher et al., 2002), which activates anthranilate to form anthraniloyl-coenzyme A, initiating the first step of the PQS biosynthesis. A pqsA mutant does not produce any akyl-quinolones (AQs) (Deziel et al., 2004). PqsB, PqsC and PqsD are probable 3-oxoacyl-(acyl carrier protein) synthases and they mediate the conversion of anthranilate into 2-heptyl-4-quinolone (HHQ) by incorporation of β-ketodecanoic acid (Deziel et al., 2004; Gallagher et al., 2002). HHQ is the precursor of PQS and can be intercellularly transmitted between P. aeruginosa cells. HHQ is converted into PQS by the action of PqsH, a putative flavin-dependent monooxygenase that purportedly hydroxylates HHQ at the 3-position (Deziel et al., 2004; Dubern and Diggle, 2008; Gallagher et al., 2002; Schertzer et al., 2009). The transcription of pqsH is controlled by LasR, implying that the PQS system is controlled by the las system (Schertzer et al., 2009). PqsL is also predicted to be a monooxygenase and is most likely to be involved in the synthesis of the AQ N-oxides, (e.g. 4-hydroxy-2-heptylquinoline-N-oxide, HQNO) (Lépine et al., 2004). Disruption in PqsL caused an overproduction of PQS (D’Argenio et al., 2002), probably owing to a blocked AQ N-oxide pathway which leads to an accumulation of HHQ (Deziel et al., 2004; Lépine et al., 2004). In certain strains of P. aeruginosa, accumulation of PQS and HHQ leads to autolysis and cell death (D’Argenio et al., 2002; D’Argenio et al., 2007; Whitchurch et al., 2005). The role of PqsE remains largely unknown, which is a probable metallo-β-lactamase. Mutation of pqsE does not affect PQS biosynthesis (Gallagher et al., 2002), but the mutants failed to respond to PQS (Diggle et al., 2003; Farrow et al., 2008; Gallagher et al., 2002), and did not express the PQS-controlled phenotypes such as pyocyanin and PA-IL lectin production. In contrast, overexpression of PqsE alone led to enhanced pyocyanin and rhamnolipid production, which is otherwise dependent on the PQS signaling system (Farrow et al., 2008). These puzzling phenomena need to be further investigated for elucidating the role of PqsE in the bacterial physiology and virulence.

PqsR is a LysR-type transcriptional regulator that binds to the promoter region of pqsABCDE operon and directly controls the expression of the operon (Cao et al., 2001; Gallagher et al., 2002). The expression of pqsR is in turn controlled by LasR/OdDHL (Camilli and Bassler, 2006). PqsR is the cognate receptor of PQS and also its co-inducer, as the activity of PqsR in inducing the expression of pqsABCDE is dramatically increased when PQS is bound by the receptor (Wade et al., 2005; Xiao et al., 2006b). HHQ was also found to be able to bind to and induce the expression of PqsR, though it does so with ~ 100-fold less potency than PQS (Wade et al., 2005; Xiao et al., 2006a). Mutation of pqsR resulted in non-production of any AQs and pyocyanin (Cao et al., 2001; Gallagher et al., 2002; Schertzer et al., 2009; von Bodman et al., 2008), indicating that PqsR is essential for executing PQS signal transduction.

The importance of pqs signaling system in the bacterial infection has been illustrated by a range of studies. Null mutation of the pqs system resulted in reduced biofilm formation and decreased production of virulence factors such as pyocyanin, elastase, PA-IL lectin and rhamnolipids (Cao et al., 2001; Diggle et al., 2003; Rahme et al., 2000; Rahme et al., 1997). PQS is also required for full virulence towards plants (Cao et al., 2001), nematodes (Gallagher et al., 2002) and mice (Cao et al., 2001; Lau et al., 2004). In burn-wound mouse models, the killing abilities of pqsA are attenuated compared to the wild type parental strain (Déziel et al., 2005; Xiao et al., 2006b). Intriguingly, the pqsH mutant did not result in a decrease in virulence in burn-wound mouse model (Xiao et al., 2006b), but displayed a reduced killing on nematodes (Gallagher et al., 2002), hence the importance of PQS in regulation of virulence remains debatable. PQS, its precursor HHQ, and the derivative HQNO (4-hydroxy-2-heptylquinoline-N-oxide), are often found in the sputum, bronchoalveolar fluid and mucopurulent fluid of cystic fibrosis sufferers (Collier et al., 2002). Taken together, this could suggest that the precursors of PQS may play an equally important role as PQS in virulence and infections.

An integrated QS system

Recently, a fourth inter-cellular communication signal has been discovered to be capable of integrating environmental stress cues with the quorum sensing network (Lee et al., 2013). Named as IQS, it belongs to a new class of quorum sensing signal molecules and was structurally established to be 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde. The genes that are involved in IQS synthesis are a non-ribosomal peptide synthase gene cluster ambBCDE. When disrupted, it caused a decrease in the production of PQS and BHL signals, as well as the virulence factors such as pyocyanin, rhamnolipids and elastase. Upon addition of 10 nmol/L IQS to the mutants, these phenotypes could be restored fully, indicating that IQS is a potent inter-cellular communication signal compared with its counterparts (Fig. 2). Further, IQS has been shown to contribute to the full virulence of P. aeruginosa in four different animal host models (mouse, zebrafish, fruitfly and nematode), highlighting the important roles of this new QS system in modulation of bacterial pathogenesis. Importantly, under phosphate depletion stress conditions, IQS was demonstrated to be able to partially take over the functions of the central las system (Lee et al., 2013), providing critical clues in understanding the puzzling phenomenon that the clinical isolates of P. aeruginosa frequently harbour mutated lasI or lasR genes (Ciofu et al., 2010; D’Argenio et al., 2007; Hoffman et al., 2009; Smith et al., 2006).

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Figure 2

Structures ofP. aeruginosaquorum sensing (QS) signals. Clockwise from left, N-(3-oxododecanoyl)-homoserine lactone (OdDHL); N-butyrylhomoserine lactone (BHL); 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS); 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (Integrated Quorum Sensing Signal, IQS)

Phosphate-depletion stress

Phosphate is essential for all living cells owing to its key roles in signal transduction reactions such as phospho-relay, and as an essential component of the energy molecule ATP, nucleotides, phospholipids and other important biomolecules. Foreseeably, bacterial pathogens may encounter strong competition for free phosphates from host cells during the process of pathogen-host interaction. Therefore, the ability to withstand phosphate starvation and the response mechanisms of harnessing phosphate from external sources is critical for P. aeruginosa survival and establishment of infections. As a result, phosphate-depletion stress has been shown to have far-reaching effects on QS signalling profiles, gene expression, physiology and virulence of bacterial pathogens (Chugani and Greenberg, 2007; Frisk et al., 2004; Jensen et al., 2006; Lee et al., 2013; Zaborin et al., 2009).

When facing with phosphate limitation, P. aeruginosa exhibits increased swarming motility and cytotoxicity towards the human bronchial epithelial cell line 16HBE14o- (Bains et al., 2012), attesting to the strong responses phosphate deprivation could elicit from the pathogen. Additionally, phosphate depletion stress was shown to prompt the up-regulation of iron chelator pyoverdine biosynthesis, which in turn, could result in the inactivation of the phosphate acquisition pathway. When the pyoverdine signalling pathway was interrupted, pyochelin biosynthesis was in turn increased as compensation (Zaborin et al., 2009). This resulted in high amounts of ferric ions to be acquired. Coupled with the dramatic increase in PQS production (part of the phosphate starvation response), the lethal PQS-Fe(III) red coloured complex was formed. When ingested, the red-spotted P. aeruginosa caused rapid mortality in C. elegans, a phenomenon known as “red death” (Zaborin et al., 2009). Such signalling cross-talk demonstrates the interconnectivity between the phosphate and iron acquisition systems in P. aeruginosa, the investment in resources the bacteria makes to maintain their homeostasis, and the deleterious effects on the host when the fine balance is tipped.

The lack of phosphate also dramatically activates the expression of pqsR and the PqsR-regulated pqsABCDE and phnAB genes. Along with the enhanced pqs system, the expression of QS-associated virulence genes responsible for the synthesis of rhamnolipids, phenazines, cyanide, exotoxin A and LasA protease are similarly induced (Bains et al., 2012; Zaborin et al., 2009). This was thought to lead to the acute mortality rate of the host organism Caenorhabditis elegans after being infected by P. aeruginosa that were grown in phosphate starvation medium (Zaborin et al., 2009). These observations correlate and could well be explained by our current knowledge on IQS. With depletion in phosphate, expression of iqs system is induced (Lee et al., 2013), which in turn triggers an up-regulation of the downstream pqs and rhl QS systems, and eventually, an observed boost in QS-associated virulence factors production and killing rates.

It is crucial to note that the two-component sensor-response regulator system PhoBR plays an indispensable role in detection and signal transduction of phosphate stress cues (Anba et al., 1990; Filloux et al., 1988; Hsieh and Wanner, 2010), as disruption of phoB completely abolished the virulence of P. aeruginosa towards C. elegans (Zaborin et al., 2009), and dramatically diminished its swarming motility and cytotoxicity (Bains et al., 2012). PhoB (and the pho regulon) was also shown to participate in the inhibition of biofilm formation, c-di-GMP signal degradation and repression of the type III secretion systems (Haddad et al., 2009), all of which could significantly affect the clinical outcome during P. aeruginosa infections (Abe et al., 2005; Costerton, 2001; Hauser et al., 2002; Hueck, 1998; Roy-Burman et al., 2001). The phoB mutant grows poorly in low phosphate medium and failed to produce the QS-dependent virulence factor pyocyanin (Lee and Zhang, unpublished data). Remarkably, PhoBR is indispensable for coordinating the las-independent, phosphate-dependent IQS signalling activation, wherein the “IQS phenotype” would be abolished in a phoB mutant (Lee et al., 2013). The PhoBR-IQS loop could also explain the observations by Jensen and co-workers, who reported that low phosphate prompted an enhancement of the rhl QS system even when las was functionally absent and this is coordinated by PhoB (Jensen et al., 2006).

Iron and PQS signaling system

Unlike phosphate, the modulatory effect of iron starvation on P. aeruginosa QS networks appears to be less direct. A deficiency in iron does lead to notable increases in the expression of genes involved in iron acquisition (ferric uptake siderophores, pyochelin and pyoverdine; ferrous iron transporters like haem and feo), exoenzymes that could cleave iron-bound host proteins (alkaline protease, lasB elastase) and other redox enzymes and toxins (exotoxin A) (Ochsner et al., 2002). Further, the iron depletion stress response was found to lead to an inhibition of oxygen transfer from the atmosphere to liquid P. aeruginosa cultures, thus protecting bacteria cells from oxidative stress. Production of the virulence factor LasB elastase is also significantly increased in these iron depletion cultures (Kim et al., 2003). Although some of the upregulated virulence factors, like alkaline protease and elastase, are known to be regulated by the QS systems of P. aeruginosa (see Table 1), a direct link between iron deprivation and up-regulation of central QS genes such as lasI, lasR, rhlI or rhlR has yet to be found. In a report by Diggle and co-workers, the PQS molecules were found to function as an iron trap when secreted into the extracellular milieu of P. aeruginosa (Diggle et al., 2007). This was hypothesized to serve the purpose of storing up free ferric ions which could subsequently be internalized into the cells by the siderophores, in order to safeguard against a sudden dip in iron concentration. Iron starvation could also trigger a Fur-dependent de-repression of the small regulatory RNAs prrF1 and prrF2 expression. PrrF1 and PrrF2 bind to and inhibit the expression of antABC genes which encode for the anthranilate degradation enzymes AntABC. Since anthranilate is the precursor of PQS biosynthesis, inhibition of its degradation could lead to accumulation of anthranilate, which consequently elevates the concentration of HHQ and PQS in the bacteria cells. This in turn might boost the PQS-PqsR signaling pathway. PqsR was also found to inhibit antABC expression, albeit in a PrrF1,2-independent manner (Oglesby et al., 2008). Taken together, the above findings seem to suggest that iron depletion stress may modulate bacterial virulence through the pqs system, which awaits further investigations.

ANR and oxygen deprivation

Low oxygen tension is a key factor affecting cyanide biosynthesis (cyanogenesis) in P. aeruginosa (Castric, 1994; Castric, 1983). The final product, hydrogen cyanide (HCN), is a highly potent extracellular virulence factor and contributes to high mortality rates during infection of host organisms (Ryall et al., 2008; Solomonson, 1981). Additionally, increase in P. aeruginosa cell density was also shown to remarkably elevate expression of hcnABC, the synthase genes for HCN, and reaches its optimum levels during the transit from exponential to stationary growth phase of the bacteria (Castric et al., 1979). This may suggest a cooperative link between oxygen deprivation and QS in the regulatory mechanism of cyanogenesis, which was subsequently demonstrated through characterization of ANR, a transcriptional regulator associated with bacterial anaerobic growth.

ANR, which is converted into its active form when oxygen tension is low, is a key regulator controlling the expression of arginine deiminase and nitrate reductase. ANR belongs to the FNR (fumarate and nitrate reductase regulator) family of transcriptional regulators and is the main transcriptional regulator that acts in parallel with the QS systems for the expression of hydrogen cyanide biosynthesis genes (Pessi and Haas, 2000). ANR, together with LasR-OdDHL or RhlR-BHL, bind to the promoter region of the hcnABC cluster, exhibiting a synergistic effect brought upon by oxygen limitation stress. Further, the PRODORIC promoter analysis programme predicted the FNR/ANR binding consensus sequences in up to 25% of the predicted QS-controlled promoters, implying that ANR might be an important co-regulator of the QS-dependent virulence genes in anaerobic environments (Schuster and Greenberg, 2006).

Starvation stress

When exposed to unfavourable environments and nutrient starvation, P. aeruginosa must rapidly cope and elicit a prompt response to modify their metabolic profiles for survival. This process is termed as the stringent response and brings about diverse effects ranging from inhibition of growth processes to cell division arrest (Joseleau-Petit et al., 1999; Svitil et al., 1993) and more importantly, a premature activation of the P. aeruginosa QS systems that is independent of cell-density (van Delden et al., 2001). The QS signals BHL and N-hexanoyl-homoserine lactone (HHL) are prematurely produced and PQS synthesis inhibited (Baysse et al., 2005). The spike in BHL QS signal is likely to result in the concomitant increase in production of downstream virulence factors elastase and rhamnolipids (Schafhauser et al., 2014).

The QS-based response is mediated by the stringent response protein RelA. In face of amino acid shortage, uncharged tRNA triggers the activity of the ribosome-associated RelA, which in turn synthesizes ppGpp (nucleotide guanosine 3’,5’-bisdiphosphate), an intracellular signal that enables the bacteria cell to self-perceive their inability in synthesis of proteins (Gentry and Cashel, 1996). When overexpressed, RelA leads to early transcriptional expression of the lasR and rhlR genes, as well as production of QS signals OdDHL and BHL (van Delden et al., 2001), hence leading to the overproduction of the aforementioned QS-dependent virulence factors. Furthermore, RelA and ppGpp was also shown to coordinate the stress response associated with alterations in membrane phospholipid composition and loss of membrane fluidity. When the phospholipid biosynthesis protein LptA was deleted, an increase in relA expression and ppGpp production was observed, which resulted in a premature activation of BHL and HHL QS signals biosynthesis (Baysse et al., 2005).

In a recent study, Schafhauser and co-workers observed that the synthesis of the starvation signal ppGpp negatively regulates the biosynthesis of HHQ and PQS signals, and is required for full expression of both the las and rhl QS systems (Schafhauser et al., 2014). In the relA and spoT double mutant that is unable to synthesize ppGpp, both the las and rhl QS systems are down-regulated, and the production of QS-dependent virulence factors rhamnolipid and elastase are reduced (Schafhauser et al., 2014). Whilst it has been previously reported that ppGpp increases the expression of LasR and RhlR and the resultant downstream factors (Baysse et al., 2005; van Delden et al., 2001), repression on the pqs system by ppGpp is somewhat unexpected. More experiments are required to investigate on the significance of this selective dampening of the pqs system.

This work was funded by the Biomedical Research Council, Agency for Science, Technology and Research (A*STAR), Singapore, and by the National Natural Science Foundation of China (Grant No. 31330002). We apologize to the scientists who made contributions to the field, but their works have not been cited due to space limitations.