P. aeruginosa, is a Gram-negative, motile, ubiquitous bacillus found in soil, fresh and sea water. It is highly versatile, able to tolerate low oxygen conditions, grow at a wide range of temperatures (4–42 °C) and survive with minimal nutrients. It is this adaptability that allows the pathogen to adhere and survive on medical equipment and other hospital surfaces initiating outbreaks of nosocomial infections characterized by general inflammation and sepsis. These infections normally occur in compromised patients including burn victims and those with neoplasia or HIV. This opportunistic pathogen is also the major cause of chronic lung infections in cystic fibrosis patients and microbial keratitis (MK) in users of extended-wear contact lenses. Though unlikely to cross-healthy, intact anatomical barriers, Pseudomonas bacteria from poorly maintained community hot tubs and swimming pools have been linked to rashes, UTIs and external ear infections in immune-competent individuals.
Research on P. aeruginosa has yielded much information on QS and biofilm formation. It uses several QS mechanisms to survive the harsh conditions on surfaces and within the host, as well as to circumvent the host immune system to cause disease. Figure 1 depicts several of these mechanisms. QS in P. aeruginosa depends on the release of a number of diffusible autoinducers which are divided into two groups based on their chemistry. The first group, the acyl homoserine lactones (AHLs) includes N-3-oxo-dodecanoyl-L-homoserine lactone (OdHL) and N-butanoyl-L-homoserine lactone (BHL), while the second group, the 4-quinolones (4Q) is represented by the Pseudomonas quinolone signal (PQS) (Brint & Ohman, 1995; Pearson et al, 1994, 1997; Pesci et al, 1999; Wilson et al, 1988). For these signals to be produced and sensed, the following QS systems are required: LasR–LasI and RhlR–RhlI for the AHLs, and PqsR/pqsABCDE for the PQS signal (Brint & Ohman, 1995; Farrow et al, 2008; Pearson et al, 1994; Pesci et al, 1999; Wade et al, 2005). LasI and RhlI synthesize OdHL and BHL, respectively, while the presence of pqsABCD is necessary for the synthesis of the PQS signal (Pesci et al, 1999; Wade et al, 2005). These signals bind to transcription regulators, and induce the expression of virulence genes such as exotoxins and proteases. OdHL binds to the LasR receptor, BHL binds to the RhlR receptor, and PQS to the PqsR (MvfR) LysR-like receptor (Brint & Ohman, 1995; Farrow et al, 2008; Pearson et al, 1994; Pesci et al, 1999; Wade et al, 2005). The activities of these QSIs are summarized in Fig 1 and Table 1.
The development of Pseudomonas biofilms relies on some of the QS signals described (Davies et al, 1998). The biofilm matrix provides a physical barrier that binds to or neutralizes host anti-microbials. Within the biofilm, Pseudomonas has a higher tolerance to heavy metals such as zinc, copper and lead than it would in its planktonic form (Whiteley et al, 2001). This tolerance prevents metal-based drugs from being able to clear chronic infections. The microenvironment in the biofilm is markedly different from that of the surrounding environment; low oxygen levels and low metabolic activity within the biofilm contribute to the disruption of the efficacy of anti-microbial drugs like tetracycline (Walters et al, 2003; Whiteley et al, 2001). Pseudomonas is capable of causing chronic (CF lung) and acute (ventilator associated/invasive) infections. Biofilms have been associated with chronic infections, while a mechanism relying on type III secretion (TSS) has been associated with acute infections. The type III secretion system (TTSS) provides bacterial pathogens, with a structure that channels effector proteins into the cytoplasm of eukaryotic host cells (Tampakaki et al, 2004). AHL-dependent QS has been reported to activate the expression of genes necessary for biofilm formation and repress genes encoding the TTSS (Bleves et al, 2005). However, a recent report by Mikkelsen et al (2009) showed that the type III effector exoenzyme S (ExoS) can also be detected in biofilm effluents but not in a planktonic cell supernatant. These results indicate that biofilms can also express the TTSS releasing effectors, in this case ExoS which is responsible for ocular damage in MK. The perceived contradictory reports by Bleeves and Mikkelsen could be reconciled by the possibility that while AHL-dependent QS may repress TTSS expression, it may not completely obliterate it, and other QS signals involved in biofilm formation could modulate the TTSS expression within biofilms.
In addition to being regulators of virulence genes and biofilm formation, the pathogen's QS signals can also modulate the host immune response. PQS and OdHL have been shown to induce apoptosis in neutrophils and macrophages during MK infections (Hoiby et al, 2001; Willcox et al, 2008; Zhu et al, 2002). Recent research into the effects of P. aeruginosa QS signals has also identified a role in the modulation of dendritic cell (DC) activity (Skindersoe et al, 2009). Skindersoe et al showed that PQS and OdHL divert TH1 cell differentiation towards TH2 cell differentiation ex vivo using cultured DCs.
Targeting QS and biofilm formation in P. aeruginosa
Conventional antibiotics work by either preventing bacterial cell division (bacteriostatic) or killing the cell (bacteridicial). However, this may increase the selective pressure towards antibiotic resistance. Targeting QS and biofilms provides an alternative that, in theory, applies a gentler evolutionary pressure towards development of drug resistance, given that QS does not control processes essential for cellular survival and/or growth. Although conventional antibiotics are used as anti-microbial drugs, some believe their function in nature at physiologically lower levels (subinhibitority anti-microbial levels) may be able to induce or interfere with QS signalling and even promote biofilm formation (Hoffman et al, 2005). Subinhibitory levels of aminoglicosides have been shown to promote biofilm development by Pseudomonas and Escherichia coli (Hoffman et al, 2005). Azithromycin (AZM) is an inhibitor of protein synthesis and is one of the few antibiotics that improves the clinical outcome of CF patients chronically infected with P. aeruginosa. Skindersoe et al showed that AZM and two other antibiotics, ceftazidime and ciprofloxacin also affect QS, possibly by altering the membrane permeability and affecting the OdHL flux. However, these antibiotics still have bacteridicial and bacteriostatic activities which may lead to the same problem of selective pressure and antibiotic resistance (Tramper-Stranders et al, 2007). These reports suggest that antibiotics may play a role in signalling in nature, as well as ‘killing’, and it will be advantageous to identify QSIs that while attenuating virulence do not affect essential bacterial processes.
Research into P. aeruginosa has provided vast information on QS and biofilms but also on potential drugs or molecules that could target these mechanisms (see Table 1). In 2006, Muh et al performed a high-throughput screening (HTS) on a library of 200,000 small compounds and identified two that were general inhibitors of QS (Muh et al, 2006). The compounds, PD12, a tetrazole with a 12-carbon alkyl tail and V-06-018, a phenyl ring with a 12-carbon alkyl tail, also inhibited the expression of the QS regulated virulence factor pyocyanin, by antagonizing LasR (the OdHL receptor). These QSIs added to the library of compounds previously identified by Smith et al (2003a, b; Suga & Smith, 2003) who showed that 2-aminocyclohexanone and 2-aminocyclopentanone are potent LasR antagonists. These QSIs and the QS signal OdHL share a chemical backbone but the QSIs have antagonistic activity on the OdHL receptor preventing the activation of downstream virulence factors. Another QSI, C-30, a derivative of a natural furanone was shown to inhibit QS and cause Pseudomonas biofilms to be susceptible to clearance by detergent and antibiotics when the biofilms were grown in its presence (Hentzer et al, 2003; Manefield et al, 2002). Although these molecules were shown to work in vitro to prevent pathogenesis, their activity in vivo in animal models remains to be addressed.
As discussed previously, QS signals can interfere with the host immune response, OdHL was shown to induce apoptosis of several cell types including macrophages and neutrophils by upregulating pro-inflammatory cytokines and chemokines (Shiner et al, 2006; Smith et al, 2001). However, how the bacterial signals were affecting the mammalian cells was unknown until 2008 when Jahoor showed that OdHL acted as an agonist to peroxisome proliferator-activated receptor β (PPARβ) and PPARδ while acting as a PPARγ antagonist. PPARγ is a trans acting repressor of the cytokine genes' transcription factor NF-κB (Pascual et al, 2005) and, by antagonizing PPARγ activity, OhDL was able to relieve the NF-κB transrepression, thus activating an inflammatory response and consequently causing tissue damage. The identification of the AHL mammalian receptors PPARβ/δ/γ also provided researchers and clinicians with potential therapeutic targets. Rosiglitazone, a PPARγ agonist was shown to block the pro-inflammatory effect of OdHL in lung epithelial cells (Jahoor et al, 2008). Identification of other PPARγ agonists may provide additional anti-inflammatory therapeutics for P. aeruginosa infections.
While targeting QS signalling is expected to have an impact on biofilm formation, several metals have direct anti-biofilm activity. Ionic silver has long been known to have anti-bacterial activity and when used in wound dressing clears planktonic P. aeruginosa infections (Melaiye et al, 2005). Chronically infected wounds, however, harbour mature biofilms that require higher silver concentrations than planktonic cells (Bjarnsholt et al, 2007). Therefore, it would be advisable that clinicians assess wounds to identify the form of colonizing Pseudomonas. If colonized by biofilms, dressing with higher ionic silver concentration should be used and chronic wounds should have their dressings frequently changed to allow for maximum silver dependent anti-microbial activity. Another metal, gallium (Ga), has also been shown to prevent the formation of biofilms that are characteristic of chronic infections (Yamamoto et al, 1994). Ga works by competing out iron (Fe) that is necessary for bacterial metabolism and also serves as a cue for biofilm formation (Banin et al, 2005, 2006). Recently, Banin et al showed that by coupling Ga to the siderophore desferrioxamine (DFO) they could form a complex (DFO–Ga) that acted like a Trojan horse (Banin et al, 2008; Patriquin et al, 2008). DFO–Ga is taken up by the bacterial cell and, once inside, it interferes with Fe metabolism and consequently biofilm formation.
Another anti-biofilm compound is nitric oxide (NO). NO is a reactive-free radical normally produced by phagocytes as part of the immune response to bacteria (Ghaffari et al, 2006). It has been shown that small molecules that release NO have anti-microbial properties and are capable of degrading biofilms (Barraud et al, 2006; De Groote & Fang, 1995). Recent studies presented a delivery system that provides a means to fine-tune the distribution of NO into biofilms (Hetrick et al, 2008, 2009). The authors use NO releasing silica nanoparticles to create a rapid diffusion that kills cells within established biofilms more effectively than a slow prolonged delivery.