RESULTS:

Antimicrobial resistance poses an escalating global threat rendering traditional drug development approaches increasingly ineffective. Thus novel alternatives to antibiotic-based therapies are needed. Exploiting pathogen cooperation as a strategy for combating resistant infections has been proposed but lacks experimental validation. Empirical findings demonstrate the successful invasion of cooperating populations by non-cooperating cheats effectively reducing virulence in vitro and in vivo. The idea of harnessing cooperative behaviours for therapeutic benefit involves exploitation of the invasive capabilities of cheats to drive medically beneficial traits into infecting populations of cells. In this study we employed Pseudomonas aeruginosa quorum sensing cheats to drive antibiotic sensitivity into both in vitro and in vivo resistant populations. We demonstrated the successful invasion of cheats followed by increased antibiotic effectiveness against cheat-invaded populations thereby establishing an experimental proof of principle for the potential application of the Trojan strategy in fighting resistant infections.

Pseudomonas aeruginosa uses quorum sensing (QS) to coordinate the expression of multiple genes necessary for establishing and maintaining infection. It has previously been shown that lasR QS mutations frequently arise in cystic fibrosis (CF) lung infections however there has been far less emphasis on determining whether other QS system mutations arise during infection or in other environments. To test this we utilized 852 publicly available sequenced P. aeruginosa genomes from the Pseudomonas International Consortium Database (IPCD) to study P. aeruginosa QS mutational signatures. To study isolates by source we focused on a subset of 654 isolates collected from CF wounds and non-infection environmental isolates where we could clearly identify their source. We also worked with a small collection of isolates in vitro to determine the impact of lasR and pqs mutations on isolate phenotypes. We found that lasR mutations are common across all environments and are not specific to infection nor a particular infection type. We also found that the pqs system proteins PqsA PqsH PqsL and MexT a protein of increasing importance to the QS field are highly variable. Conversely RsaL a negative transcriptional regulator of the las system was found to be highly conserved suggesting selective pressure to repress las system activity. Overall our findings suggest that QS mutations in P. aeruginosa are common and not limited to the las system; however LasR is unique in the frequency of putative loss-of-function mutations.

In the opportunistic pathogen Pseudomonas aeruginosa quorum sensing (QS) is a social trait that is exploitable by non-cooperating cheats. Previously it has been shown that by linking QS to the production of both public and private goods cheats can be prevented from invading populations of cooperators and this was described by Dandekar et al. (Science 2012;338:264–266) as ‘a metabolic incentive to cooperate’. We hypothesized that P. aeruginosa could evolve novel cheating strategies to circumvent private goods metabolism by rewiring its combinatorial response to two QS signals (3O-C12-HSL and C4-HSL). We performed a selection experiment that cycled P. aeruginosa between public and private goods growth media and evolved an isolate that rewired its control of cooperative protease expression from a synergistic (AND-gate) response to dual-signal input to a 3O-C12-HSL-only response. We show that this isolate circumvents metabolic incentives to cooperate and acts as a combinatorial signalling cheat with higher fitness in competition with its ancestor. Our results show three important principles: first combinatorial QS allows for diverse social strategies to emerge; second restrictions levied by private goods are not sufficient to explain the maintenance of cooperation in natural populations; and third modifying combinatorial QS responses could result in important physiological outcomes in bacterial populations.

Here we highlight the development of a simple and high-throughput mung bean model to study virulence in the opportunistic pathogen Pseudomonas aeruginosa. The model is easy to set up and infection and virulence can be monitored for up to 10 days. In a first test of the model we found that mung bean seedlings infected with PAO1 showed poor development of roots and high mortality rates compared to uninfected controls. We also found that a quorum-sensing (QS) mutant was significantly less virulent when compared with the PAO1 wild-type. Our work introduces a new tool for studying virulence in P. aeruginosa that will allow for high-throughput virulence studies of mutants and testing of the in vivo efficacy of new therapies at a time when new antimicrobial drugs are desperately needed.

Burkholderia pseudomallei is the causative agent of melioidosis a fatal human tropical disease. The non-specific DNA-binding protein DpsA plays a key role in protecting B. pseudomallei from oxidative stress mediated for example by organic hydroperoxides. The regulation of dpsA expression is poorly understood but one possibility is that it is regulated in a cell population density-dependent manner via N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) since a lux-box motif has been located within the dpsA promoter region. Using liquid chromatography and tandem mass spectrometry it was first established that B. pseudomallei strain PP844 synthesizes AHLs. These were identified as N-octanoylhomoserine lactone (C8-HSL) N-(3-oxooctanoyl)homoserine lactone (3-oxo-C8-HSL) N-(3-hydroxyoctanoyl)-homoserine lactone (3-hydroxy-C8-HSL) N-decanoylhomoserine lactone (C10-HSL) N-(3-hydroxydecanoyl) homoserine lactone (3-hydroxy-C10-HSL) and N-(3-hydroxydodecanoyl)homoserine lactone (3-hydroxy-C12-HSL). Mutation of the genes encoding the LuxI homologue BpsI or the LuxR homologue BpsR resulted in the loss of C8-HSL and 3-oxo-C8-HSL synthesis demonstrating that BpsI was responsible for directing the synthesis of these AHLs only and that bpsI expression and hence C8-HSL and 3-oxo-C8-HSL production depends on BpsR. In bpsI bpsR and bpsIR mutants dpsA expression was substantially down-regulated. Furthermore dpsA expression in Escherichia coli required both BpsR and C8-HSL. bpsIR-deficient mutants exhibited hypersensitivity to the organic hydroperoxide tert-butyl hydroperoxide by displaying a reduction in cell viability which was restored by provision of exogenous C8-HSL (bpsI mutant only) by complementation with the bpsIR genes or by overexpression of dpsA. These data indicate that in B. pseudomallei QS regulates the response to oxidative stress at least in part via the BpsR/C8-HSL-dependent regulation of DpsA.

Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes. Two such regulatory genes ptxR and ptxS which are divergently transcribed from each other have been described previously. While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins the mechanism that regulates the expression of either ptxR or ptxS in P. aeruginosa is not known. The presence of a P. aeruginosa protein complex that specifically binds to a segment within this region was determined. In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P. aeruginosa HU and MvaT. Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region. A PAO1 isogenic mutant defective in mvaT PAOΔmvaT was constructed and characterized. The lysate of PAOΔmvaT failed to bind to the 150 bp probe. The effect of mvaT on ptxS and ptxR expression was examined using real-time PCR experiments. The expression of ptxS was lower in PAOΔmvaT than in PAO1 but no difference was detected in ptxR expression. These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.