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Pseudomonas aeruginosa forms biofilms in the cystic fibrosis lung. Quorum sensing (QS) controls biofilm maturation, immune evasion, antibiotic tolerance and virulence factor production. Garlic shows QS inhibitory activity in vitro and in animal models. We report the first clinical trial in man of a QS inhibitor.We randomized 34 patients to garlic or olive oil capsules (both 656 mg daily). Clinical outcomes and safety bloods were measured at baseline and after 8 weeks treatment. In this exploratory study, analysis was per protocol.Eight patients withdrew, leaving 26 for analysis (13 garlic). With placebo, there was a greater decline in mean (SD) percentage change from baseline FEV(1) [-3.6% (11.3)] than with garlic [-2.0% (12.3)]. This was not significant (mean difference = 1.6, 95% CI -12.7 to 15.9, P = 0.8). The mean (SD) increase in weight was greater with garlic [1.0% (2.0)] than with placebo [0.6% (2.0)]--non-significant (mean difference = 0.4%, 95% CI -1.3 to 2.0, P = 0.6). The median (range) change in clinical score with garlic was -1 (-3 to 5) and 1 (-1 to 4) with placebo (negative score means improvement). This was non-significant [median difference = -1 (-3 to 0), P = 0.16]. In the garlic group, seven patients had IV antibiotics versus five placebo. There was a highly significant correlation between plasma and sputum measurements of the QS molecule 3-oxo-C12-HSL (Pearson correlation coefficient = 0.914, P = 0.004). At the end of treatment five patients in each group had abnormal liver function or triglycerides and five garlic patients (one placebo) reported minor adverse effects.Garlic capsules were well tolerated. Although there was no significant effect of garlic compared to placebo in this pilot study, there was a suggestion of improvement with garlic which should be investigated in a larger trial.

In this paper we propose an extension of the modelling framework based on P systems which explicitly includes modularity. Modularisation in cellular systems can be produced by chemical specificity and spatial localisation in different compartments. Both these features can be easily specified and analysed in P systems using sets of rewriting rules to describe chemical specificity and membranes to represent spatial localisation. Our methodology enables the assembly of cell systems biology models by combining modules which represents functional subsystems. A case study consisting of a bacterial colony system is presented to illustrate our approach.

In this work we present a new methodology for structure and parameter estimation in cell systems biology modelling. Our modelling framework is based on P systems, an unconventional computational paradigm that abstracts from the structure and functioning of the living cell. The process of designing models, consisting of both the optimisation of the modular structure and of the stochastic kinetic parameters, is performed using a memetic algorithm. Specically, we use a nested evolutionary algorithm where the first layer evolves rule structures while the inner layer, implemented also as a genetic algorithm (GA), fine tunes the parameters of the model. Our approach consists of an incremental methodology. Starting from very simple P system modules specifying basic molecular interactions, more complicated modules are produced to model more complex molecular systems. These newly found modules are in turn added to the library of available P systems modules so as to be used subsequently to develop more intricate and circuitous cellular models. The effectiveness of the algorithm was tested on three case studies, namely, molecular complexation, enzymatic reactions and autoregulation in transcriptional networks.

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA–E operon (termed hhqA–E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.

Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

Pseudomonas aeruginosa regulates the production of many exoproteins and secondary metabolites via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-L-homoserine lactone (C4-HSL). In this study, we found that transcription of the quorum sensing-regulated genes lecA (coding for PA-IL lectin), lasB (coding for elastase), and rpoS appeared to be growth phase dependent and their expression could not be advanced to the logarithmic phase in cells growing in batch culture by the addition of exogenous C4-HSL and 3O-C12-HSL. To identify novel regulators responsible for this growth phase dependency, a P. aeruginosa lecA::lux reporter strain was subjected to random transposon mutagenesis. A number of mutants affected in lecA expression were found that exhibited altered production of multiple quorum sensing-dependent phenotypes. While some mutations were mapped to new loci such as clpA and mvaT and a putative efflux system, a number of mutations were also mapped to known regulators such as lasR, rhlR, and rpoS. MvaT was identified as a novel global regulator of virulence gene expression, as a mutation in mvaT resulted in enhanced lecA expression and pyocyanin production. This mutant also showed altered swarming ability and production of the LasB and LasA proteases, 3O-C12-HSL, and C4-HSL. Furthermore, addition of exogenous 3O-C12-HSL and C4-HSL to the mvaT mutant significantly advanced lecA expression, suggesting that MvaT is involved in the growth phase-dependent regulation of the lecA gene.

Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either N-acylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant protein revealed that LuxS levels increased throughout growth even though AI-2 activity was only maximally detected at the mid-exponential phase of growth and disappeared by the onset of stationary phase. Similar results were obtained with E. coli DH5 transformed with luxS, suggesting that AI-2 production is not limited by a lack of LuxS protein. Analysis of Rgp and Kgp protease activities revealed that the P. gingivalis luxS mutant produced around 45% less Rgp and 30% less Kgp activity than the parent strain. In addition, the luxS mutant exhibited a fourfold reduction in haemagglutinin titre. However, these reductions in virulence determinant levels were insufficient to attenuate the luxS mutant in a murine lesion model of P. gingivalis infection.