Reciprocal regulation by the CepIR and CciIR quorum sensing systems in Burkholderia cenocepacia

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia </it>belongs to a group of closely related organisms called the <it>B. cepacia </it>complex (Bcc) which are important opportunistic human pathogens. <it>B. cenocepacia </it>utilizes a mechanism of cell-cell communication called quorum sensing to control gene expression including genes involved in virulence. The <it>B. cenocepacia </it>quorum sensing network includes the CepIR and CciIR regulatory systems.</p> <p>Results</p> <p>Global gene expression profiles during growth in stationary phase were generated using microarrays of <it>B. cenocepacia cepR</it>, <it>cciR </it>and <it>cepRcciIR </it>mutants. This is the first time CciR was shown to be a global regulator of quorum sensing gene expression. CepR was primarily responsible for positive regulation of gene expression while CciR generally exerted negative gene regulation. Many of the genes that were regulated by both quorum sensing systems were reciprocally regulated by CepR and CciR. Microarray analysis of the <it>cepRcciIR </it>mutant suggested that CepR is positioned upstream of CciR in the quorum sensing hierarchy in <it>B. cenocepacia</it>. A comparison of CepIR-regulated genes identified in previous studies and in the current study showed a substantial amount of overlap validating the microarray approach. Several novel quorum sensing-controlled genes were confirmed using qRT-PCR or promoter::<it>lux </it>fusions. CepR and CciR inversely regulated flagellar-associated genes, the nematocidal protein AidA and a large gene cluster on Chromosome 3. CepR and CciR also regulated genes required for iron transport, synthesis of extracellular enzymes and surface appendages, resistance to oxidative stress, and phage-related genes.</p> <p>Conclusion</p> <p>For the first time, the influence of CciIR on global gene regulation in <it>B. cenocepacia </it>has been elucidated. Novel genes under the control of the CepIR and CciIR quorum sensing systems in <it>B. cenocepacia </it>have been identified. The two quorum sensing systems exert reciprocal regulation of many genes likely enabling fine-tuned control of quorum sensing gene expression in <it>B. cenocepacia </it>strains carrying the cenocepacia island.</p

Quorum sensing as a mechanism to harness the wisdom of the crowds

Bacteria release and sense small molecules called autoinducers in a process known as quorum sensing. The prevailing interpretation of quorum sensing is that by sensing autoinducer concentrations, bacteria estimate population density to regulate the expression of functions that are only beneficial when carried out by a sufficiently large number of cells. However, a major challenge to this interpretation is that the concentration of autoinducers strongly depends on the environment, often rendering autoinducer-based estimates of cell density unreliable. Here we propose an alternative interpretation of quorum sensing, where bacteria, by releasing and sensing autoinducers, harness social interactions to sense the environment as a collective. Using a computational model we show that this functionality can explain the evolution of quorum sensing and arises from individuals improving their estimation accuracy by pooling many imperfect estimates – analogous to the ‘wisdom of the crowds’ in decision theory. Importantly, our model reconciles the observed dependence of quorum sensing on both population density and the environment and explains why several quorum sensing systems regulate the production of private goods.</p

Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa

Numerous species of bacteria use an elegant regulatory mechanism known as quorum sensing to control the expression of specific genes in a cell-density dependent manner. In Gram-negative bacteria, quorum sensing systems function through a cell-to-cell signal molecule (autoinducer) that consists of a homoserine lactone with a fatty acid side chain. Such is the case in the opportunistic human pathogen Pseudomonas aeruginosa, which contains two quorum sensing systems (las and rhl) that operate via the autoinducers, N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-Lhomoserine lactone. The study of these signal molecules has shown that they bind to and activate transcriptional activator proteins that specifically induce numerous P. aeruginosa virulence genes. We report here that P. aeruginosa produces another signal molecule, 2-heptyl-3-hydroxy-4-quinolone, which has been designated as the Pseudomonas quinolone signal. It was found that this unique cell-to-cell signal controlled the expression of lasB, which encodes for the major virulence factor, LasB elastase. We also show that the synthesis and bioactivity of Pseudomonas quinolone signal were mediated by the P. aeruginosa las and rhl quorum sensing systems, respectively. The demonstration that 2-heptyl-3- hydroxy-4-quinolone can function as an intercellular signal sheds light on the role of secondary metabolites and shows that P. aeruginosa cell-to-cell signaling is not restricted to acyl-homoserine lactones. Originally published Proc. Natl. Acad. Sci, Vol. 96, No. 20, Sep. 199

New vaccines for infectious diseases: immunological targeting of the quorum sensing system of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen of animals and humans causing medical complications in burns, wounds, and cystic fibrosis. P. aeruginosa is efficient at adapting its virulence phenotype depending on the site of infection. Emerging multi-drug resistant strains and a limited number of effective anti-pseudomonal antibiotics renders P. aeruginosa infections increasingly difficult to treat. To address this need, this thesis considers targeting bacterial quorum sensing, which regulates the production of virulence factors, as an alternative prophylactic strategy. The P. aeruginosa quorum sensing system is compromised of three interlinked but independent systems, Las, Rhl and Pqs, which produce and utilise quorum sensing system molecules, 3OC12-HSL, C4-HSL and PQS respectively. Immunological targeting of the quorum sensing system molecule 3OC12-HSL, through active immunisation (vaccines), inhibits the Las system, resulting in a longer life expectancy in mice infected with P. aeruginosa in vivo. However, P. aeruginosa has the capacity to develop resistance, through compensatory mechanisms, towards quorum sensing inhibition that targets the Las system only. This emphasises the need to target all three quorum sensing systems, Las, Rhl, and the Pqs, in order to inhibit quorum sensing. The present study focuses particularly on the development of a multi-component anti-quorum sensing system vaccine that would target the three main quorum sensing system molecules, effectively compromising the quorum sensing system and minimising compensatory mechanisms. This involved the synthesis of haptens based on the quorum sensing system molecules, which were used to haptenise the immunogenic carrier keyhole limpet haemocyanin. Syntheses of the haptens, AP1 (derivative of 3OC8-HSL) and AP2 (derivative of PQS), were conducted using adapted published methods. The resulting conjugates, AP1-keyhole limpet haemocyanin and AP2-keyhole limpet haemocyanin were immunogenic in mice and rabbits. The specific anti-hapten polyclonal antibodies that were generated demonstrated cross-reactivity with the natural quorum sensing system molecules of P. aeruginosa that translated in significant and specific anti-quorum sensing system molecule activity in bioluminescence reporter assays. Anti-AP1 polyclonal antibodies were able to reduce biofilm formation at high concentrations, however, significant reduction of biofilm formation was seen when the anti-hapten antibodies were used in combination. In this study, it has been demonstrated that the inhibition of the quorum sensing system should include the three systems, Las, Rhl and PQS, and that this can be done by a multi-component anti-quorum sensing system vaccine. These data suggest that a multi-component anti-quorum sensing system vaccine takes us a step forward to a viable prophylaxis against P. aeruginosa for susceptible patients

Synthesis and biological activity of novel quorum sensing compounds

Bacteria communicate with chemical signals in a process known as quorum sensing. This population density-dependent process involves the bacterial production, release and detection of structurally specific small molecules and enables the bacterial pathogen to regulate its virulence on a population-wide level. Using a variety of chemical and biological techniques, I have studied various quorum sensing systems in several bacteria, including Vibrio cholera and Pseudomonas aeruginosa. A key principle of this research involves the design, synthesis and testing of novel compounds for their biological activity. These molecules are typically based off of an initial lead target, which is often identified from a high-throughput screen and serves as a template for further optimization. Specifically, I have researched quorum sensing compounds that affect Hfq-RNA interactions in V. cholera, the LasR receptor in P. aeuruginosa and HapR in V. cholera. Taken together, the results of these studies provide a basis for future investigations involving quorum sensing, and demonstrate how organic chemistry can be employed to study these fascinating biochemical systems

Towards a P Systems Pseudomonas Quorum Sensing Model

Pseudomonas aeruginosa is an opportunistic bacterium that exploits quorum sensing communication to synchronize individuals in a colony and this leads to an increase in the effectiveness of its virulence. In this paper we derived a mechanistic P systems model to describe the behavior of a single bacterium and we discuss a possible approach, based on an evolutionary algorithm, to tune its parameters that will allow a quantitative simulation of the system.Kingdom's Engineering and Physical Sciences Research Council EP/D021847/

Examining effects of the DNA regulator Lrp on quorum sensing gene expression in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that has the capacity to express multiple virulence factors that are regulated through an extensive quorum sensing network. Three major quorum sensing systems have been identified in Pseudomonas species: the acyl homoserine lactones of las and rhl, and the Pseudomonas Quinolone Signal (PQS). We seek to investigate the involvement of a global regulator, Lrp with the expression of these three networks. Specifically, we will compare expression levels of las, rhl, and pqs in wild type P. aeruginosa (MPAO1) with an lrp transposon insertion mutant using quantitative PCR. Through this comparative qPCR analysis, we hope to support the identification of novel roles of the Lrp DNA regulator involvement in cross-talk with the quorum sensing pathways that has not been previously recognized. Due to the virulence of Pseudomonas aeruginosa, if Lrp can be identified as a factor in the regulation of the quorum sensing networks, it could potentially be used as a therapeutic target in the disruption of the production of many virulence factors such biofilms, siderophores, toxins and motility which are all regulated by the quorum sensing networks

Microfluidics-based liquid chromatography/mass spectrometry multiple reaction monitoring approach for the relative quantification of Burkholderia cenocepacia secreted virulence factors

Rationale: Burkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking. Methods: Based on results of a label-free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors. Results: Increase in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non-physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF. Conclusions: Microfluidics-based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens

Stochastic Approaches in P Systems for Simulating Biological Systems

Different stochastic strategies for modeling biological systems with P systems are reviewed in this paper, such as the multi-compartmental approach and dynamical probabilistic P systems. The respective results obtained from the simulations of a test case study (the quorum sensing phenomena in Vibrio Fischeri colonies) are shown, compared and discussed

New vaccines for infectious diseases: immunological targeting of the quorum sensing system of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen of animals and humans causing medical complications in burns, wounds, and cystic fibrosis. P. aeruginosa is efficient at adapting its virulence phenotype depending on the site of infection. Emerging multi-drug resistant strains and a limited number of effective anti-pseudomonal antibiotics renders P. aeruginosa infections increasingly difficult to treat. To address this need, this thesis considers targeting bacterial quorum sensing, which regulates the production of virulence factors, as an alternative prophylactic strategy. The P. aeruginosa quorum sensing system is compromised of three interlinked but independent systems, Las, Rhl and Pqs, which produce and utilise quorum sensing system molecules, 3OC12-HSL, C4-HSL and PQS respectively. Immunological targeting of the quorum sensing system molecule 3OC12-HSL, through active immunisation (vaccines), inhibits the Las system, resulting in a longer life expectancy in mice infected with P. aeruginosa in vivo. However, P. aeruginosa has the capacity to develop resistance, through compensatory mechanisms, towards quorum sensing inhibition that targets the Las system only. This emphasises the need to target all three quorum sensing systems, Las, Rhl, and the Pqs, in order to inhibit quorum sensing. The present study focuses particularly on the development of a multi-component anti-quorum sensing system vaccine that would target the three main quorum sensing system molecules, effectively compromising the quorum sensing system and minimising compensatory mechanisms. This involved the synthesis of haptens based on the quorum sensing system molecules, which were used to haptenise the immunogenic carrier keyhole limpet haemocyanin. Syntheses of the haptens, AP1 (derivative of 3OC8-HSL) and AP2 (derivative of PQS), were conducted using adapted published methods. The resulting conjugates, AP1-keyhole limpet haemocyanin and AP2-keyhole limpet haemocyanin were immunogenic in mice and rabbits. The specific anti-hapten polyclonal antibodies that were generated demonstrated cross-reactivity with the natural quorum sensing system molecules of P. aeruginosa that translated in significant and specific anti-quorum sensing system molecule activity in bioluminescence reporter assays. Anti-AP1 polyclonal antibodies were able to reduce biofilm formation at high concentrations, however, significant reduction of biofilm formation was seen when the anti-hapten antibodies were used in combination. In this study, it has been demonstrated that the inhibition of the quorum sensing system should include the three systems, Las, Rhl and PQS, and that this can be done by a multi-component anti-quorum sensing system vaccine. These data suggest that a multi-component anti-quorum sensing system vaccine takes us a step forward to a viable prophylaxis against P. aeruginosa for susceptible patients