Promoter Specificity Elements in Pseudomonas aeruginosa Quorum-Sensing-Controlled Genes

The LasR-dependent and RhlR-dependent quorum-sensing systems are global regulators of gene expression in Pseudomonas aeruginosa. Previous studies have demonstrated that promoter elements of the quorum-sensing-controlled genes lasB and hcnABC are important in density-dependent regulation. We have identified LasR- and RhlR-dependent determinants in promoters of quorum-sensing-controlled genes qsc102, qsc117 (acpP), and qsc131 (phzA to -G) by in silico, deletion, point-mutational, and primer extension analyses. Each of these genes (in addition to lasI and rsaL) is activated by LasR, and qsc117 and qsc131 also respond to RhlR. Point mutations in the promoters of the LasR-specific gene, qsc102, relax specificity so that this promoter can respond to RhlR in addition to LasR. Our findings indicate that quorum-sensing-controlled promoters in P. aeruginosa are either specific for LasR or respond to both LasR and RhlR and that critical bases in the promoter elements determine specificity.     

Interspecies communication between Burkholderia cepacia and Pseudomonas aeruginosa

Burkholderia cepacia and Pseudomonas aeruginosa are opportunistic pathogens that commonly cause pulmonary infections in cystic fibrosis patients and occasionally co-infect patients' lungs. Both organisms possess quorum-sensing systems dependent on N-acyl homoserine lactone (N-acyl-HSL). Cross-feeding assays demonstrated that P. aeruginosa and B. cepacia were able to utilize heterologous N-acyl-HSL signaling molecules. The ability of quorum-sensing genes from one species to complement the respective quorum-sensing mutations in the heterologous species was also examined. These studies suggest that B. cepacia CepR can use N-acyl-HSLs synthesized by RhlI and LasI and that P. aeruginosa LasR and RhlR can use N-acyl-HSLs synthesized by CepI. It is possible that a mixed bacterial population of B. cepacia and P. aeruginosa can coordinately regulate some of their virulence factors and influence the progression of lung disease due to infection with these organisms.     

The Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo

Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P. aeruginosa had on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P. aeruginosa to colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice. In this model, 3O-C(12)-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1alpha (IL-1alpha) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1beta, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections. Therefore, the quorum-sensing systems of P. aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.     

Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which "natural" conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.     

Inhibitors of the Pseudomonas aeruginosa quorum‐sensing regulator,QscR

QscR is a quorum‐sensing (QS) signal receptor that controls expression of virulence genes in the prevalent opportunistic pathogen, Pseudomonas aeruginosa. Unlike the previously reported LuxR‐type QS receptor proteins, that is, LasR and TraR, QscR can be obtained as an apo‐protein that can reversibly form an active complex in vitro with its cognate signal molecule, 3‐oxododecanoyl‐homoserine lactone (3OC12‐HSL), and subsequently bind to target promoter DNA sequences. To search for potential QS inhibitors, an in vitro gel retardation assay was developed using the purified QscR. Both the in vitro assay and the in vivo cell‐based assay using QscR‐overproducing recombinant strains were applied in the screening process. Furanones were chosen for testing the activity as QS inhibitors because they have been reported to strongly inhibit expression of QS‐related genes in Agrobacterium tumefaciens. Among more than a hundred furanones tested, three compounds showed strong and dose‐dependent inhibitory effects on QscR in both assays. One compound in particular, designated as F2, could completely inhibit the 3OC12‐HSL‐dependent QscR activity in vitro at a concentration of 50‐fold molar excess over 3OC12‐HSL. However, with the furanones F3 and F4, which are structurally similar to F2 but with a nitro group instead of the amine moiety, significantly decreased activities were observed. These results suggest that (i) the in vitro assay is a sensitive and reliable tool for screening QS inhibitors, and (ii) furanones are potentially important QS inhibitors for many LuxR‐type receptor proteins. Biotechnol. Bioeng. 2010; 106: 119–126. © 2010 Wiley Periodicals, Inc.