Pseudomonas aeruginosa biofilm formation on endotracheal tubes requires multiple two-component systems.

Introduction. Indwelling medical devices such as endotracheal tubes (ETTs), urinary catheters, vascular access devices, tracheostomies and feeding tubes are often associated with hospital-acquired infections. Bacterial biofilm formed on the ETTs in intubated patients is a significant risk factor associated with ventilator-associated pneumonia. Pseudomonas aeruginosa is one of the four frequently encountered bacteria responsible for causing pneumonia, and the biofilm formation on ETTs. However, understanding of biofilm formation on ETT and interventions to prevent biofilm remains lagging. The ability to sense and adapt to external cues contributes to their success. Thus, the biofilm formation is likely to be influenced by the two-component systems (TCSs) that are composed of a membrane-associated sensor kinase and an intracellular response regulator.Aim. This study aims to establish an in vitro method to analyse the P. aeruginosa biofilm formation on ETTs, and identify the TCSs that contribute to this process.Methodology. In total, 112 P. aeruginosa PA14 TCS mutants were tested for their ability to form biofilm on ETTs, their effect on quorum sensing (QS) and motility.Results. Out of 112 TCS mutants studied, 56 had altered biofilm biomass on ETTs. Although the biofilm formation on ETTs is QS-dependent, none of the 56 loci controlled quorum signal. Of these, 18 novel TCSs specific to ETT biofilm were identified, namely, AauS, AgtS, ColR, CopS, CprR, NasT, KdpD, ParS, PmrB, PprA, PvrS, RcsC, PA14_11120, PA14_32580, PA14_45880, PA14_49420, PA14_52240, PA14_70790. The set of 56 included the GacS network, TCS proteins involved in fimbriae synthesis, TCS proteins involved in antimicrobial peptide resistance, and surface-sensing. Additionally, several of the TCS-encoding genes involved in biofilm formation on ETTs were found to be linked to flagellum-dependent swimming motility.Conclusions. Our study established an in vitro method for studying P. aeruginosa biofilm formation on the ETT surfaces. We also identified novel ETT-specific TCSs that could serve as targets to prevent biofilm formation on indwelling devices frequently used in clinical settings.

Legionella quorum sensing meets cyclic-di-GMP signaling.

Bacterial gene regulation occurs through complex networks, wherein linear systems respond to intracellular or extracellular cues and engage on vivid crosstalk. The ubiquitous water-borne bacterium Legionella pneumophila colonizes various distinct environmental niches ranging from biofilms to protozoa, and - as an 'accidental' pathogen - the human lung. Consequently, L. pneumophila gene regulation evolved to integrate a broad spectrum of different endogenous and exogenous signals. Endogenous signals produced and detected by L. pneumophila comprise the quorum sensing autoinducer LAI-1 (3-hydroxypentadecane-4-one) and c-di-GMP. As an exogenous cue, nitric oxide controls the c-di-GMP regulatory network of L. pneumophila. The Legionella quorum sensing (Lqs) system regulates virulence, motility and natural competence of L. pneumophila. The Lqs system is linked to c-di-GMP signaling through the pleiotropic transcription factor LvbR, which also regulates the architecture of L. pneumophila biofilms. In this review, we highlight recent insights into the crosstalk of Legionella quorum sensing and c-di-GMP signaling.

Bacteria-Host Crosstalk: Sensing of the Quorum in the Context of Pseudomonas aeruginosa Infections.

Cell-to-cell signaling via small molecules is an essential process to coordinate behavior in single species within a community, and also across kingdoms. In this review, we discuss the quorum sensing (QS) systems used by the opportunistic pathogen Pseudomonas aeruginosa to sense bacterial population density and fitness, and regulate virulence, biofilm development, metabolite acquisition, and mammalian host defense. We also focus on the role of N-acylhomoserine lactone-dependent QS signaling in the modulation of innate immune responses connected together via calcium signaling, homeostasis, mitochondrial and cytoskeletal dynamics, and governing transcriptional and proteomic responses of host cells. A future perspective emphasizes the need for multidisciplinary efforts to bring current knowledge of QS into a more detailed understanding of the communication between bacteria and host, as well as into strategies to prevent and treat P. aeruginosa infections and reduce the rate of antibiotic resistance.

Long-term effects of N-acyl-homoserine lactone-based quorum sensing on the characteristics of ANAMMOX granules in high-loaded reactors.

Laboratory experiments were carried out to determine the long-term effects of N-acyl-homoserine lactone (AHL)-based quorum sensing on the characteristics of ANAMMOX granules in high-loaded reactors. Results clearly showed that adding 30 mg L-1 N-octanoyl-DL-homoserinelactone (C8-HSL) at the initial stage (1-40 d) of the experiment had long-term positive effects on the settleability of granules and controlled the sludge floatation effectively. C8-HSL decreased the content of bound extracellular polymeric substances (B-EPS) and the ratio of protein to carbohydrate (PN/PS) by 17% and 48%, respectively and increased the relative hydrophobicity (RH) of the granules by 28%. The results of batch tests indicated that C8-HSL significantly reduced the content of loosely-bound EPS (LB-EPS) in the B-EPS, which was responsible for variations in granule settleability and stability. Thus, the settleability of the granules was improved significantly due to addition of C8-HSL, contributing to operational stability and the high TN removal efficiency of the reactor. On day 150, when the nitrogen loading rates of all reactors were 13.4 kg TN m-3 d-1, the nitrogen removal rate and nitrogen removal efficiency of the reactor with C8-HSL (R3) were up to 11.2 kg TN m-3 d-1 and 88%, respectively. N-hexanoyl-DL-homoserine lactone (C6-HSL) improved activity of the granules, while N-dodecanoyl-DL-homoserine lactone (C12-HSL) had no effect on the characteristics of the granules. The long-term effects of C8-HSL on the settleability of granules may be attributed to sustainable release of endogenous signals induced by exogenous signal.

Stochastic Turing patterns in a synthetic bacterial population.

The origin of biological morphology and form is one of the deepest problems in science, underlying our understanding of development and the functioning of living systems. In 1952, Alan Turing showed that chemical morphogenesis could arise from a linear instability of a spatially uniform state, giving rise to periodic pattern formation in reaction-diffusion systems but only those with a rapidly diffusing inhibitor and a slowly diffusing activator. These conditions are disappointingly hard to achieve in nature, and the role of Turing instabilities in biological pattern formation has been called into question. Recently, the theory was extended to include noisy activator-inhibitor birth and death processes. Surprisingly, this stochastic Turing theory predicts the existence of patterns over a wide range of parameters, in particular with no severe requirement on the ratio of activator-inhibitor diffusion coefficients. To explore whether this mechanism is viable in practice, we have genetically engineered a synthetic bacterial population in which the signaling molecules form a stochastic activator-inhibitor system. The synthetic pattern-forming gene circuit destabilizes an initially homogenous lawn of genetically engineered bacteria, producing disordered patterns with tunable features on a spatial scale much larger than that of a single cell. Spatial correlations of the experimental patterns agree quantitatively with the signature predicted by theory. These results show that Turing-type pattern-forming mechanisms, if driven by stochasticity, can potentially underlie a broad range of biological patterns. These findings provide the groundwork for a unified picture of biological morphogenesis, arising from a combination of stochastic gene expression and dynamical instabilities.

Synergistic activity of cosecreted natural products from amoebae-associated bacteria.

Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant Pseudomonas strain, isolated from the bacterivorous amoeba Dictyostelium discoideum. Genome analysis of this bacterium revealed the presence of two biosynthetic gene clusters that were found adjacent to each other on a contiguous stretch of the bacterial genome. Although one cluster codes for the polyketide synthase producing the known antibiotic mupirocin, the other cluster encodes a nonribosomal peptide synthetase leading to the unreported cyclic lipopeptide jessenipeptin. We describe its complete structure elucidation, as well as its synergistic activity against methicillin-resistant Staphylococcus aureus, when in combination with mupirocin. Both biosynthetic gene clusters are regulated by quorum-sensing systems, with 3-oxo-decanoyl homoserine lactone (3-oxo-C10-AHL) and hexanoyl homoserine lactone (C6-AHL) being the respective signal molecules. This study highlights the regulation, richness, and complex interplay of bacterial natural products that emerge in the context of microbial competition.

Microbial homoserine lactones (AHLs) are effectors of root morphological changes in barley.

While colonizing the rhizosphere, bacterial intra- and inter-specific communication is accomplished by N-Acyl-homoserine-lactones (AHLs) in a density-dependent manner. Moreover, plants are naturally exposed to AHLs and respond with tissue-specificity. In the present study, we investigated the influence of N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL) and N-dodecanoyl-d/l-homoserine lactone (C12-HSL) on growth and root development in barley (Hordeum vulgare L.), and identified initial reactions in root cells after AHL exposures using physiological, staining, and electrophysiological methods. Treatment with short- and long-chain AHLs modulated plant growth and branched root architecture and induced nitric oxide (NO) accumulation in the calyptra and root elongation zone of excised roots in an AHL derivative-independent way. Additionally, C6- and C8-HSL treatments stimulated K+ uptake in root cells only at certain concentrations, whereas all tested concentrations of C12-HSL induced K+ uptake. In further experiments, C8-HSL promoted membrane hyperpolarization in epidermal root cells. Thus, we conclude AHLs promote plant growth and lateral root formation, and cause NO accumulation as an early response to AHLs. Furthermore, the AHL-mediated membrane hyperpolarization is leading to increased K+ uptake of the root tissue.

Pseudomonas aeruginosa quorum-sensing molecule homoserine lactone modulates inflammatory signaling through PERK and eI-F2α.

Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (HSL-C12) as a quorum-sensing molecule to regulate bacterial gene expression. Because HSL-C12 is membrane permeant, multiple cell types in P. aeruginosa-infected airways may be exposed to HSL-C12, especially adjacent to biofilms where local (HSL-C12) may be high. Previous reports showed that HSL-C12 causes both pro- and anti-inflammatory effects. To characterize HSL-C12's pro- and anti-inflammatory effects in host cells, we measured protein synthesis, NF-κB activation, and KC (mouse IL-8) and IL-6 mRNA and protein secretion in wild-type mouse embryonic fibroblasts (MEF). To test the role of the endoplasmic reticulum stress inducer, PERK we compared these responses in PERK(-/-) and PERK-corrected PERK(-/-) MEF. During 4-h treatments of wild-type MEF, HSL-C12 potentially activated NF-κB p65 by preventing the resynthesis of IκB and increased transcription of KC and IL-6 genes (quantitative PCR). HSL-C12 also inhibited secretion of KC and/or IL-6 into the media (ELISA) both in control conditions and also during stimulation by TNF-α. HSL-C12 also activated PERK (as shown by increased phosphorylation of eI-F2α) and inhibited protein synthesis (as measured by incorporation of [(35)S]methionine by MEF). Comparisons of PERK(-/-) and PERK-corrected MEF showed that HSL-C12's effects were explained in part by activation of PERK→phosphorylation of eI-F2α→inhibition of protein synthesis→reduced IκBα production→activation of NF-κB→increased transcription of the KC gene but reduced translation and secretion of KC. HSL-C12 may be an important modulator of early (up to 4 h) inflammatory signaling in P. aeruginosa infections.

Pseudomonas aeruginosa homoserine lactone triggers apoptosis and Bak/Bax-independent release of mitochondrial cytochrome C in fibroblasts.

Pseudomonas aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12-triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild-type (WT) and Bax(-/-) /Bak(-/-) (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψmito ), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax-dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψmito and release of cytochrome C and cell death. Re-expression of Bax or Bak in DKO MEF did not alter the WT-like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax-independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψmito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.

The bacterial quorum-sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone reciprocally modulates pro- and anti-inflammatory cytokines in activated macrophages.

The bacterial molecule N-3-oxo-dodecanoyl-l-homoserine lactone (C12) has critical roles in both interbacterial communication and interkingdom signaling. The ability of C12 to downregulate production of the key proinflammatory cytokine TNF-α in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that, in contrast to TNF-α suppression, C12 amplifies production of the major anti-inflammatory cytokine IL-10 in LPS-stimulated murine RAW264.7 macrophages, as well as peritoneal macrophages. Furthermore, C12 increased IL-10 mRNA levels and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NF-κB DNA-binding levels and prolonged p38 MAPK phosphorylation in RAW264.7 macrophages, suggesting that increased transcriptional activity of NF-κB and/or p38-activated transcription factors serves to upregulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria downregulate immune responses to flourish and establish a chronic infection.

Chemical constituents from a soil-derived actinomycete, Actinomadura miaoliensis BCRC 16873, and their inhibitory activities on lipopolysaccharide-induced tumor necrosis factor production.

An investigation on the secondary metabolites from the BuOH extract of the fermentation broth of the thermotolerant polyester-degrading actinomycete Actinomadura miaoliensis BCRC 16873 was carried out. One previously undescribed α-pyrone (=pyran-2-one) derivative, designated as miaolienone (1), and a new butanolide, miaolinolide (2), together with 13 known compounds, 3-15, were obtained. Their structures were established on the basis of extensive 1D- and 2D-NMR analyses in combination with HR-MS experiments. In addition, the isolated compounds 1-15 were evaluated for the inhibitory effects of the isolates on the production of tumor necrosis factor (TNF-α) induced by lipopolysaccharide (LPS). Among the isolates, 1 and 2 significantly inhibited TNF-α production in U937 cells in vitro, and the IC(50) values were 0.59 and 0.76 µM, respectively. Compounds 3-5 displayed moderate inhibitory activities on LPS-induced TNF-α production.

The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro.

Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalian cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration process.

N-3-oxo-decanoyl-L-homoserine-lactone activates auxin-induced adventitious root formation via hydrogen peroxide- and nitric oxide-dependent cyclic GMP signaling in mung bean.

N-Acyl-homoserine-lactones (AHLs) are bacterial quorum-sensing signaling molecules that regulate population density. Recent evidence demonstrates their roles in plant defense responses and root development. Hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and cyclic GMP (cGMP) are essential messengers that participate in various plant physiological processes, but how these messengers modulate the plant response to N-acyl-homoserine-lactone signals remains poorly understood. Here, we show that the N-3-oxo-decanoyl-homoserine-lactone (3-O-C10-HL), in contrast to its analog with an unsubstituted branch chain at the C3 position, efficiently stimulated the formation of adventitious roots and the expression of auxin-response genes in explants of mung bean (Vigna radiata) seedlings. This response was mimicked by the exogenous application of auxin, H(2)O(2), NO, or cGMP homologs but suppressed by treatment with scavengers or inhibitors of H(2)O(2), NO, or cGMP metabolism. The 3-O-C10-HL treatment enhanced auxin basipetal transport; this effect could be reversed by treatment with H(2)O(2) or NO scavengers but not by inhibitors of cGMP synthesis. Inhibiting 3-O-C10-HL-induced H(2)O(2) or NO accumulation impaired auxin- or 3-O-C10-HL-induced cGMP synthesis; however, blocking cGMP synthesis did not affect auxin- or 3-O-C10-HL-induced H(2)O(2) or NO generation. Additionally, cGMP partially rescued the inhibitory effect of H(2)O(2) or NO scavengers on 3-O-C10-HL-induced adventitious root development and auxin-response gene expression. These results suggest that 3-O-C10-HL, unlike its analog with an unmodified branch chain at the C3 position, can accelerate auxin-dependent adventitious root formation, possibly via H(2)O(2)- and NO-dependent cGMP signaling in mung bean seedlings.

[Quorum sensing involved in the regulation of secondary metabolism in streptomycetes--a review].

Quorum sensing as an extracellular signal transduction system is distributed widely among many bacteria to coordinate their behaviors or actions by mediating gene expression, and plays key roles in many physiological processes and pathogenicity. Quorum sensing was also observed among many streptomycetes, as an important regulatory mechanism of secondary metabolite biosynthesis and/or cell differentiation, and displayed certain diversity of the autoinducer structures and action mechanisms. The participation of A-factor-driven quorum sensing systems in the secondary metabolism has been extensively studied, and triggered the identification of a major signal class featured with gamma-butyrolactone core. Additionally, PI-factor, M-factor and certain small antibiotic molecules recently found in streptomycetes clearly could play important roles in the biosynthetic pathways of some antibiotics, and might represent extracellular autoinducer classes with novel structures. Meanwhile, some specific products of streptomycetes including cholesterol oxidase and glycerol have been identified to function as cell-signaling molecules which modulate the secondary metabolic activities in streptomycetes, probably by the mode of quorum sensing. Here, we reviewed research advances on quorum sensing systems involved in the accumulation of secondary metabolites in streptomycetes, mainly focusing on the clarification of their action modes and structural diversity of autoinducers. We also prospected the research trends in this field and application of autoinducers through quorum-sensing in metabolic engineering of natural products.

The Pseudomonas aeruginosa autoinducer 3O-C12 homoserine lactone provokes hyperinflammatory responses from cystic fibrosis airway epithelial cells.

The discovery of novel antiinflammatory targets to treat inflammation in the cystic fibrosis (CF) lung stands to benefit patient populations suffering with this disease. The Pseudomonas aeruginosa quorum sensing autoinducer N-3-oxododecanoyl homoserine lactone (3O-C12) is an important bacterial virulence factor that has been reported to induce proinflammatory cytokine production from a variety of cell types. The goal of this study was to examine the ability of 3O-C12 to induce proinflammatory cytokine production in normal and CF bronchial epithelial cells, and better understand the cellular mechanisms by which this cytokine induction occurs. 3O-C12 was found to induce higher levels of IL-6 production in the CF cell lines IB3-1 and CuFi, compared to their corresponding control cell lines C38 and NuLi. Systems biology and network analysis revealed a high predominance of over-represented innate immune pathways bridged together by calcium-dependant transcription factors governing the transcriptional responses of A549 airway cells to stimulation with 3O-C12. Using calcium-flux assays, 3O-C12 was found to induce larger and more sustained increases in intracellular calcium in IB3-1 cells compared to C38, and blocking this calcium flux with BAPTA-AM reduced the production of IL-6 by IB3-1 to the levels produced by C38. These data suggest that 3O-C12 induces proinflammatory cytokine production in airway epithelial cells in a calcium-dependent manner, and that dysregulated calcium storage or signalling in CF cells results in an increased production of proinflammatory cytokines.

Quorum sensing regulation in Aeromonas hydrophila.

We present detailed results on the C4-HSL-mediated quorum sensing (QS) regulatory system of the opportunistic Gram-negative bacterium Aeromonas hydrophila. This bacterium contains a particularly simple QS system that allows for a detailed modeling of kinetics. In a model system (i.e., the Escherichia coli monitor strain MH205), the C4-HSL production of A. hydrophila is interrupted by fusion of gfp(ASV). In the present in vitro study, we measure the response of the QS regulatory ahyRI locus in the monitor strain to predetermined concentrations of C4-HSL signal molecules. A minimal kinetic model describes the data well. It can be solved analytically, providing substantial insight into the QS mechanism: at high concentrations of signal molecules, a slow decay of the activated regulator sets the timescale for the QS regulation loop. Slow saturation ensures that, in an A. hydrophila cell, the QS system is activated only by signal molecules produced by other A. hydrophila cells. Separate information on the ahyR and ahyI loci can be extracted, thus allowing the probe to be used in identifying the target when testing QS inhibitors.

The coronin family of proteins.

The coronins, first described in Dictyostelium discoideum in 1991, have meanwhile been detected in all eukaryotes except plants. They belong to the superfamily of WD40-repeat proteins and represent a large family of proteins, which are often involved in cytoskeletal functions. Phylogenetic studies clearly distinguish 12 subfamilies of which six exclusively occur in vertebrates. In the present book we have made a sincere attempt to provide a comprehensive overview on all aspects of coronin proteins including history, structure, subcellular localization and function in different organisms. In addition, we also included a general overview on the WD40 family of proteins and the structurally related Kelch family. The book should be of interest for scientists outside the field, but is more importantly intended as a fast and competent guide for newcomers as well as doctoral and postdoctoral scientists to coronin research in all its facets.

A brief history of the coronin family.

What I'd like to do in this chapter is to share with you my recollections from the earliest days of coronin research and then to provide an overview of the still-developing story of this fascinating family of proteins.