Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1

A systematic analysis of the type III secretion (T3S) genes of Pseudomonas aeruginosa strain PAO1 revealed that they are under quorum-sensing control. This observation was supported by the down-regulation of the T3S regulon in the presence of RhlR-C4HSL and the corresponding advanced secretion of ExoS in a rhlI mutant.     

Quorum sensing in Pseudomonas aeruginosa biofilms

In nature, the bulk of bacterial biomass is believed to exist as an adherent community of cells called a biofilm. Pseudomonas aeruginosa has become a model organism for studying this mode of growth. Over the past decade, significant strides have been made towards understanding biofilm development in P. aeruginosa and we now have a clearer picture of the mechanisms involved. Available evidence suggests that construction of these sessile communities proceeds by many different pathways, rather than a specific programme of biofilm development. A cell-to-cell communication mechanism known as quorum sensing (QS) has been found to play a role in P. aeruginosa biofilm formation. Because both QS and biofilms are impacted by the surrounding environment, understanding the full involvement of cell-to-cell signalling in establishing these complex communities represents a challenge. Nevertheless, under set conditions, several links between QS and biofilm formation have been recognized, which is the focus of this review. A role for antibiotics as alternative QS signalling molecules influencing biofilm development is also discussed.     

Quorum Sensing Inhibition in Pseudomonas aeruginosa PAO1 by Antagonistic Compound Phenylacetic Acid

In Pseudomonas aeruginosa, quorum sensing (QS) autoinducer known as acyl homoserine lactone (AHL) acts as a key regulator in the expression of pathogenic characters. In this work, the efficiency of phenylacetic acid (PAA) in reducing the production of AHL-dependent factors in P. aeruginosa PAO1 was studied. PAA at a concentration of 200?μg?ml(-1) displayed significant reduction in QS-dependent pyocyanin, exopolysaccharide, and protease and elastase production in PAO1. In swimming inhibition assay, PAA-treated PAO1 cells exhibited poor motility in swimming agar plate. In in vivo analysis, PAO1-preinfected Caenorhabditis elegans showed enhanced survival when treated with PAA. PAA at the QS inhibitory concentration showed no growth inhibitory activity on PAO1. Results of the present study revealed the potential of PAA as antipathogenic compound to prevent QS-dependent pathogenicity of P. aeruginosa.     

Biofilm and Quorum sensing mediated pathogenicity in Pseudomonas aeruginosa

The emergence of multidrug resistance has become an alarming and lifethreatening phenomenon for humans. Various mechanisms are involved in the development of resistance in bacteria towards antimicrobial compounds and immune system. Bacterial biofilm is a complicated, selfdefensive, rigid structure of bacteria crowded together to develop a selfrecessive nature, which enhances the ability to cause infections much easier in the living host. P. aeruginosa biofilm formation is supported by extracellular polymeric substances (EPS) such as exopolysaccharides, extracellular DNA (eDNA), proteins and biomolecules. Published evidences suggest that biofilm formation can also be the result of several other mechanisms such as cell signaling or communication. Bacterial biofilm is also regulated by strong intercellular communication known as Quorum Sensing (QS). It is a cellular communication mechanism involving autoinducers and regulators. In P. aeruginosa, Acyl Homoserine Lactone, the prime signaling molecule, controls approximately 300 genes responsible for various cellular functions, including its pathogenesis. The surrounding environment and metabolism have a specific effect on the biofilm and QS, thus, understanding the involvement of QS in the biofilm developing mechanism is still complicated and complex to understand. Therefore, this review will include basic knowledge of the biofilmforming mechanism and other regulatory factors involved in causing infections and diseases in the host organisms.     

Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.     

Utilization of Acyl-Homoserine Lactone Quorum Signals for Growth by a Soil Pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42°C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other γ-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.     

Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals

A recent study suggests that the opportunistic pathogen Pseudomonas aeruginosa can actively monitor the host immune system. The P. aeruginosa outer membrane protein OprF was found to bind specifically to the cytokine interferon-gamma (IFN-gamma), and this interaction upregulated production of virulence factors through a cell-cell communication system known as quorum sensing (QS). Taken together with previous findings that P. aeruginosa QS can alter the host immune response (e.g. by activation of IFN-gamma), these data illustrate an exciting new element of bacteria-host interactions in which the P. aeruginosa quorum-sensing system both senses and modulates the host immune state.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.The metabolically versatile Pseudomonas aeruginosa is an opportunistic pathogen of plants, animals, and humans and is ubiquitously distributed in soil and aquatic habitats. The common reference strain is P. aeruginosa PAO1, a spontaneous chloramphenicol-resistant mutant of the original PAO strain (earlier called “P. aeruginosa strain 1”) that had been isolated in 1954 from a wound in Melbourne, Australia (9, 10). This PAO1 strain from Bruce Holloway''s laboratory has become the reference strain for Pseudomonas genetics and functional analyses of the physiology and metabolism of this gammaproteobacterium. A genetic map of its chromosome was generated by exploiting the mechanisms of gene exchange in bacteria, i.e., transduction and conjugation (11). With the advent of pulsed-field gel electrophoresis (PFGE), a physical map of the PAO1 genome was constructed (32) and later merged with the genetic map information (12). By 2000 the PAO1 strain had been completely sequenced (36). Thereafter, the genome annotation has been continually updated and the database content and functionality have been expanded to facilitate accelerated discovery of P. aeruginosa drug targets and vaccine candidates (38). Two near-saturation libraries of transposon insertion mutants have been constructed in P. aeruginosa PAO1 as a global resource for the scientific community (14, 22).Comparison of the genome sequence with the physical map revealed a large, 2.2-Mb inversion between the sequenced PAO1-UW strain (36) and the original PAO1 strain (9, 10), indicating that PAO1 sublines maintained worldwide in numerous laboratories and strain collections had diversified their genomic sequence. Mutational events were already reported in the 1970s (10), and more recently sequence variations of MexT, which regulates the MexEF-OprN multidrug efflux system, were described (18, 24). Furthermore, a PAO1 subline from a German strain collection (PAO1-D) and another, independent PAO1 subline from a Japanese strain collection (PAO1-J) that had been stored by research groups in Germany and Japan, respectively, were found to be quorum-sensing-negative mutants that carried point mutations in the regulatory gene lasR (6). In addition, spontaneous secretion-defective vfr mutants from a PAO1 population were observed after several cycles of static growth (2). Similarly, we noted a difference in virulence in a mouse infection model (see below) between the MPAO1 and PAO1-DSM sublines that had been utilized for the construction of the transposon library (14) and the physical map (32), respectively. PAO1-DSM was indistinguishable in its SpeI-DpnI-SwaI-PacI physical map from the PAO1 subline that had been stored in the Holloway laboratory (12). Hence, we decided to compare the genomic sequence of the initially sequenced PAO1 subline PAO1-UW (36) with that of MPAO1 and PAO1-DSM. Combined physical mapping and DNA sequencing-by-synthesis revealed numerous single-nucleotide polymorphisms (SNPs) and insertions-deletions (indels) in the chromosomes that were associated with differences in fitness, antimicrobial susceptibility, and virulence of the sublines.     

Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm

The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of Pseudomonas aeruginosa PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to approximately 80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed cells. Changes in gene expression associated with dispersion of P. aeruginosa biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of P. aeruginosa from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of P. aeruginosa correlates with a specific dispersal phenotype.     

Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

Studies on the Pseudomonas aeruginosa PAO1 bacteriophage receptors

Receptor for phage PIK specific for Pseudomonas aeruginosa strain PAO1 was studied. Phage PIK was strongly inactivated by lipopolysaccharide (LPS) in vitro, exhibiting a PhI50 of 4.8 micrograms/ml. Further it was noted that this inactivation by LPS was reduced to 50% by several mono- and disaccharides when tested in vitro. D-glucosamine, D-mannose and L-rhamnose were found to be most effective at the concentration of 0.045 M, 0.25 M and 0.35 M respectively. This suggests the possibility that phage PIK receptor in LPS contains D-mannose, L-rhamnose and D-glucosamine. Either one of the former two could be located at a terminal position alpha-linked to the adjacent residue or located internally in the polysaccharide chain linked through its C-4 position. A theoretical approach to the interpretation of phage cell interaction was also investigated.     

Quorum sensing inhibition in Pseudomonas aeruginosa biofilms: new insights through network mining

Quorum sensing plays a pivotal role in Pseudomonas aeruginosa’s virulence. This paper reviews experimental results on antimicrobial strategies based on quorum sensing inhibition and discusses current targets in the regulatory network that determines P. aeruginosa biofilm formation and virulence. A bioinformatics framework combining literature mining with information from biomedical ontologies and curated databases was used to create a knowledge network of potential anti-quorum sensing agents for P. aeruginosa. A total of 110 scientific articles, corresponding to 1,004 annotations, were so far included in the network and are analysed in this work. Information on the most studied agents, QS targets and methods is detailed. This knowledge network offers a unique view of existing strategies for quorum sensing inhibition and their main regulatory targets and may be used to readily access otherwise scattered information and to help generate new testable hypotheses. This knowledge network is publicly available at http://pcquorum.org/.     

Aspergillus ochraceopetaliformis SSP13 modulates quorum sensing regulated virulence and biofilm formation in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen causing the majority of acute and persistent infections in human beings. The ability to form biofilm adds a new dimension to its resistance to conventional therapeutic agents. In the present study, down-regulation of quorum sensing regulated virulence and biofilm development resulting from exposure to Aspergillus ochraceopetaliformis SSP13 extract was investigated. The in vitro results inferred impairment in the production of LasA protease, LasB elastase, chitinase, pyocyanin, exopolysaccharides and rhamnolipids. In addition, motility and biofilm formation by P. aeruginosa PAO1 was significantly altered. The in vitro results were further supported by molecular docking studies of the metabolites obtained from GC-MS analysis depicting the quorum sensing attenuation by targeting the receptor proteins LasR and RhlR. The in vitro and in silico studies suggested new avenues for the development of bioactive metabolites from A. ochraceopetaliformis SSP13 extract as potential anti-infective agents.