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Sample records for aeruginosa pao1 genome

  1. Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains.

    PubMed

    Klockgether, Jens; Munder, Antje; Neugebauer, Jens; Davenport, Colin F; Stanke, Frauke; Larbig, Karen D; Heeb, Stephan; Schöck, Ulrike; Pohl, Thomas M; Wiehlmann, Lutz; Tümmler, Burkhard

    2010-02-01

    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. PMID:20023018

  2. Genes related to chromate resistance by Pseudomonas aeruginosa PAO1.

    PubMed

    Rivera, Sonia L; Vargas, Eréndira; Ramírez-Díaz, Martha I; Campos-García, Jesús; Cervantes, Carlos

    2008-08-01

    Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon-insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted in the mutants with the PAO1 genome revealed that the genes affected in three mutant strains were oprE (ORF PA0291), rmlA (ORF PA5163), and ftsK (ORF PA2615), respectively. A relationship of these genes with chromate tolerance has not been previously reported. No other phenotypic changes were observed in the oprE mutant but its resistance to chromate was not fully restored by expressing the ChrA protein, which extrudes chromate ions from the cytoplasm to the periplasmic space. These data suggest that OprE participates in the efflux of chromate from the periplasm to the outside. Increased susceptibility of the rmlA mutant to the metals cadmium and mercury and to the anion-superoxide generator paraquat suggests a protective role of LPS against chromate toxicity. A higher susceptibility of the ftsK mutant to compounds affecting DNA structure (ciprofloxacin, tellurite, mitomycin C) suggests a role of FtsK in the recombinational repair of DNA damage caused by chromate. In conclusion, the P. aeruginosa genome contains diverse genes related to its intrinsic resistance to chromate. Systems pertaining to the outer membrane (OprE), the cell wall (LPS), and the cytoplasm (FtsK) were identified in this work as involved in chromate protection mechanisms. PMID:18446454

  3. Fructooligosacharides Reduce Pseudomonas aeruginosa PAO1 Pathogenicity through Distinct Mechanisms

    PubMed Central

    Ortega-González, Mercedes; Sánchez de Medina, Fermín; Molina-Santiago, Carlos; López-Posadas, Rocío; Pacheco, Daniel; Krell, Tino; Martínez-Augustin, Olga; Abdelali, Daddaoua

    2014-01-01

    Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-α. Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF-κB pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed. PMID:24465697

  4. Fructooligosacharides reduce Pseudomonas aeruginosa PAO1 pathogenicity through distinct mechanisms.

    PubMed

    Ortega-González, Mercedes; Sánchez de Medina, Fermín; Molina-Santiago, Carlos; López-Posadas, Rocío; Pacheco, Daniel; Krell, Tino; Martínez-Augustin, Olga; Abdelali, Daddaoua

    2014-01-01

    Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-α. Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF-κB pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed. PMID:24465697

  5. Transcriptome analysis of Pseudomonas aeruginosa PAO1 grown at both body and elevated temperatures.

    PubMed

    Chan, Kok-Gan; Priya, Kumutha; Chang, Chien-Yi; Abdul Rahman, Ahmad Yamin; Tee, Kok Keng; Yin, Wai-Fong

    2016-01-01

    Functional genomics research can give us valuable insights into bacterial gene function. RNA Sequencing (RNA-seq) can generate information on transcript abundance in bacteria following abiotic stress treatments. In this study, we used the RNA-seq technique to study the transcriptomes of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 following heat shock. Samples were grown at both the human body temperature (37 °C) and an arbitrarily-selected temperature of 46 °C. In this work using RNA-seq, we identified 133 genes that are differentially expressed at 46 °C compared to the human body temperature. Our work identifies some key P. aeruginosa PAO1 genes whose products have importance in both environmental adaptation as well as in vivo infection in febrile hosts. More importantly, our transcriptomic results show that many genes are only expressed when subjected to heat shock. Because the RNA-seq can generate high throughput gene expression profiles, our work reveals many unanticipated genes with further work to be done exploring such genes products. PMID:27547539

  6. Transcriptome analysis of Pseudomonas aeruginosa PAO1 grown at both body and elevated temperatures

    PubMed Central

    Priya, Kumutha; Chang, Chien-Yi; Abdul Rahman, Ahmad Yamin; Tee, Kok Keng; Yin, Wai-Fong

    2016-01-01

    Functional genomics research can give us valuable insights into bacterial gene function. RNA Sequencing (RNA-seq) can generate information on transcript abundance in bacteria following abiotic stress treatments. In this study, we used the RNA-seq technique to study the transcriptomes of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 following heat shock. Samples were grown at both the human body temperature (37 °C) and an arbitrarily-selected temperature of 46 °C. In this work using RNA-seq, we identified 133 genes that are differentially expressed at 46 °C compared to the human body temperature. Our work identifies some key P. aeruginosa PAO1 genes whose products have importance in both environmental adaptation as well as in vivo infection in febrile hosts. More importantly, our transcriptomic results show that many genes are only expressed when subjected to heat shock. Because the RNA-seq can generate high throughput gene expression profiles, our work reveals many unanticipated genes with further work to be done exploring such genes products. PMID:27547539

  7. Pseudomonas aeruginosa PAO-1 Lipopolysaccharide-Diphtheria Toxoid Conjugate Vaccine: Preparation, Characterization and Immunogenicity

    PubMed Central

    Najafzadeh, Faezeh; Shapouri, Reza; Rahnema, Mehdi; Rokhsartalab Azar, Shadi; Kianmehr, Anvarsadat

    2015-01-01

    Background: Treatment of Pseudomonas aeruginosa PAO-1 infections through immunological means has been proved to be efficient and protective. Objectives: The purpose of this study was to produce a conjugate vaccine composed of detoxified lipopolysaccharide (D-LPS) P. aeruginosa and diphtheria toxoid (DT). Materials and Methods: Firstly, LPS was purified and characterized from P. aeruginosa PAO1 and then detoxified. D-LPS was covalently coupled to DT as a carrier protein via amidation method with adipic acid dihydrazide (ADH) as a spacer molecule and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) as a linker. The molar ratio of LPS to DT in the prepared conjugate was 3:1. The immunogenicity of D-LPS-DT conjugate vaccine in mice model was evaluated as well. Results: The conjugate was devoid of endotoxin activity and 0.125 U/mL of D-LPS was acceptable for immunization. D-LPS-DT conjugate was nonpyrogenic for rabbits and nontoxic for mice. Mice immunization with D-LPS-DT conjugate vaccine elicited the fourfold higher IgG antibody compared to D-LPS. Anti-LPS IgG antibody was predominantly IgG1 subclass and then IgG3, IgG2a and IgG2b, respectively. Conclusions: Vaccine based on the conjugation of P. aeruginosa PAO-1 LPS with DT increased anti-LPS antibodies and had a significant potential to protect against Pseudomonas infections. PMID:26301059

  8. Extracts of Cordia gilletii de wild (Boraginaceae) quench the quorum sensing of Pseudomonas aeruginosa PAO1

    PubMed Central

    Okusa, Philippe N.; Rasamiravaka, Tsiry; Vandeputte, Olivier; Stévigny, Caroline; Jaziri, Mondher El; Duez, Pierre

    2014-01-01

    Aim: The fight against infectious diseases and antimicrobial resistances needs the exploration of new active compounds with new proprieties like disrupting quorum sensing (QS) mechanisms, which is a cell-to-cell communication that regulates bacterial virulence factors. In this work, leaves and root barks extracts of a Congolese medicinal plant, Cordia gilletii, were investigated for their effect on the production of Pseudomonas aeruginosa major virulence factors regulated by QS. Materials and Methods: The effect of C. gilletii extracts on virulence factors of P. aeruginosa PAO1 was studied by the evaluation of the production of pyocyanine, elastase and biofilm; and by the measurement of the expression of QS-related genes. Results: The dichloromethane extract from root barks was found to quench the production of pyocyanin, a QS-dependent virulence factor in P. aeruginosa PAO1. Moreover, this extract specifically inhibits the expression of several QS-regulated genes (i.e. lasB, rhlA, lasI, lasR, rhlI, and rhlR) and reduces biofilm formation by PAO1. Conclusion: This study contributes to explain the efficacy of C. gilletii in the traditional treatment of infectious diseases caused by P. aeruginosa. PMID:26401363

  9. Pseudomonas aeruginosa PAO1 exopolysaccharides are important for mixed species biofilm community development and stress tolerance

    PubMed Central

    Periasamy, Saravanan; Nair, Harikrishnan A. S.; Lee, Kai W. K.; Ong, Jolene; Goh, Jie Q. J.; Kjelleberg, Staffan; Rice, Scott A.

    2015-01-01

    Pseudomonas aeruginosa PAO1 produces three polysaccharides, alginate, Psl, and Pel that play distinct roles in attachment and biofilm formation for monospecies biofilms. Considerably less is known about their role in the development of mixed species biofilm communities. This study has investigated the roles of alginate, Psl, and Pel during biofilm formation of P. aeruginosa in a defined and experimentally informative mixed species biofilm community, consisting of P. aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Loss of the Psl polysaccharide had the biggest impact on the integration of P. aeruginosa in the mixed species biofilms, where the percent composition of the psl mutant was significantly lower (0.06%) than its wild-type (WT) parent (2.44%). In contrast, loss of the Pel polysaccharide had no impact on mixed species biofilm development. Loss of alginate or its overproduction resulted in P. aeruginosa representing 8.4 and 18.11%, respectively, of the mixed species biofilm. Dual species biofilms of P. aeruginosa and K. pneumoniae were not affected by loss of alginate, Pel, or Psl, while the mucoid P. aeruginosa strain achieved a greater biomass than its parent strain. When P. aeruginosa was grown with P. protegens, loss of the Pel or alginate polysaccharides resulted in biofilms that were not significantly different from biofilms formed by the WT PAO1. In contrast, overproduction of alginate resulted in biofilms that were comprised of 35–40% of P. aeruginosa, which was significantly higher than the WT (5–20%). Loss of the Psl polysaccharide significantly reduced the percentage composition of P. aeruginosa in dual species biofilms with P. protegens (<1%). Loss of the Psl polysaccharide significantly disrupted the communal stress resistance of the three species biofilms. Thus, the polysaccharide composition of an individual species significantly impacts mixed species biofilm development and the emergent properties of such communities. PMID

  10. Determining Multiple Responses of Pseudomonas aeruginosa PAO1 to an Antimicrobial Agent, Free Nitrous Acid.

    PubMed

    Gao, Shu-Hong; Fan, Lu; Peng, Lai; Guo, Jianhua; Agulló-Barceló, Míriam; Yuan, Zhiguo; Bond, Philip L

    2016-05-17

    Free nitrous acid (FNA) has recently been demonstrated as an antimicrobial agent on a range of micro-organisms, especially in wastewater-treatment systems. However, the antimicrobial mechanism of FNA is largely unknown. Here, we report that the antimicrobial effects of FNA are multitargeted. The response of a model denitrifier, Pseudomnas aeruginosa PAO1 (PAO1), common in wastewater treatment, was investigated in the absence and presence of inhibitory level of FNA (0.1 mg N/L) under anaerobic denitrifying conditions. This was achieved through coupling gene expression analysis, by RNA sequencing, and with a suite of physiological analyses. Various transcripts exhibited significant changes in abundance in the presence of FNA. Respiration was likely inhibited because denitrification activity was severely depleted, and decreased transcript levels of most denitrification genes occurred. As a consequence, the tricarboxylic acid (TCA) cycle was inhibited due to the lowered cellular redox state in the FNA-exposed cultures. Meanwhile, during FNA exposure, PAO1 rerouted its carbon metabolic pathway from the TCA cycle to pyruvate fermentation with acetate as the end product as a possible survival mechanism. Additionally, protein synthesis was significantly decreased, and ribosome preservation was evident. These findings improve our understanding of PAO1 in response to FNA and contribute toward the potential application for use of FNA as an antimicrobial agent. PMID:27116299

  11. Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

    PubMed Central

    Lotlikar, Shalaka R.; Hnatusko, Shane; Dickenson, Nicholas E.; Choudhari, Shyamal P.; Picking, Wendy L.

    2013-01-01

    Bacterial β-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO2. They maintain the intracellular balance of CO2/bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative β-CAs that share 28–45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn2+ and catalyse hydration of CO2 to bicarbonate. PAO102 (psCA1) was 19–26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II β-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO2; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO2. Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism. PMID:23728627

  12. Vanadate and triclosan synergistically induce alginate production by Pseudomonas aeruginosa strain PAO1

    PubMed Central

    Damron, F. Heath; Davis, Michael R.; Withers, T. Ryan; Ernst, Robert K.; Goldberg, Joanna B.; Yu, Guangli; Yu, Hongwei D.

    2011-01-01

    Summary Alginate overproduction by P. aeruginosa strains, also known as mucoidy, is associated with chronic lung infections in cystic fibrosis (CF). It is not clear how alginate induction occurs in the wild type (wt) mucA strains. When grown on Pseudomonas isolation agar (PIA), P. aeruginosa strains PAO1 and PA14 are nonmucoid producing minimal amounts of alginate. Here we report the addition of ammonium metavanadate (AMV), a phosphatase inhibitor, to PIA (PIA-AMV) induced mucoidy in both these laboratory strains and early lung colonizing nonmucoid isolates with a wt mucA. This phenotypic switch was reversible depending on the availability of vanadate salts and triclosan, a component of PIA. Alginate induction in PAO1 on PIA-AMV was correlated with increased proteolytic degradation of MucA, and required envelope proteases AlgW or MucP, and a two-component phosphate regulator, PhoP. Other changes included the addition of palmitate to lipid A, a phenotype also observed in chronic CF isolates. Proteomic analysis revealed the upregulation of stress chaperones, which was confirmed by increased expression of the chaperone/protease MucD. Altogether, these findings suggest a model of alginate induction and the PIA-AMV medium may be suitable for examining early lung colonization phenotypes in CF before the selection of the mucA mutants. PMID:21631603

  13. Pseudomonas aeruginosa PAO1 virulence factors and poplar tree response in the rhizosphere

    PubMed Central

    Attila, Can; Ueda, Akihiro; Cirillo, Suat L. G.; Cirillo, Jeffrey D.; Chen, Wilfred; Wood, Thomas K.

    2008-01-01

    Summary Whole‐transcriptome analysis was used here for the first time in the rhizosphere to discern the genes involved in the pathogenic response of Pseudomonas aeruginosa PAO1 as well as to discern the response of the poplar tree. Differential gene expression shows that 185 genes of the bacterium and 753 genes of the poplar tree were induced in the rhizosphere. Using the P. aeruginosatranscriptome analysis, isogenic knockout mutants, and two novel plant assays (poplar and barley), seven novel PAO1 virulence genes were identified (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150 and PA4295). The uncharacterized putative haemolysin repressor, PA2463, upon inactivation, resulted in greater poplar virulence and elevated haemolysis while this mutant remained competitive in the rhizosphere. In addition, disruption of the haemolysin gene itself (PA2462) reduced the haemolytic activity of P. aeruginosa, caused less cytotoxicity and reduced barley virulence, as expected. Inactivating PA1385, a putative glycosyl transferase, reduced both poplar and barley virulence. Furthermore, disrupting PA2663, a putative membrane protein, reduced biofilm formation by 20‐fold. Inactivation of PA3476 (rhlI) increased virulence with barley as well as haemolytic activity and cytotoxicity, so quorum sensing is important in plant pathogenesis. Hence, this strategy is capable of elucidating virulence genes for an important pathogen. PMID:21261818

  14. Gene PA2449 Is Essential for Glycine Metabolism and Pyocyanin Biosynthesis in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lundgren, Benjamin R.; Thornton, William; Dornan, Mark H.; Villegas-Peñaranda, Luis Roberto; Boddy, Christopher N.

    2013-01-01

    Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in Pseudomonas aeruginosa PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in P. aeruginosa PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria. PMID:23457254

  15. Nanomechanical Response of Pseudomonas aeruginosa PAO1 Bacterial Cells to Cationic Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Walters, Grant; Dutcher, John

    2013-03-01

    We have used an atomic force microscopy (AFM)-based creep deformation technique to study changes to the viscoelastic properties of individual Gram-negative Pseudomonas aeruginosa PAO1 cells as a function of time of exposure to two cationic peptides: polymyxin B (PMB), a cyclic antimicrobial peptide, and the structurally-related compound, polymyxin B nonapeptide (PMBN). The measurements provide a direct measure of the mechanical integrity of the bacterial cell envelope, and the results can be understood in terms of simple viscoelastic models of arrangements of springs and dashpots, which can be ascribed to different components within the bacterial cell. Time-resolved creep deformation experiments reveal abrupt changes to the viscoelastic properties of P. aeruginosa bacterial cells after exposure to both PMB and PMBN, with quantitatively different changes for the two cationic peptides. These measurements provide new insights into the kinetics and mechanism of action of antimicrobial peptides on bacterial cells.

  16. A theoretical and experimental proteome map of Pseudomonas aeruginosa PAO1

    PubMed Central

    Lecoutere, Elke; Verleyen, Peter; Haenen, Steven; Vandersteegen, Katrien; Noben, Jean-Paul; Robben, Johan; Schoofs, Liliane; Ceyssens, Pieter-Jan; Volckaert, Guido; Lavigne, Rob

    2012-01-01

    A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spots were visualized, and 181 protein spots were characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same protein may be detected in two or more neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks. PMID:22950023

  17. Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress.

    PubMed

    Orlandi, Viviana T; Bolognese, Fabrizio; Chiodaroli, Luca; Tolker-Nielsen, Tim; Barbieri, Paola

    2015-12-01

    Pseudomonas aeruginosa is an opportunistic pathogen known to be resistant to different classes of antibiotics and disinfectants. P. aeruginosa also displays a certain degree of tolerance to photodynamic therapy (PDT), an alternative antimicrobial approach exploiting a photo-oxidative stress induced by exogenous photosensitizers and visible light. To evaluate whether P. aeruginosa pigments can contribute to its relative tolerance to PDT, we analysed the response to this treatment of isogenic transposon mutants of P. aeruginosa PAO1 with altered pigmentation. In general, in the presence of pigments a higher tolerance to PDT-induced photo-oxidative stress was observed. Hyperproduction of pyomelanin makes the cells much more tolerant to stress caused by either radicals or singlet oxygen generated by different photosensitizers upon photoactivation. Phenazines, pyocyanin and phenazine-1-carboxylic acid, produced in different amounts depending on the cultural conditions, are able to counteract both types of PDT-elicited reactive oxygen species. Hyperproduction of pyoverdine, caused by a mutation in a quorum-sensing gene, rendered P. aeruginosa more tolerant to a photosensitizer that generates mainly singlet oxygen, although in this case the observed tolerance to photo-oxidative stress cannot be exclusively attributed to the presence of the pigment. PMID:26419906

  18. Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1

    PubMed Central

    Ravichandran, Ayshwarya; Wong, Chui Ching; Swarup, Sanjay

    2015-01-01

    Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness. PMID:25894344

  19. Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370.

    PubMed

    Zaitseva, Julia; Granik, Vladimir; Belik, Alexandr; Koksharova, Olga; Khmel, Inessa

    2009-06-01

    Antibacterial drugs in the nitrofuran series, such as nitrofurazone, furazidin, nitrofurantoin and nifuroxazide, as well as the nitric oxide generators sodium nitroprusside and isosorbide mononitrate in concentrations that do not suppress bacterial growth, were shown to increase the capacity of pathogenic bacteria Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370 to form biofilms. At 25-100microg/ml, nitrofurans 2-2.5-fold enhanced biofilm formation of P. aeruginosa PAO1, and NO donors 3-6-fold. For B. cenocepacia 370, the enhancement was 2-5-fold (nitrofurans) and 4.5-fold (sodium nitroprusside), respectively. PMID:19460431

  20. Major proteomic changes associated with amyloid-induced biofilm formation in Pseudomonas aeruginosa PAO1.

    PubMed

    Herbst, Florian-Alexander; Søndergaard, Mads T; Kjeldal, Henrik; Stensballe, Allan; Nielsen, Per H; Dueholm, Morten S

    2015-01-01

    The newly identified functional amyloids in Pseudomonas (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen P. aeruginosa; however, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap-mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloids were found to not solely act as inert structural biofilm components. Their presence induced major changes in the global proteome of the bacterium. These included the lowered abundance of classical virulence factors such as elastase B and the secretion system of alkaline protease A. Amyloid-mediated biofilm formation furthermore increased abundance of the alginate and pyoverdine synthesis machinery, which turned P. aeruginosa PAO1 into an unexpected mucoid phenotype. The results imply a significant impact of functional amyloids on the physiology of P. aeruginosa with subsequent implications for biofilm formation and chronic infections. PMID:25317949

  1. Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1.

    PubMed

    Nithya, Chari; Begum, Mansur Farzana; Pandian, Shunmugiah Karutha

    2010-09-01

    According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 microg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70-74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat. PMID:20665017

  2. Kinetic modeling of rhamnolipid production by Pseudomonas aeruginosa PAO1 including cell density-dependent regulation.

    PubMed

    Henkel, Marius; Schmidberger, Anke; Vogelbacher, Markus; Kühnert, Christian; Beuker, Janina; Bernard, Thomas; Schwartz, Thomas; Syldatk, Christoph; Hausmann, Rudolf

    2014-08-01

    The production of rhamnolipid biosurfactants by Pseudomonas aeruginosa is under complex control of a quorum sensing-dependent regulatory network. Due to a lack of understanding of the kinetics applicable to the process and relevant interrelations of variables, current processes for rhamnolipid production are based on heuristic approaches. To systematically establish a knowledge-based process for rhamnolipid production, a deeper understanding of the time-course and coupling of process variables is required. By combining reaction kinetics, stoichiometry, and experimental data, a process model for rhamnolipid production with P. aeruginosa PAO1 on sunflower oil was developed as a system of coupled ordinary differential equations (ODEs). In addition, cell density-based quorum sensing dynamics were included in the model. The model comprises a total of 36 parameters, 14 of which are yield coefficients and 7 of which are substrate affinity and inhibition constants. Of all 36 parameters, 30 were derived from dedicated experimental results, literature, and databases and 6 of them were used as fitting parameters. The model is able to describe data on biomass growth, substrates, and products obtained from a reference batch process and other validation scenarios. The model presented describes the time-course and interrelation of biomass, relevant substrates, and products on a process level while including a kinetic representation of cell density-dependent regulatory mechanisms. PMID:24770383

  3. Identification of Novel Genes Responsible for Overexpression of ampC in Pseudomonas aeruginosa PAO1

    PubMed Central

    Tsutsumi, Yuko; Tomita, Haruyoshi

    2013-01-01

    The development of resistance to antipseudomonal penicillins and cephalosporins mediated by the chromosomal ampC gene in Pseudomonas aeruginosa is of clinical importance. We isolated piperacillin-resistant mutants derived from P. aeruginosa PAO1 and analyzed two mutants that had an insertion in mpl and nuoN. One mutant, YT1677, was resistant to piperacillin and ceftazidime and had an insertion in mpl, which encodes UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase. The other mutant, YT7988, showed increased MICs of piperacillin, ceftazidime, cefepime, and cefoperazone, and the insertion was mapped to nuoN, which encodes NADH dehydrogenase I chain N. Complementation experiments demonstrated that these mutations resulted in higher levels of resistance to β-lactams. The expression of genes reported to be involved in β-lactam resistance was examined by real-time PCR in YT1677 and YT7988 mutants. Overexpression was observed for only ampC, and other genes were expressed normally. Deletion of the ampR gene in YT1677 and YT7988 resulted in decreased expression of ampC, indicating that the mutations in YT1677 and YT7988 affected the expression of ampC through the function of AmpR. PMID:24041903

  4. Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides Requires cprA, a Gene That Is Disrupted in the PAO1 Strain

    PubMed Central

    Gutu, Alina D.; Rodgers, Nicole S.; Park, Jihye

    2015-01-01

    The arn locus, found in many Gram-negative bacterial pathogens, mediates resistance to polymyxins and other cationic antimicrobial peptides through 4-amino-l-arabinose modification of the lipid A moiety of lipopolysaccharide. In Pseudomonas aeruginosa, several two-component regulatory systems (TCSs) control the arn locus, which is necessary but not sufficient for these resistance phenotypes. A previous transposon mutagenesis screen to identify additional polymyxin resistance genes that these systems regulate implicated an open reading frame designated PA1559 in the genome of the P. aeruginosa PAO1 strain. Resequencing of this chromosomal region and bioinformatics analysis for a variety of P. aeruginosa strains revealed that in the sequenced PAO1 strain, a guanine deletion at the end of PA1559 results in a frameshift and truncation of a full-length open reading frame that also encompasses PA1560 in non-PAO1 strains, such as P. aeruginosa PAK. Deletion analysis in the PAK strain showed that this full-length open reading frame, designated cprA, is necessary for polymyxin resistance conferred by activating mutations in the PhoPQ, PmrAB, and CprRS TCSs. The cprA gene was also required for PmrAB-mediated resistance to other cationic antimicrobial peptides in the PAK strain. Repair of the mutated cprA allele in the PAO1 strain restored polymyxin resistance conferred by an activating TCS mutation. The deletion of cprA did not affect the arn-mediated lipid A modification, indicating that the CprA protein is necessary for a different aspect of polymyxin resistance. This protein has a domain structure with a strong similarity to the extended short-chain dehydrogenase/reductase family that comprises isomerases, lyases, and oxidoreductases. These results suggest a new avenue through which to pursue targeted inhibition of polymyxin resistance. PMID:26100714

  5. Structural insights into YfiR sequestering by YfiB in Pseudomonas aeruginosa PAO1

    PubMed Central

    Li, Shanshan; Li, Tingting; Xu, Yueyang; Zhang, Qionglin; Zhang, Wei; Che, Shiyou; Liu, Ruihua; Wang, Yingying; Bartlam, Mark

    2015-01-01

    YfiBNR is a tripartite signalling system in Pseudomonas aeruginosa that modulates intracellular c-di-GMP levels in response to signals received in the periplasm. YfiB is an outer membrane lipoprotein and presumed sensor protein that sequesters the repressor protein YfiR. To provide insights into YfiBNR function, we have determined three-dimensional crystal structures of YfiB and YfiR from P. aeruginosa PAO1 alone and as a 1:1 complex. A YfiB(27–168) construct is predominantly dimeric, whereas a YfiB(59–168) is monomeric, indicating that YfiB can dimerize via its N-terminal region. YfiR forms a stable complex with YfiB(59–168), while the YfiR binding interface is obstructed by the N-terminal region in YfiB(27–168). The YfiB-YfiR complex reveals a conserved interaction surface on YfiR that overlaps with residues predicted to interact with the periplasmic PAS domain of YfiN. Comparison of native and YfiR-bound structures of YfiB suggests unwinding of the N-terminal linker region for attachment to the outer membrane. A model is thus proposed for YfiR sequestration at the outer membrane by YfiB. Our work provides the first detailed insights into the interaction between YfiB and YfiR at the molecular level and is a valuable starting point for further functional and mechanistic studies of the YfiBNR signalling system. PMID:26593397

  6. In vitro antibiofilm activity of the freshwater bryozoan Hyalinella punctata: a case study of Pseudomonas aeruginosa PAO1.

    PubMed

    Pejin, Boris; Ciric, Ana; Karaman, Ivo; Horvatovic, Mladen; Glamoclija, Jasmina; Nikolic, Milos; Sokovic, Marina

    2016-08-01

    The antibiofilm and possible antiquorum sensing effects against the strain Pseudomonas aeruginosa PAO1 of five crude extracts of the freshwater bryozoan Hyalinella punctata (Hancock, 1850) were evaluated in vitro for the first time. H. punctata ethyl acetate extract (HpEtAc) exhibited the highest antibiofilm activity reducing the biofilm formation of P. aeruginosa PAO1 in the range of 80.63-88.13%. While all tested extracts reduced the twitching motility of the aforementioned bacterial strain, HpEtAc showed to be the most effective. Finally, at a concentration of 0.5 MIC, the same extract mostly inhibited the production of pyocyanin by P. aeruginosa PAO1 (71.53%). In comparison both with the positive controls used (streptomycin and ampicillin, 67.13 and 69.77%, respectively), HpEtAc was found to inhibit pyocyanin in a higher extent. An extensive chemical characterisation of this particular extract may result in isolation and identification of novel lead compounds targeting P. aeruginosa, an opportunistic human pathogen. PMID:26264659

  7. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution. PMID:26957519

  8. FUNCTIONAL ANALYSIS OF GENES FOR BIOSYNTHESIS OF PYOCYANIN AND PHENAZINE-1-CARBOXAMIDE FROM PSEUDOMONAS AERUGINOSA PAO1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from P. fluorescens and P. aureofaciens. Functional studies in phenazine-nonpr...

  9. The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting

    PubMed Central

    Sana, Thibault G.; Berni, Benjamin; Bleves, Sophie

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases such as chronic lung colonization in cystic fibrosis patients and acute infections in hospitals. The capacity of P. aeruginosa to be pathogenic toward several hosts is notably due to different secretion systems. Amongst them, P. aeruginosa encodes three Type Six Secretion Systems (T6SS), named H1- to H3-T6SS, that act against either prokaryotes and/or eukaryotic cells. They are independent from each other and inject diverse toxins that interact with different components in the host cell. Here we summarize the roles of these T6SSs in the PAO1 strain, as well as the toxins injected and their targets. While H1-T6SS is only involved in antiprokaryotic activity through at least seven different toxins, H2-T6SS and H3-T6SS are also able to target prokaryotic as well as eukaryotic cells. Moreover, recent studies proposed that H2- and H3-T6SS have a role in epithelial cells invasion by injecting at least three different toxins. The diversity of T6SS effectors is astounding and other effectors still remain to be discovered. In this review, we present a table with other putative P. aeruginosa strain PAO1 T6SS-dependent effectors. Altogether, the T6SSs of P. aeruginosa are important systems that help fight other bacteria for their ecological niche, and are important in the pathogenicity process. PMID:27376031

  10. Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1.

    PubMed

    Yamano, Y; Nishikawa, T; Komatsu, Y

    1993-05-01

    The porin oprE gene of Pseudomonas aeruginosa PAO1 was isolated. Its nucleotide sequence indicated that the structural gene of 1383 nucleotide residues encodes a precursor consisting of 460 amino acid residues with a signal peptide of 29 amino acid residues, which was confirmed by the N-terminal 23-amino-acid sequence and the reaction with anti-OprE polyclonal antiserum. Anaerobiosis induced OprE production at the transcription level. The transcription start site was determined to be 40 nucleotides upstream from the ATG initiation codon. The control region contained an appropriately situated E sigma 54 recognition site and the putative second half of an ANR box. The amino acid sequence of OprE had some clusters of sequence homologous with that of OprD of P. aeruginosa, which might be responsible for the outer membrane permeability of imipenem and basic amino acids. PMID:8394980

  11. Inhibition of Pseudomonas aeruginosa PAO1 adhesion to and invasion of A549 lung epithelial cells by natural extracts.

    PubMed

    Ahmed, Ghada F; Elkhatib, Walid F; Noreddin, Ayman M

    2014-01-01

    Pseudomonas aeruginosa colonizes the lungs in cystic fibrosis (CF) and mechanically ventilated patients by binding to the cellular receptors on the surface of the lung epithelium. Studies have shown that blocking this interaction could be achieved with sub-minimum inhibitory concentrations of antibiotics such as ciprofloxacin. The development of bacterial resistance is a probable drawback of such an intervention. The use of natural extracts to interfere with bacterial adhesion and invasion has recently gained substantial attention and is hypothesized to inhibit bacterial binding and consequently prevent or reduce pathogenicity. This study used an A549 lung epithelial cell infection model, and the results revealed that a combination of aqueous cranberry extract with ciprofloxacin could completely prevent the adhesion and invasion of P. aeruginosa PAO1 compared to the untreated control. All of the natural extracts (cranberry, dextran, and soybean extracts) and ciprofloxacin showed a significant reduction (P<0.0001) in P. aeruginosa PAO1 adhesion to and invasion of lung epithelial cells relative to the control. The cranberry, dextran, and soybean extracts could substantially increase the anti-adhesion and anti-invasion effects of ciprofloxacin to the averages of 100% (P<0.0001), 80% (P<0.0001), and 60% (P<0.0001), respectively. Those extracts might result in a lower rate of the development of bacterial resistance; they are relatively safe and inexpensive agents, and utilizing such extracts, alone or in combination with ciprofloxacin, as potential anti-adhesion and anti-invasion remedies, could be valuable in preventing or reducing P. aeruginosa lung infections. PMID:24894307

  12. Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase.

    PubMed

    Van Alst, Nadine E; Sherrill, Lani A; Iglewski, Barbara H; Haidaris, Constantine G

    2009-10-01

    Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant DeltanarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  13. In silico analysis and molecular modeling of RNA polymerase, sigma S (RpoS) protein in Pseudomonas aeruginosa PAO1

    PubMed Central

    Sedighi, Mansour; Moghoofei, Mohsen; Kouhsari, Ebrahim; Pournajaf, Abazar; Emadi, Behzad; Tohidfar, Masoud; Gholami, Mehrdad

    2015-01-01

    Background: Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection). Methods: The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. Results: The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace. Conclusion: Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions. PMID:26989748

  14. Evaluation of Pseudomonas aeruginosa (PAO1) adhesion to human alveolar epithelial cells A549 using SYTO 9 dye.

    PubMed

    Larrosa, Mar; Truchado, Pilar; Espín, Juan Carlos; Tomás-Barberán, Francisco A; Allende, Ana; García-Conesa, María Teresa

    2012-06-01

    Pseudomonas aeruginosa is an aerobic Gram-negative bacterium characterized by a natural resistance to several antibiotics. It is a major cause of nosocomial infections in patients with compromised host defence mechanisms mainly related to the respiratory tract. P. aeruginosa infection first step is the adhesion of the bacteria to the host cells and thus, the development of techniques that can easily assess adhesion of bacteria strains and of bacteria isolated from biological samples is fundamental. The aim of our work was to develop a fast and effective method to evaluate the adhesion of P. aeruginosa to bronchial epithelial cells. To meet our goal we optimized a staining protocol using the vital dye SYTO 9 and P. aeruginosa PAO1. We established the appropriate dying conditions as well as the stability of the stained bacteria. Adhesion was first measured using the traditional plate counting method and then, adhesion values were compared to those obtained using a fluorescence microplate reader and epifluorescence microscopy. Our results show that the use of SYTO 9 does not interfere with the bacteria viability, bacteria cell growth, and adhesion of P. aeruginosa to A549 epithelial cells. Both the fluorescence microplate reader and the epifluorescence microscopy gave similar results to those attained with the plate counting method, however, the epifluorescence microscopy also allowed for simultaneous discrimination of damaging effects on the human cells. Overall, our data indicate that the use of SYTO 9 combined with a fluorescence microplate reader or an epifluorescence microscope provides a rapid method to evaluate the adhesion of P. aeruginosa to human epithelial cells. However, to show unequivocally that a specific drug or compound has a truly inhibitory effect on the bacterial adhesion without affecting the number of human cells, the epifluorescence microscopy is recommended. PMID:22464926

  15. A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aeruginosa PAO1 using quantitative mass spectrometry.

    PubMed

    Park, Amber J; Murphy, Kathleen; Krieger, Jonathan R; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M

    2014-04-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein-protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  16. A Temporal Examination of the Planktonic and Biofilm Proteome of Whole Cell Pseudomonas aeruginosa PAO1 Using Quantitative Mass Spectrometry*

    PubMed Central

    Park, Amber J.; Murphy, Kathleen; Krieger, Jonathan R.; Brewer, Dyanne; Taylor, Paul; Habash, Marc; Khursigara, Cezar M.

    2014-01-01

    Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein–protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa

  17. Back to the future: evolving bacteriophages to increase their effectiveness against the pathogen Pseudomonas aeruginosa PAO1

    PubMed Central

    Betts, Alex; Vasse, Marie; Kaltz, Oliver; Hochberg, Michael E

    2013-01-01

    Antibiotic resistance is becoming increasingly problematic for the treatment of infectious disease in both humans and livestock. The bacterium Pseudomonas aeruginosa is often found to be resistant to multiple antibiotics and causes high patient mortality in hospitals. Bacteriophages represent a potential option to combat pathogenic bacteria through their application in phage therapy. Here, we capitalize on previous studies showing how evolution may increase phage infection capacity relative to ancestral genotypes. We passaged four different phage isolates (podoviridae, myoviridae) through six serial transfers on the ancestral strain of Pseudomonas aeruginosa PAO1. We first demonstrate that repeated serial passage on ancestral bacteria increases infection capacity of bacteriophage on ancestral hosts and on those evolved for one transfer. This result is confirmed when examining the ability of evolved phage to reduce ancestral host population sizes. Second, through interaction with a single bacteriophage for 24 h, P. aeruginosa can evolve resistance to the ancestor of that bacteriophage; this also provides these evolved bacteria with cross-resistance to the other three bacteriophages. We discuss how the evolutionary training of phages could be employed as effective means of combatting bacterial infections or disinfecting surfaces in hospital settings, with reduced risk of bacterial resistance compared with conventional methods. PMID:24187587

  18. Back to the future: evolving bacteriophages to increase their effectiveness against the pathogen Pseudomonas aeruginosa PAO1.

    PubMed

    Betts, Alex; Vasse, Marie; Kaltz, Oliver; Hochberg, Michael E

    2013-11-01

    Antibiotic resistance is becoming increasingly problematic for the treatment of infectious disease in both humans and livestock. The bacterium Pseudomonas aeruginosa is often found to be resistant to multiple antibiotics and causes high patient mortality in hospitals. Bacteriophages represent a potential option to combat pathogenic bacteria through their application in phage therapy. Here, we capitalize on previous studies showing how evolution may increase phage infection capacity relative to ancestral genotypes. We passaged four different phage isolates (podoviridae, myoviridae) through six serial transfers on the ancestral strain of Pseudomonas aeruginosa PAO1. We first demonstrate that repeated serial passage on ancestral bacteria increases infection capacity of bacteriophage on ancestral hosts and on those evolved for one transfer. This result is confirmed when examining the ability of evolved phage to reduce ancestral host population sizes. Second, through interaction with a single bacteriophage for 24 h, P. aeruginosa can evolve resistance to the ancestor of that bacteriophage; this also provides these evolved bacteria with cross-resistance to the other three bacteriophages. We discuss how the evolutionary training of phages could be employed as effective means of combatting bacterial infections or disinfecting surfaces in hospital settings, with reduced risk of bacterial resistance compared with conventional methods. PMID:24187587

  19. Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines

    PubMed Central

    Vázquez-Rivera, Dolores; González, Omar; Guzmán-Rodríguez, Jaquelina; Díaz-Pérez, Alma L.; Ochoa-Zarzosa, Alejandra; López-Bucio, José; Meza-Carmen, Víctor; Campos-García, Jesús

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen of plants and animals, which produces virulence factors in order to infect or colonize its eukaryotic hosts. Cyclodipeptides (CDPs) produced by P. aeruginosa exhibit cytotoxic properties toward human tumor cells. In this study, we evaluated the effect of a CDP mix, comprised of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) that were isolated from P. aeruginosa, on two human cancer cell lines. Our results demonstrated that the CDP mix promoted cell death in cultures of the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.53 and 0.66 mg/mL, for HeLa and Caco-2 cells, respectively. Flow cytometric analysis, using annexin V and propidium iodide as apoptosis and necrosis indicators, respectively, clearly showed that HeLa and Caco-2 cells exhibited apoptotic characteristics when treated with the CDP mix at a concentration <0.001 mg/mL. IC50 values for apoptotic cells in HeLa and Caco-2 cells were 6.5 × 10−5 and 1.8 × 10−4 mg/mL, respectively. Our results indicate that an apoptotic pathway is involved in the inhibition of cell proliferation caused by the P. aeruginosa CDP mix. PMID:25821788

  20. Extensive Reduction of Cell Viability and Enhanced Matrix Production in Pseudomonas aeruginosa PAO1 Flow Biofilms Treated with a d-Amino Acid Mixture

    PubMed Central

    Sanchez, Zoe; Tani, Akio

    2013-01-01

    Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a d-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells. PMID:23220960

  1. Dictyostelium discoideum as a surrogate host-microbe model for antivirulence screening in Pseudomonas aeruginosa PAO1.

    PubMed

    Bravo-Toncio, Catalina; Álvarez, Javiera A; Campos, Francisca; Ortíz-Severín, Javiera; Varas, Macarena; Cabrera, Ricardo; Lagos, Carlos F; Chávez, Francisco P

    2016-05-01

    The interest of the pharmaceutical industry in developing new antibiotics is decreasing, as established screening systems which identify compounds that kill or inhibit the growth of bacteria can no longer be used. Consequently, antimicrobial screening using classical minimum inhibitory concentration (MIC) measurements is becoming obsolete. The discovery of antimicrobial agents that specifically target a bacterial pathogen without affecting the host and its beneficial bacteria is a promising strategy. However, few host-microbe models are available for in vivo screening of novel antivirulence molecules. Here we designed high-throughput developmental assays in the social amoeba Dictyostelium discoideum to measure Pseudomonas aeruginosa virulence and to screen for novel antivirulence molecules without side effects to the host and its beneficial bacteria Klebsiella aerogenes. Thirty compounds were evaluated that had been previously selected by virtual screening for inhibitors of P. aeruginosa PAO1 polyphosphate kinase 1 (PaPPK1) and diverse compounds with combined PPK1 inhibitory and antivirulence activities were identified. This approach demonstrates that D. discoideum is a suitable surrogate host for preliminary high-throughput screening of antivirulence agents and that PPK1 is a suitable target for developing novel antivirulence compounds that can be further validated in mammalian models. PMID:27066943

  2. Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1.

    PubMed

    Winder, C L; Al-Adham, I S; Abdel Malek, S M; Buultjens, T E; Horrocks, A J; Collier, P J

    2000-08-01

    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). PMID:10971761

  3. Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1

    PubMed Central

    Zincke, Diansy; Balasubramanian, Deepak; Silver, Lynn L.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe and life-threatening infections that are highly impervious to treatment. This microbe readily exhibits intrinsic and acquired resistance to varied antimicrobial drugs. Resistance to penicillin-like compounds is commonplace and provided by the chromosomal AmpC β-lactamase. A second, chromosomally encoded β-lactamase, PoxB, has previously been reported in P. aeruginosa. In the present work, the contribution of this class D enzyme was investigated using a series of clean in-frame ampC, poxB, and oprD deletions, as well as complementation by expression under the control of an inducible promoter. While poxB deletions failed to alter β-lactam sensitivities, expression of poxB in ampC-deficient backgrounds decreased susceptibility to both meropenem and doripenem but had no effect on imipenem, penicillin, and cephalosporin MICs. However, when expressed in an ampCpoxB-deficient background, that additionally lacked the outer membrane porin-encoding gene oprD, PoxB significantly increased the imipenem as well as the meropenem and doripenem MICs. Like other class D carbapenem-hydrolyzing β-lactamases, PoxB was only poorly inhibited by class A enzyme inhibitors, but a novel non-β-lactam compound, avibactam, was a slightly better inhibitor of PoxB activity. In vitro susceptibility testing with a clinical concentration of avibactam, however, failed to reduce PoxB activity against the carbapenems. In addition, poxB was found to be cotranscribed with an upstream open reading frame, poxA, which itself was shown to encode a 32-kDa protein of yet unknown function. PMID:26621621

  4. GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1

    PubMed Central

    Sarwar, Zaara; Lundgren, Benjamin R.; Grassa, Michael T.; Wang, Michael X.; Gribble, Megan; Moffat, Jennifer F.

    2016-01-01

    ABSTRACT Glycine serves as a major source of single carbon units for biochemical reactions within bacterial cells. Utilization of glycine is tightly regulated and revolves around a key group of proteins known as the glycine cleavage system (GCS). Our lab previously identified the transcriptional regulator GcsR (PA2449) as being required for catabolism of glycine in the opportunistic pathogen Pseudomonas aeruginosa PAO1. In an effort to clarify and have an overall better understanding of the role of GcsR in glycine metabolism, a combination of transcriptome sequencing and electrophoretic mobility shift assays was used to identify target genes of this transcriptional regulator. It was found that GcsR binds to an 18-bp consensus sequence (TGTAACG-N4-CGTTCCG) upstream of the gcs2 operon, consisting of the gcvH2, gcvP2, glyA2, sdaA, and gcvT2 genes. The proteins encoded by these genes, namely, the GCS (GcvH2-GcvP2-GcvT2), serine hydroxymethyltransferase (GlyA2), and serine dehydratase (SdaA), form a metabolic pathway for the conversion of glycine into pyruvate, which can enter the central metabolism. GcsR activates transcription of the gcs2 operon in response to glycine. Interestingly, GcsR belongs to a family of transcriptional regulators known as TyrR-like enhancer-binding proteins (EBPs). Until this study, TyrR-like EBPs were only known to function in regulating aromatic amino acid metabolism. GcsR is the founding member of a new class of TyrR-like EBPs that function in the regulation of glycine metabolism. Indeed, homologs of GcsR and its target genes are present in almost all sequenced genomes of the Pseudomonadales order, suggesting that this genetic regulatory mechanism is a common theme for pseudomonads. IMPORTANCE Glycine is required for various cellular functions, including cell wall synthesis, protein synthesis, and the biosynthesis of several important metabolites. Regulating levels of glycine metabolism allows P. aeruginosa to maintain the metabolic flux

  5. Growth-dependent alterations in production of serotype-specific and common antigen lipopolysaccharides in Pseudomonas aeruginosa PAO1.

    PubMed Central

    McGroarty, E J; Rivera, M

    1990-01-01

    Pseudomonas aeruginosa PAO1 was grown in various media and at different temperatures, and the heterogeneity of the extracted lipopolysaccharide (LPS) was characterized by polyacrylamide gel electrophoresis. The size distributions of the serotype-specific LPS and the common antigen LPS were analyzed on Western blots (immunoblots). Cells grown at high, near-growth-limiting temperatures, at low pH, in low concentrations of phosphate, or in high concentrations of NaCl, MgCl2, glycerol, or sucrose produced decreased amounts of the very long chain population of O-antigen LPS molecules. Lower temperatures and lowered glycerol, lowered sucrose, low sulfate, lower salt concentrations, and elevated pH did not significantly affect the level of this LPS population. The size and amount of common antigen LPS was either unaffected or increased slightly when the cells were grown under the above stress conditions. Cells grown under normal, nonstressed conditions were agglutinated only by serotype-specific antibodies. In contrast, cells grown under stress conditions, in which the long-O-polymer LPS was absent, were agglutinated by both serotype-specific and common antigen-specific antibodies. The results indicate that the long O polymers cover and mask the shorter common antigen. However, specific growth conditions limit the production of the long O polymer, allowing the exposure and reactivity of the common antigen on the cell surface. Images PMID:2108085

  6. Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of Pseudomonas aeruginosa PAO1

    PubMed Central

    Yang, Zhe; Lu, Chung-Dar

    2007-01-01

    The arginine transaminase (ATA) pathway represents one of the multiple pathways for l-arginine catabolism in Pseudomonas aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates l-arginine and pyruvate into 2-ketoarginine and l-alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and l-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD+ and l-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With l-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated Vmax and kcat values of 54.6 ± 2.5 μmol/min/mg and 38.6 ± 1.8 s−1. The apparent Km and catalytic efficiency (kcat/Km) were 1.6 ± 0.1 mM and 24.1 mM−1 s−1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM−1 s−1 for l-arginine. When l-lysine was used as the substrate, MS analysis suggested Δ1-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism. PMID:17416668

  7. Influence of ferric iron on gene expression and rhamnolipid synthesis during batch cultivation of Pseudomonas aeruginosa PAO1.

    PubMed

    Schmidberger, Anke; Henkel, Marius; Hausmann, Rudolf; Schwartz, Thomas

    2014-08-01

    Bioprocesses based on sustainable resources and rhamnolipids in particular have become increasingly attractive in recent years. These surface-active glycolipids with various chemical and biological properties have diverse biotechnological applications and are naturally produced by Pseudomonas aeruginosa. Their production, however, is tightly governed by a complex growth-dependent regulatory network, one of the major obstacles in the way to upscale production. P. aeruginosa PAO1 was grown in shake flask cultures using varying concentrations of ferric iron. Gene expression was assessed using quantitative PCR. A strong increase in relative expression of the genes for rhamnolipid synthesis, rhlA and rhlC, as well as the genes of the pqs quorum sensing regulon was observed under iron-limiting conditions. Iron repletion on the other hand caused a down-regulation of those genes. Furthermore, gene expression of different iron regulation-related factors, i.e. pvdS, fur and bqsS, was increased in response to iron limitation. Ensuing from these results, a batch cultivation using production medium without any addition of iron was conducted. Both biomass formation and specific growth rates were not impaired compared to normal cultivation conditions. Expression of rhlA, rhlC and pvdS, as well as the gene for the 3-oxo-C12-HSL synthetase, lasI, increased until late stationary growth phase. After this time point, their expression steadily decreased. Expression of the C4-HSL synthetase gene, rhlI, on the other hand, was found to be highly increased during the entire process. PMID:24752844

  8. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    PubMed

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production. PMID:26922727

  9. Induction of mexCD-oprJ operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1.

    PubMed

    Morita, Yuji; Murata, Takeshi; Mima, Takehiko; Shiota, Sumiko; Kuroda, Teruo; Mizushima, Tohru; Gotoh, Naomasa; Nishino, Takeshi; Tsuchiya, Tomofusa

    2003-04-01

    Induction of the MexCD-OprJ multidrug efflux pump was investigated in wild-type Pseudomonas aeruginosa PAO1. MexCD-OprJ was induced by clinically important disinfectants such as benzalkonium chloride and chlorhexidine gluconate, and by some cytotoxic agents such as tetraphenylphosphonium chloride, ethidium bromide and rhodamine 6G. MexCD-OprJ was not induced by norfloxacin, tetracycline, chloramphenicol, streptomycin, erythromycin or carbenicillin, although they are substrates for the pump. Cells of PAO1 showed increased resistance to norfloxacin when grown in the presence of the inducers of the mexCD-oprJ operon mentioned above. These results indicate that MexCD-OprJ plays an important role in intrinsic multidrug resistance in wild-type P. aeruginosa in hospitals where disinfectants are used frequently. PMID:12654738

  10. The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1.

    PubMed

    Lin, Jinshui; Cheng, Juanli; Chen, Keqi; Guo, Chenghao; Zhang, Weipeng; Yang, Xu; Ding, Wei; Ma, Li; Wang, Yao; Shen, Xihui

    2015-01-01

    The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria. Three separate T6SSs called H1-, H2-, and H3-T6SS have been discovered in Pseudomonas aeruginosa PAO1. Recent studies suggest that, in contrast to the H1-T6SS that targets prokaryotic cells, H2- and H3-T6SS are involved in interactions with both prokaryotic and eukaryotic cells. However, the detailed functions of T6SS components are still uncharacterized. The intracellular multiplication factor (IcmF) protein is conserved in type VI secretion systems (T6SS) of all different bacterial pathogens. Bioinformatic analysis revealed that IcmF3 in P. aeruginosa PAO1 is different from other IcmF homologs and may represent a new branch of these proteins with distinct functions. Herein, we have investigated the function of IcmF3 in this strain. We have shown that deletion of the icmF3 gene in P. aeruginosa PAO1 is associated with pleiotropic phenotypes. The icmF3 mutant has variant colony morphology and an hypergrowth phenotype in iron-limiting medium. Surprisingly, this mutant is also defective for the production of pyoverdine, as well as defects in swimming motility and virulence in a C. elegans worm model. The icmF3 mutant exhibits higher conjugation frequency than the wild type and increased biofilm formation on abiotic surfaces. Additionally, expression of two phenazine biosynthetic loci is increased in the icmF3 mutant, leading to the overproduction of pyocyanin. Finally, the mutant exhibits decreased susceptibility to aminoglycosides such as tobramycin and gentamicin. And the detected phenotypes can be restored completely or partially by trans complementation of wild type icmF3 gene. The pleiotropic effects observed upon icmF3 deletion demonstrate that icmF3 plays critical roles in both pathogenesis and environmental adaptation in P. aeruginosa PAO1. PMID:26484316

  11. Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates.

    PubMed Central

    Kureishi, A; Diver, J M; Beckthold, B; Schollaardt, T; Bryan, L E

    1994-01-01

    The Pseudomonas aeruginosa DNA gyrase gyrA gene was cloned and sequenced from strain PAO1. An open reading frame of 2,769 bp was found; it coded for a protein of 923 amino acids with an estimated molecular mass of 103 kDa. The derived amino acid sequence shared 67% identity with Escherichia coli GyrA and 54% identity with Bacillus subtilis GyrA, although conserved regions were present throughout the sequences, particularly toward the N terminus. Complementation of an E. coli mutant with a temperature-sensitive gyrA gene with the PAO1 gyrA gene showed that the gene is expressed in E. coli and is able to functionally complement the E. coli DNA gyrase B subunit. Expression of PAO1 gyrA in E. coli or P. aeruginosa with mutationally altered gyrA genes caused a reversion to wild-type quinolone susceptibility, indicating that the intrinsic susceptibility of the PAO1 GyrA to quinolones is comparable to that of the E. coli enzyme. PCR was used to amplify 360 bp of P. aeruginosa gyrA encompassing the so-called quinolone resistance-determining region from ciprofloxacin-resistant clinical isolates from patients with cystic fibrosis. Mutations were found in three of nine isolates tested; these mutations caused the following alterations in the sequence of GyrA: Asp at position 87 (Asp-87) to Asn, Asp-87 to Tyr, and Thr-83 to Ile. The resistance mechanisms in the other six isolates are unknown. The results of the study suggested that mechanisms other than a mutational alteration in gyrA are the most common mechanism of ciprofloxacin resistance in P. aeruginosa from the lungs of patients with cystic fibrosis. Images PMID:7811002

  12. The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lin, Jinshui; Cheng, Juanli; Chen, Keqi; Guo, Chenghao; Zhang, Weipeng; Yang, Xu; Ding, Wei; Ma, Li; Wang, Yao; Shen, Xihui

    2015-01-01

    The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria. Three separate T6SSs called H1-, H2-, and H3-T6SS have been discovered in Pseudomonas aeruginosa PAO1. Recent studies suggest that, in contrast to the H1-T6SS that targets prokaryotic cells, H2- and H3-T6SS are involved in interactions with both prokaryotic and eukaryotic cells. However, the detailed functions of T6SS components are still uncharacterized. The intracellular multiplication factor (IcmF) protein is conserved in type VI secretion systems (T6SS) of all different bacterial pathogens. Bioinformatic analysis revealed that IcmF3 in P. aeruginosa PAO1 is different from other IcmF homologs and may represent a new branch of these proteins with distinct functions. Herein, we have investigated the function of IcmF3 in this strain. We have shown that deletion of the icmF3 gene in P. aeruginosa PAO1 is associated with pleiotropic phenotypes. The icmF3 mutant has variant colony morphology and an hypergrowth phenotype in iron-limiting medium. Surprisingly, this mutant is also defective for the production of pyoverdine, as well as defects in swimming motility and virulence in a C. elegans worm model. The icmF3 mutant exhibits higher conjugation frequency than the wild type and increased biofilm formation on abiotic surfaces. Additionally, expression of two phenazine biosynthetic loci is increased in the icmF3 mutant, leading to the overproduction of pyocyanin. Finally, the mutant exhibits decreased susceptibility to aminoglycosides such as tobramycin and gentamicin. And the detected phenotypes can be restored completely or partially by trans complementation of wild type icmF3 gene. The pleiotropic effects observed upon icmF3 deletion demonstrate that icmF3 plays critical roles in both pathogenesis and environmental adaptation in P. aeruginosa PAO1. PMID:26484316

  13. Molecular Characterization and Regulation of the aguBA Operon, Responsible for Agmatine Utilization in Pseudomonas aeruginosa PAO1

    PubMed Central

    Nakada, Yuji; Jiang, Ying; Nishijyo, Takayuki; Itoh, Yoshifumi; Lu, Chung-Dar

    2001-01-01

    Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (Mr 32,759; 292 amino acids) displayed sequence similarity (≤60% identity) to enzymes of the β-alanine synthase/nitrilase family. While the deduced AguA protein (Mr 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (Mr 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function. PMID:11673419

  14. Molecular characterization and regulation of the aguBA operon, responsible for agmatine utilization in Pseudomonas aeruginosa PAO1.

    PubMed

    Nakada, Y; Jiang, Y; Nishijyo, T; Itoh, Y; Lu, C D

    2001-11-01

    Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (M(r) 32,759; 292 amino acids) displayed sequence similarity (< or =60% identity) to enzymes of the beta-alanine synthase/nitrilase family. While the deduced AguA protein (M(r) 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (M(r) 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function. PMID:11673419

  15. Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

    PubMed Central

    Sarabhai, Sajal; Sharma, Prince; Capalash, Neena

    2013-01-01

    Background Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors. Methods and Results Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C12HSL and C4HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C4HSL. F7 also showed antagonistic activity against 3-oxo-C12HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract. Conclusions This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced

  16. Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1.

    PubMed

    Kim, Eun-Jin; Sabra, Wael; Zeng, An-Ping

    2003-09-01

    Pseudomonas aeruginosa PAO1 was recently found to exhibit two remarkable physiological responses to oxidative stress: (1) a strong reduction in the efficiency of oxygen transfer from the gas phase into the liquid phase, thus causing oxygen limitation in the culture and (2) formation of a clear polysaccharide capsule on the cell surface. In this work, it has been shown that the iron concentration in the culture plays a crucial role in evoking these phenomena. The physiological responses of two P. aeruginosa PAO1 isolates (NCCB 2452 and ATCC 15692) were examined in growth media with varied iron concentrations. In a computer-controlled bioreactor cultivation system for controlled dissolved oxygen tension (pO2), a strong correlation between the exhaustion of iron and the onset of oxygen limitation was observed. The oxygen transfer rate of the culture, characterized by the volumetric oxygen transfer coefficient, kLa, significantly decreased under iron-limited conditions. The formation of alginate and capsule was more strongly affected by iron concentration than by oxygen concentration. The reduction of the oxygen transfer rate and the subsequent oxygen limitation triggered by iron deficiency may represent a new and efficient way for P. aeruginosa PAO1 to adapt to growth conditions of iron limitation. Furthermore, the secretion of proteins into the culture medium was strongly enhanced by iron limitation. The formation of the virulence factor elastase and the iron chelators pyoverdine and pyochelin also significantly increased under iron-limited conditions. These results have implications for lung infection of cystic fibrosis patients by P. aeruginosa in view of the prevalence of iron limitation at the site of infection and the respiratory failure leading to death. PMID:12949186

  17. Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in Pseudomonas aeruginosa Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA

    PubMed Central

    Malhotra, Sonal; Silo-Suh, Laura A.; Mathee, Kalai; Ohman, Dennis E.

    2000-01-01

    Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation. Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The oprF-lacZ fusion showed little difference in levels of expression in the two strains. However, the dsbA-lacZ fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase

  18. Regulation of Motility and Phenazine Pigment Production by FliA Is Cyclic-di-GMP Dependent in Pseudomonas aeruginosa PAO1

    PubMed Central

    Lo, Yi-Ling; Shen, Lunda; Chang, Chih-Hsuan; Bhuwan, Manish; Chiu, Cheng-Hsun; Chang, Hwan-You

    2016-01-01

    The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa. PMID:27175902

  19. Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1

    PubMed Central

    Yang, Wen; Li, Kan; Bai, Yuwei; Xu, Yueyang; Jin, Jin; Wang, Yingying; Bartlam, Mark

    2015-01-01

    Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and α4-α5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation. PMID:25909591

  20. Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1.

    PubMed

    Cui, Qinna; Lv, Huinan; Qi, Zhuangzhuang; Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

    2016-01-01

    Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

  1. Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1

    PubMed Central

    Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

    2016-01-01

    Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

  2. Structural Analysis of WbpE from Pseudomonas aeruginosa PAO1: A Nucleotide Sugar Aminotransferase Involved in O-Antigen Assembly

    SciTech Connect

    Larkin, A.; Olivier, N; Imperiali, B

    2010-01-01

    In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD, and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5{prime}-phosphate (PLP) and product UDP-GlcNAc(3NH{sub 2})A as the external aldimine at 1.9 {angstrom} resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the {alpha}-carboxylate moiety of {alpha}-ketoglutarate, is also uniquely positioned to bestow specificity for the 6{double_prime}-carboxyl group of GlcNAc(3NH2)A through a salt bridge. This finding is intriguing because while an analogous basic residue is present in WbpE homologues that do not process 6{double_prime}-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C6{double_prime} atom. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2{double_prime}, C3{double_prime}, and C6{double_prime} positions and provides insight into a novel target for treatment of P

  3. Proteomic profiling of Pseudomonas aeruginosa AES-1R, PAO1 and PA14 reveals potential virulence determinants associated with a transmissible cystic fibrosis-associated strain

    PubMed Central

    2012-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood. Results A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute) isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 P. aeruginosa proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins. Conclusions Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit P. aeruginosa infections in CF patients. PMID:22264352

  4. Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1

    NASA Astrophysics Data System (ADS)

    Islam, Salim T.; Huszczynski, Steven M.; Nugent, Timothy; Gold, Alexander C.; Lam, Joseph S.

    2013-12-01

    O antigen (O-Ag) in many bacteria is synthesized via the Wzx/Wzy-dependent pathway in which Wzy polymerizes lipid-linked O-Ag subunits to modal lengths regulated by Wzz. Characterization of 83 site-directed mutants of Wzy from Pseudomonas aeruginosa PAO1 (WzyPa) in topologically-mapped periplasmic (PL) and cytoplasmic loops (CL) verified the functional importance of PL3 and PL5, with the former shown to require overall cationic properties. Essential Arg residues in the RX10G motifs of PL3 and PL5 were found to be conserved in putative homologues of WzyPa, as was the overall sequence homology between these two periplasmic loops in each protein. Amino acid substitutions in CL6 were found to alter Wzz-mediated O-antigen modality, with evidence suggesting that these changes may perturb the C-terminal WzyPa tertiary structure. Together, these data suggest that the catch-and-release mechanism of O-Ag polymerization is widespread among bacteria and that regulation of polymer length is affected by interaction of Wzz with Wzy.

  5. Function-Biased Choice of Additives for Optimization of Protein Crystallization: The Case of the Putative Thioesterase PA5185 from Pseudomonas aeruginosa PAO1

    SciTech Connect

    Chruszcz, Maksymilian; Zimmerman, Matthew D.; Wang, Shuren; Koclega, Katarzyna D.; Zheng, Heping; Evdokimova, Elena; Kudritska, Marina; Cymborowski, Marcin; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2009-09-15

    The crystal structure of PA5185, a putative thioesterase from Pseudomonas aeruginosa strain PAO1, was solved using multi-wavelength anomalous diffraction to 2.4 {angstrom}. Analysis of the structure and information about the putative function of the protein were used to optimize crystallization conditions. The crystal growth was optimized by applying additives with chemical similarity to a fragment of a putative PA5185 substrate (CoA or its derivative). Using new crystallization conditions containing this function-biased set of additives, several new crystal forms were produced, and structures of three of them (in three different space groups) were determined. One of the new crystal forms had an improved resolution limit of 1.9 {angstrom}, and another displayed an alternative conformation of the highly conserved loop containing Asn26, which could play a physiological role. Surprisingly, none of the additives were ordered in the crystal structures. Application of function-biased additives could be used as a standard optimization protocol for producing improved diffraction, or new crystal forms, which may lead to better understanding of the biological functions of proteins.

  6. Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli.

    PubMed

    Lee, Jae Taek; Lee, Seung Sik; Mondal, Suvendu; Tripathi, Bhumi Nath; Kim, Siu; Lee, Keun Woo; Hong, Sung Hyun; Bai, Hyoung-Woo; Cho, Jae-Young; Chung, Byung Yeoup

    2016-08-31

    Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of Ser(78) to Cys(78) resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of Cys(78) in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone. PMID:27457208

  7. Enhancement of the Chaperone Activity of Alkyl Hydroperoxide Reductase C from Pseudomonas aeruginosa PAO1 Resulting from a Point-Specific Mutation Confers Heat Tolerance in Escherichia coli

    PubMed Central

    Lee, Jae Taek; Lee, Seung Sik; Mondal, Suvendu; Tripathi, Bhumi Nath; Kim, Siu; Lee, Keun Woo; Hong, Sung Hyun; Bai, Hyoung-Woo; Cho, Jae-Young; Chung, Byung Yeoup

    2016-01-01

    Alkyl hydroperoxide reductase subunit C from Pseudomonas aeruginosa PAO1 (PaAhpC) is a member of the 2-Cys peroxiredoxin family. Here, we examined the peroxidase and molecular chaperone functions of PaAhpC using a site-directed mutagenesis approach by substitution of Ser and Thr residues with Cys at positions 78 and 105 located between two catalytic cysteines. Substitution of Ser with Cys at position 78 enhanced the chaperone activity of the mutant (S78C-PaAhpC) by approximately 9-fold compared with that of the wild-type protein (WT-PaAhpC). This increased activity may have been associated with the proportionate increase in the high-molecular-weight (HMW) fraction and enhanced hydrophobicity of S78C-PaAhpC. Homology modeling revealed that mutation of Ser78 to Cys78 resulted in a more compact decameric structure than that observed in WT-PaAhpC and decreased the atomic distance between the two neighboring sulfur atoms of Cys78 in the dimer-dimer interface of S78C-PaAhpC, which could be responsible for the enhanced hydrophobic interaction at the dimer-dimer interface. Furthermore, complementation assays showed that S78C-PaAhpC exhibited greatly improved the heat tolerance, resulting in enhanced survival under thermal stress. Thus, addition of Cys at position 78 in PaAhpC modulated the functional shifting of this protein from a peroxidase to a chaperone. PMID:27457208

  8. Regulation and characterization of the dadRAX locus for D-amino acid catabolism in Pseudomonas aeruginosa PAO1.

    PubMed

    He, Weiqing; Li, Congran; Lu, Chung-Dar

    2011-05-01

    D-amino acids are essential components for bacterial peptidoglycan, and these natural compounds are also involved in cell wall remodeling and biofilm disassembling. In Pseudomonas aeruginosa, the dadAX operon, encoding the D-amino acid dehydrogenase DadA and the amino acid racemase DadX, is essential for D- and L-Ala catabolism, and its expression requires a transcriptional regulator, DadR. In this study, purified recombinant DadA alone was sufficient to demonstrate the proposed enzymatic activity with very broad substrate specificity; it utilizes all D-amino acids tested as substrates except D-Glu and D-Gln. DadA also showed comparable k(cat) and K(m) values on D-Ala and several D-amino acids. dadRAX knockout mutants were constructed and subjected to analysis of their growth phenotypes on amino acids. The results revealed that utilization of L-Ala, L-Trp, D-Ala, and a specific set of D-amino acids as sole nitrogen sources was abolished in the dadA mutant and/or severely hampered in the dadR mutant while growth yield on D-amino acids was surprisingly improved in the dadX mutant. The dadA promoter was induced by several L-amino acids, most strongly by Ala, and only by D-Ala among all tested D-amino acids. Enhanced growth of the dadX mutant on D-amino acids is consistent with the finding that the dadA promoter was constitutively induced in the dadX mutant, where exogenous D-Ala but not L-Ala reduced the expression. Binding of DadR to the dadA regulatory region was demonstrated by electromobility shift assays, and the presence of L-Ala but not D-Ala increased affinity by 3-fold. The presence of multiple DadR-DNA complexes in the dadA regulatory region was demonstrated in vitro, and the formation of these nucleoprotein complexes exerted a complicated impact on promoter activation in vivo. In summary, the results from this study clearly demonstrate DadA to be the enzyme solely responsible for the proposed D-amino acid dehydrogenase activity of broad substrate

  9. The Combined Structural and Kinetic Characterization of a Bacterial Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes NMO Class I and II*

    PubMed Central

    Salvi, Francesca; Agniswamy, Johnson; Yuan, Hongling; Vercammen, Ken; Pelicaen, Rudy; Cornelis, Pierre; Spain, Jim C.; Weber, Irene T.; Gadda, Giovanni

    2014-01-01

    Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBankTM as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with kcatapp/Kmapp of 12 × 106 m−1 s−1, kcatapp of 1300 s−1, and Kmapp of 110 μm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO. PMID:25002579

  10. Functional analysis and regulation of the divergent spuABCDEFGH-spuI operons for polyamine uptake and utilization in Pseudomonas aeruginosa PAO1.

    PubMed

    Lu, Chung-Dar; Itoh, Yoshifumi; Nakada, Yuji; Jiang, Ying

    2002-07-01

    A multiple-gene locus for polyamine uptake and utilization was discovered in Pseudomonas aeruginosa PAO1. This locus contained nine genes designated spuABCDEFGHI (spu for spermidine and putrescine utilization). The physiological functions of the spu genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn5 transposon-mediated spu knockout mutants. Growth and uptake experiments support that the spuDEFGH genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that spuC encodes putrescine aminotransferase with pyruvate as the amino group receptor. Although spuA and spuB mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated. Assays of lacZ fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the spuABCDEFGH operon and the divergently transcribed spuI gene of unknown function. Since the observed induction effect of agmatine was abolished in an aguA mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the spuA-spuI divergent promoters. S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites. Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region. Finally, the absence of the putrescine-inducible spuA expression and putrescine aminotransferase (spuC) formation in the cbrB mutant indicated that the spu operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator. PMID:12081945

  11. Quorum quenching activity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn., and its effect on quorum sensing regulated biofilm in Pseudomonas aeruginosa PAO1.

    PubMed

    Rajesh, P S; Ravishankar Rai, V

    2014-01-01

    Quorum sensing mechanism allows the microorganisms to resist the antibiotic treatment by forming biofilms. Quorum quenching is one of the mechanisms to control the development of drug resistance in microbes. Endophyte bacteria are beneficial to plant growth as they support the immune system against the pathogen attack. The endophytic bacteria present in Pterocarpus santalinus were screened for the presence of N-acyl homoserine lactones (AHLs) degrading bacteria using biosensor strains and further confirmed by quantifying the violacein production. Cell-free lysate of endophytic bacteria, Bacillus firmus PT18 and Enterobacter asburiae PT39 exhibited potent AHL degrading ability by inhibiting about 80% violacein production in biosensor strain. Furthermore, when the cell-free lysate was applied to Pseudomonas aeruginosa PAO1 and PAO1-JP2 biofilm it resulted in significant (p<0.01) inhibition of biofilm formation. The biofilm inhibition was confirmed by visualization of biofilm slides under fluorescence microscopy, which showed decrease in total biomass formation in treated slides. Isolation and amplification of the gene (aiiA) indicated that the presence of AHL lactonase in cell-free lysate and sequence alignment indicated that AiiA contains a "HXHXDH" zinc-binding motif that is being conserved in several groups of metallohydrolases. Therefore, the study shows the potential of AHLs degradation by AHL lactonase present in cell-free lysate of isolated endophytic bacteria and inhibition of quorum sensing regulated biofilm formation in P. aeruginosa PAO1. PMID:24268182

  12. Involvement of NarK1 and NarK2 Proteins in Transport of Nitrate and Nitrite in the Denitrifying Bacterium Pseudomonas aeruginosa PAO1

    PubMed Central

    Sharma, Vandana; Noriega, Chris E.; Rowe, John J.

    2006-01-01

    Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ΔnarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ΔnarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions. PMID:16391109

  13. Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1.

    PubMed

    Choi, Sung-Chan; Zhang, Can; Moon, Sooyoung; Oh, Young-Sook

    2014-09-01

    4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development. PMID:25085732

  14. Identification of greA encoding a transcriptional elongation factor as a member of the carA-orf-carB-greA operon in Pseudomonas aeruginosa PAO1.

    PubMed Central

    Lu, C D; Kwon, D H; Abdelal, A T

    1997-01-01

    A homolog of the transcriptional elongation factor, GreA, was identified in Pseudomonas aeruginosa PAO1. The deduced amino acid sequence for GreA from this organism exhibits 65.2% identity to its counterpart in Escherichia coli K-12. The nucleotide sequence of greA from P. aeruginosa overlaps by four bases the 3' terminus of carB which encodes the large subunit of carbamoylphosphate synthetase. S1 nuclease experiments showed that level of the greA transcript is elevated approximately 10-fold under conditions of pyrimidine limitation, consistent with the conclusion that transcription is initiated from the previously identified pyrimidine-sensitive promoter upstream of the carA-orf-carB-greA operon. Transcriptional fusion experiments showed the presence of an additional weak promoter within the carB sequence. A greA insertional mutant of Pseudomonas aerugionsa was constructed by gene replacement. The mutant derivative grew well in rich medium but did not grow in minimal medium supplemented by arginine and nucleosides. The greA phenotype was suppressed by secondary mutations at a relatively high rate, consistent with the notion of an important physiological role for GreA. PMID:9139926

  15. Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-antigen Assembly in Pseudomonas aeruginosa PAO1

    PubMed Central

    Larkin, Angelyn; Imperiali, Barbara

    2009-01-01

    The B-band O-antigen of the lipopolysaccharide found in the opportunistic pathogen Pseudomonas aeruginosa PAO1 (serotype O5) comprises a repeating trisaccharide unit that is critical for virulence and protection from host defense systems. One of the carbohydrates in this repeating unit, the rare diacetylated aminuronic acid derivative 2,3-diacetamido-2,3-dideoxy-β-d-mannuronic acid (ManNAc(3NAc)A), is thought to be produced by five enzymes (WbpA, WbpB, WbpE, WbpD and WbpI) in a stepwise manner starting from UDP-GlcNAc. Although the genes responsible for the biosynthesis of this sugar are known, only two of the five encoded proteins (WbpA and WbpI) have been thoroughly investigated. In this report, we describe the cloning, overexpression, purification and biochemical characterization of the three central enzymes in this pathway, WbpB, WbpE, and WbpD. Using a combination of capillary electrophoresis, RP-HPLC and NMR spectroscopy, we show that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH2)A in a coupled reaction via a unique NAD+ recycling pathway. In addition, we confirm that WbpD catalyzes the acetylation of UDP-GlcNAc(3NH2)A to give UDP-GlcNAc(3NAc)A. Notably, WbpA, WbpB, WbpE, WbpD and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel, thereby providing supplies of this complex glycosyl donor for future studies of LPS assembly. This work completes the biochemical characterization of the enzymes in this pathway and provides novel targets for potential therapeutics to combat infections with drug resistant P. aeruginosa strains. PMID:19348502

  16. Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-d-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-d-Rhamnosyltransferase WbpZ

    PubMed Central

    Wang, Shuo; Hao, Youai; Lam, Joseph S.; Vlahakis, Jason Z.; Szarek, Walter A.; Vinnikova, Anna; Veselovsky, Vladimir V.

    2015-01-01

    ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of d-rhamnose (d-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-d-Rha and enzymatically synthesized GDP-d-[3H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one d-Rha residue from GDP-d-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring d-mannose (d-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-d-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-rhamnosyltransferase that has significant activity of GDP-d-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a d-Rha-transferase and a critical step in the development of CPA synthesis inhibitors. IMPORTANCE This is the first characterization of a d-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen. PMID:25845842

  17. Complete Genome Sequence of the Multiresistant Taxonomic Outlier Pseudomonas aeruginosa PA7

    PubMed Central

    Roy, Paul H.; Tetu, Sasha G.; Larouche, André; Elbourne, Liam; Tremblay, Simon; Ren, Qinghu; Dodson, Robert; Harkins, Derek; Shay, Ryan; Watkins, Kisha; Mahamoud, Yasmin; Paulsen, Ian T.

    2010-01-01

    Pseudomonas aeruginosa PA7 is a non-respiratory human isolate from Argentina that is multiresistant to antibiotics. We first sequenced gyrA, gyrB, parC, parE, ampC, ampR, and several housekeeping genes and found that PA7 is a taxonomic outlier. We report here the complete sequence of the 6,588,339 bp genome, which has only about 95% overall identity to other strains. PA7 has multiple novel genomic islands and a total of 51 occupied regions of genomic plasticity. These islands include antibiotic resistance genes, parts of transposons, prophages, and a pKLC102-related island. Several PA7 genes not present in PAO1 or PA14 are putative orthologues of other Pseudomonas spp. and Ralstonia spp. genes. PA7 appears to be closely related to the known taxonomic outlier DSM1128 (ATCC9027). PA7 lacks several virulence factors, notably the entire TTSS region corresponding to PA1690-PA1725 of PAO1. It has neither exoS nor exoU and lacks toxA, exoT, and exoY. PA7 is serotype O12 and pyoverdin type II. Preliminary proteomic studies indicate numerous differences with PAO1, some of which are probably a consequence of a frameshift mutation in the mvfR quorum sensing regulatory gene. PMID:20107499

  18. The Accessory Genome of Pseudomonas aeruginosa

    PubMed Central

    Kung, Vanderlene L.; Ozer, Egon A.; Hauser, Alan R.

    2010-01-01

    Summary: Pseudomonas aeruginosa strains exhibit significant variability in pathogenicity and ecological flexibility. Such interstrain differences reflect the dynamic nature of the P. aeruginosa genome, which is composed of a relatively invariable “core genome” and a highly variable “accessory genome.” Here we review the major classes of genetic elements comprising the P. aeruginosa accessory genome and highlight emerging themes in the acquisition and functional importance of these elements. Although the precise phenotypes endowed by the majority of the P. aeruginosa accessory genome have yet to be determined, rapid progress is being made, and a clearer understanding of the role of the P. aeruginosa accessory genome in ecology and infection is emerging. PMID:21119020

  19. Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa

    SciTech Connect

    Saiman, L.; Cacalano, G.; Prince, A. )

    1990-08-01

    Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.

  20. Integrated whole-genome screening for Pseudomonas aeruginosa virulence genes using multiple disease models reveals that pathogenicity is host specific.

    PubMed

    Dubern, Jean-Frédéric; Cigana, Cristina; De Simone, Maura; Lazenby, James; Juhas, Mario; Schwager, Stephan; Bianconi, Irene; Döring, Gerd; Eberl, Leo; Williams, Paul; Bragonzi, Alessandra; Cámara, Miguel

    2015-11-01

    Pseudomonas aeruginosa is a multi-host opportunistic pathogen causing a wide range of diseases because of the armoury of virulence factors it produces, and it is difficult to eradicate because of its intrinsic resistance to antibiotics. Using an integrated whole-genome approach, we searched for P. aeruginosa virulence genes with multi-host relevance. We constructed a random library of 57 360 Tn5 mutants in P. aeruginosa PAO1-L and screened it in vitro for those showing pleiotropic effects in virulence phenotypes (reduced swarming, exo-protease and pyocyanin production). A set of these pleiotropic mutants were assayed for reduced toxicity in Drosophila melanogaster, Caenorhabditis elegans, human cell lines and mice. Surprisingly, the screening revealed that the virulence of the majority of P. aeruginosa mutants varied between disease models, suggesting that virulence is dependent on the disease model used and hence the host environment. Genomic analysis revealed that these virulence-related genes encoded proteins from almost all functional classes, which were conserved among P. aeruginosa strains. Thus, we provide strong evidence that although P. aeruginosa is capable of infecting a wide range of hosts, many of its virulence determinants are host specific. These findings have important implication when searching for novel anti-virulence targets to develop new treatments against P. aeruginosa. PMID:25845292

  1. Genetically and Phenotypically Distinct Pseudomonas aeruginosa Cystic Fibrosis Isolates Share a Core Proteomic Signature

    PubMed Central

    Penesyan, Anahit; Kumar, Sheemal S.; Kamath, Karthik; Shathili, Abdulrahman M.; Venkatakrishnan, Vignesh; Krisp, Christoph; Packer, Nicolle H.; Molloy, Mark P.; Paulsen, Ian T.

    2015-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of “ready-made” nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such

  2. Identification of virulence genes in a pathogenic strain of Pseudomonas aeruginosa by representational difference analysis.

    PubMed

    Choi, Ji Young; Sifri, Costi D; Goumnerov, Boyan C; Rahme, Laurence G; Ausubel, Frederick M; Calderwood, Stephen B

    2002-02-01

    Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA. PMID:11807055

  3. Genome Analysis of Environmental and Clinical P. aeruginosa Isolates from Sequence Type-1146

    PubMed Central

    Sánchez, David; Gomila, Margarita; Bennasar, Antonio; Lalucat, Jorge; García-Valdés, Elena

    2014-01-01

    The genomes of Pseudomonas aeruginosa isolates of the new sequence type ST-1146, three environmental (P37, P47 and P49) and one clinical (SD9) isolates, with differences in their antibiotic susceptibility profiles have been sequenced and analysed. The genomes were mapped against P. aeruginosa PAO1-UW and UCBPP-PA14. The allelic profiles showed that the highest number of differences were in “Related to phage, transposon or plasmid” and “Secreted factors” categories. The clinical isolate showed a number of exclusive alleles greater than that for the environmental isolates. The phage Pf1 region in isolate SD9 accumulated the highest number of nucleotide substitutions. The ORF analysis of the four genomes assembled de novo indicated that the number of isolate-specific genes was higher in isolate SD9 (132 genes) than in isolates P37 (24 genes), P47 (16 genes) and P49 (21 genes). CRISPR elements were found in all isolates and SD9 showed differences in the spacer region. Genes related to bacteriophages F116 and H66 were found only in isolate SD9. Genome comparisons indicated that the isolates of ST-1146 are close related, and most genes implicated in pathogenicity are highly conserved, suggesting a genetic potential for infectivity in the environmental isolates similar to the clinical one. Phage-related genes are responsible of the main differences among the genomes of ST-1146 isolates. The role of bacteriophages has to be considered in the adaptation processes of isolates to the host and in microevolution studies. PMID:25329302

  4. Genome comparison of Pseudomonas aeruginosa large phages.

    PubMed

    Hertveldt, Kirsten; Lavigne, Rob; Pleteneva, Elena; Sernova, Natalia; Kurochkina, Lidia; Korchevskii, Roman; Robben, Johan; Mesyanzhinov, Vadim; Krylov, Victor N; Volckaert, Guido

    2005-12-01

    Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences. PMID:16256135

  5. Genomic Rearrangements and Functional Diversification of lecA and lecB Lectin-Coding Regions Impacting the Efficacy of Glycomimetics Directed against Pseudomonas aeruginosa.

    PubMed

    Boukerb, Amine M; Decor, Aude; Ribun, Sébastien; Tabaroni, Rachel; Rousset, Audric; Commin, Loris; Buff, Samuel; Doléans-Jordheim, Anne; Vidal, Sébastien; Varrot, Annabelle; Imberty, Anne; Cournoyer, Benoit

    2016-01-01

    LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewis(a) rather than Lewis(x). Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewis(a) showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great

  6. Genomic Rearrangements and Functional Diversification of lecA and lecB Lectin-Coding Regions Impacting the Efficacy of Glycomimetics Directed against Pseudomonas aeruginosa

    PubMed Central

    Boukerb, Amine M.; Decor, Aude; Ribun, Sébastien; Tabaroni, Rachel; Rousset, Audric; Commin, Loris; Buff, Samuel; Doléans-Jordheim, Anne; Vidal, Sébastien; Varrot, Annabelle; Imberty, Anne; Cournoyer, Benoit

    2016-01-01

    LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewisa rather than Lewisx. Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewisa showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great variations

  7. The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa

    PubMed Central

    Wagner, Andreas; MacLean, R. Craig

    2016-01-01

    Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs. PMID:27149698

  8. Subtilase SprP exerts pleiotropic effects in Pseudomonas aeruginosa.

    PubMed

    Pelzer, Alexander; Polen, Tino; Funken, Horst; Rosenau, Frank; Wilhelm, Susanne; Bott, Michael; Jaeger, Karl-Erich

    2014-02-01

    The open reading frame PA1242 in the genome of Pseudomonas aeruginosa PAO1 encodes a putative protease belonging to the peptidase S8 family of subtilases. The respective enzyme termed SprP consists of an N-terminal signal peptide and a so-called S8 domain linked by a domain of unknown function (DUF). Presumably, this DUF domain defines a discrete class of Pseudomonas proteins as homologous domains can be identified almost exclusively in proteins of the genus Pseudomonas. The sprP gene was expressed in Escherichia coli and proteolytic activity was demonstrated. A P. aeruginosa ∆sprP mutant was constructed and its gene expression pattern compared to the wild-type strain by genome microarray analysis revealing altered expression levels of 218 genes. Apparently, SprP is involved in regulation of a variety of different cellular processes in P. aeruginosa including pyoverdine synthesis, denitrification, the formation of cell aggregates, and of biofilms. PMID:24376018

  9. Evolutionary insight from whole-genome sequencing of Pseudomonas aeruginosa from cystic fibrosis patients.

    PubMed

    Marvig, Rasmus Lykke; Sommer, Lea M; Jelsbak, Lars; Molin, Søren; Johansen, Helle Krogh

    2015-01-01

    The opportunistic pathogen Pseudomonas aeruginosa causes chronic airway infections in patients with cystic fibrosis (CF), and it is directly associated with the morbidity and mortality connected with this disease. The ability of P. aeruginosa to establish chronic infections in CF patients is suggested to be due to the large genetic repertoire of P. aeruginosa and its ability to genetically adapt to the host environment. Here, we review the recent work that has applied whole-genome sequencing to understand P. aeruginosa population genomics, within-host microevolution and diversity, mutational mechanisms, genetic adaptation and transmission events. Finally, we summarize the advances in relation to medical applications and laboratory evolution experiments. PMID:25865196

  10. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa

    PubMed Central

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa. PMID:27194047

  11. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa.

    PubMed

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa. PMID:27194047

  12. Network-assisted investigation of virulence and antibiotic-resistance systems in Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Hwang, Sohyun; Kim, Chan Yeong; Ji, Sun-Gou; Go, Junhyeok; Kim, Hanhae; Yang, Sunmo; Kim, Hye Jin; Cho, Ara; Yoon, Sang Sun; Lee, Insuk

    2016-05-01

    Pseudomonas aeruginosa is a Gram-negative bacterium of clinical significance. Although the genome of PAO1, a prototype strain of P. aeruginosa, has been extensively studied, approximately one-third of the functional genome remains unknown. With the emergence of antibiotic-resistant strains of P. aeruginosa, there is an urgent need to develop novel antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores P. aeruginosa gene function in systems-level models. Here, we present a genome-wide functional network of P. aeruginosa genes, PseudomonasNet, which covers 98% of the coding genome, and a companion web server to generate functional hypotheses using various network-search algorithms. We demonstrate that PseudomonasNet-assisted predictions can effectively identify novel genes involved in virulence and antibiotic resistance. Moreover, an antibiotic-resistance network based on PseudomonasNet reveals that P. aeruginosa has common modular genetic organisations that confer increased or decreased resistance to diverse antibiotics, which accounts for the pervasiveness of cross-resistance across multiple drugs. The same network also suggests that P. aeruginosa has developed mechanism of trade-off in resistance across drugs by altering genetic interactions. Taken together, these results clearly demonstrate the usefulness of a genome-scale functional network to investigate pathogenic systems in P. aeruginosa.

  13. Draft Genome Sequence of a Klebsiella pneumoniae Carbapenemase-Positive Sequence Type 111 Pseudomonas aeruginosa Strain

    PubMed Central

    Dotson, Gabrielle A.; Dekker, John P.; Palmore, Tara N.; Segre, Julia A.

    2016-01-01

    Here, we report the draft genome sequence of a sequence type 111 Pseudomonas aeruginosa strain isolated in 2014 from a patient at the NIH Clinical Center. This P. aeruginosa strain exhibits pan-drug resistance and harbors the blaKPC-2 gene, encoding the Klebsiella pneumoniae carbapenemase enzyme, on a plasmid. PMID:26868386

  14. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1.

    PubMed

    Dyson, Zoe A; Seviour, Robert J; Tucci, Joseph; Petrovski, Steve

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  15. Genome Sequences of Pseudomonas oryzihabitans Phage POR1 and Pseudomonas aeruginosa Phage PAE1

    PubMed Central

    Dyson, Zoe A.; Seviour, Robert J.; Tucci, Joseph

    2016-01-01

    We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa. The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA. PMID:27313312

  16. Draft Genome Sequence of Quorum-Sensing and Quorum-Quenching Pseudomonas aeruginosa Strain MW3a

    PubMed Central

    Wong, Cheng Siang; Yin, Wai-Fong; Chan, Xin Yue

    2014-01-01

    Pseudomonas aeruginosa has a broad range of habitation, from aquatic environments to human lungs. The coexistence of quorum-sensing and quorum-quenching activities occurs in P. aeruginosa strain MW3a. In this work, we present the draft genome sequence of P. aeruginosa MW3a, an interesting bacterium isolated from a marine environment. PMID:24744329

  17. A Tribute to Disorder in the Genome of the Bloom-Forming Freshwater Cyanobacterium Microcystis aeruginosa

    PubMed Central

    Humbert, Jean-François; Barbe, Valérie; Latifi, Amel; Gugger, Muriel; Calteau, Alexandra; Coursin, Therese; Lajus, Aurélie; Castelli, Vanina; Oztas, Sophie; Samson, Gaëlle; Longin, Cyrille; Medigue, Claudine; de Marsac, Nicole Tandeau

    2013-01-01

    Microcystis aeruginosa is one of the most common bloom-forming cyanobacteria in freshwater ecosystems worldwide. This species produces numerous secondary metabolites, including microcystins, which are harmful to human health. We sequenced the genomes of ten strains of M. aeruginosa in order to explore the genomic basis of their ability to occupy varied environments and proliferate. Our findings show that M. aeruginosa genomes are characterized by having a large open pangenome, and that each genome contains similar proportions of core and flexible genes. By comparing the GC content of each gene to the mean value of the whole genome, we estimated that in each genome, around 11% of the genes seem to result from recent horizontal gene transfer events. Moreover, several large gene clusters resulting from HGT (up to 19 kb) have been found, illustrating the ability of this species to integrate such large DNA molecules. It appeared also that all M. aeruginosa displays a large genomic plasticity, which is characterized by a high proportion of repeat sequences and by low synteny values between the strains. Finally, we identified 13 secondary metabolite gene clusters, including three new putative clusters. When comparing the genomes of Microcystis and Prochlorococcus, one of the dominant picocyanobacteria living in marine ecosystems, our findings show that they are characterized by having almost opposite evolutionary strategies, both of which have led to ecological success in their respective environments. PMID:23950996

  18. Draft Genome Assembly of Pseudomonas aeruginosa Quality Control Reference Strain Boston 41501.

    PubMed

    Minogue, T D; Daligault, H E; Davenport, K W; Broomall, S M; Bruce, D C; Chain, P S; Coyne, S R; Gibbons, H S; Jaissle, J; Chertkov, O; Freitas, T; Rosenzweig, C N; Xu, Y; Johnson, S L

    2014-01-01

    We present the scaffolded genome assembly of Pseudomonas aeruginosa Boston 41501, now publicly available in GenBank (JOVK00000000) in 10 contigs placed into a single scaffold. The 6.82-Mbp genome contains 66.1% G+C content and 6,295 coding sequences, including type 4 pilus and type 3 secretion system production genes. PMID:25278526

  19. Draft Genome Assembly of Pseudomonas aeruginosa Quality Control Reference Strain Boston 41501

    PubMed Central

    Minogue, T. D.; Daligault, H. E.; Davenport, K. W.; Broomall, S. M.; Bruce, D. C.; Chain, P. S.; Coyne, S. R.; Gibbons, H. S.; Jaissle, J.; Chertkov, O.; Freitas, T.; Rosenzweig, C. N.; Xu, Y.

    2014-01-01

    We present the scaffolded genome assembly of Pseudomonas aeruginosa Boston 41501, now publicly available in GenBank (JOVK00000000) in 10 contigs placed into a single scaffold. The 6.82-Mbp genome contains 66.1% G+C content and 6,295 coding sequences, including type 4 pilus and type 3 secretion system production genes. PMID:25278526

  20. Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis.

    PubMed Central

    Hla, S W; Hui, K P; Tan, W C; Ho, B

    1996-01-01

    The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis. PMID:8904417

  1. Construction and Analysis of Photolyase Mutants of Pseudomonas aeruginosa and Pseudomonas syringae: Contribution of Photoreactivation, Nucleotide Excision Repair, and Mutagenic DNA Repair to Cell Survival and Mutability following Exposure to UV-B Radiation

    PubMed Central

    Kim, Jae J.; Sundin, George W.

    2001-01-01

    Based on nucleotide sequence homology with the Escherichia coli photolyase gene (phr), the phr sequence of Pseudomonas aeruginosa PAO1 was identified from the genome sequence, amplified by PCR, cloned, and shown to complement a known phr mutation following expression in Escherichia coli SY2. Stable, insertional phr mutants containing a tetracycline resistance gene cassette were constructed in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 by homologous recombination and sucrose-mediated counterselection. These mutants showed a decrease in survival compared to the wild type of as much as 19-fold after irradiation at UV-B doses of 1,000 to 1,550 J m−2 followed by a recovery period under photoreactivating conditions. A phr uvrA mutant of P. aeruginosa PAO1 was markedly sensitive to UV-B irradiation exhibiting a decrease in survival of 6 orders of magnitude following a UV-B dose of 250 J m−2. Complementation of the phr mutations in P. aeruginosa PAO1 and P. syringae pv. syringae FF5 using the cloned phr gene from strain PAO1 resulted in a restoration of survival following UV-B irradiation and recovery under photoreactivating conditions. The UV-B survival of the phr mutants could also be complemented by the P. syringae mutagenic DNA repair determinant rulAB. Assays for increases in the frequency of spontaneous rifampin-resistant mutants in UV-B-irradiated strains containing rulAB indicated that significant UV-B mutability (up to a 51-fold increase compared to a nonirradiated control strain) occurred even in the wild-type PAO1 background in which rulAB only enhanced the UV-B survival by 2-fold under photoreactivating conditions. The frequency of occurrence of spontaneous nalidixic acid-resistant mutants in the PAO1 uvrA and uvrA phr backgrounds complemented with rulAB were 3.8 × 10−5 and 2.1 × 10−3, respectively, following a UV-B dose of 1,550 J m−2. The construction and characterization of phr mutants in the present study will facilitate the

  2. Draft Genome Sequence of a Clinically Isolated Extensively Drug-Resistant Pseudomonas aeruginosa Strain

    PubMed Central

    Manivannan, Bhavani; Mahalingam, Niranjana; Jadhao, Sudhir; Mishra, Amrita; Nilawe, Pravin

    2016-01-01

    We present the draft genome assembly of an extensively drug-resistant (XDR) Pseudomonas aeruginosa strain isolated from a patient with a history of genito urinary tuberculosis. The draft genome is 7,022,546 bp with a G+C content of 65.48%. It carries 7 phage genomes, genes for quorum sensing, biofilm formation, virulence, and antibiotic resistance. PMID:27013045

  3. Identification of Novel Genomic Islands in Liverpool Epidemic Strain of Pseudomonas aeruginosa Using Segmentation and Clustering.

    PubMed

    Jani, Mehul; Mathee, Kalai; Azad, Rajeev K

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen implicated in a myriad of infections and a leading pathogen responsible for mortality in patients with cystic fibrosis (CF). Horizontal transfers of genes among the microorganisms living within CF patients have led to highly virulent and multi-drug resistant strains such as the Liverpool epidemic strain of P. aeruginosa, namely the LESB58 strain that has the propensity to acquire virulence and antibiotic resistance genes. Often these genes are acquired in large clusters, referred to as "genomic islands (GIs)." To decipher GIs and understand their contributions to the evolution of virulence and antibiotic resistance in P. aeruginosa LESB58, we utilized a recursive segmentation and clustering procedure, presented here as a genome-mining tool, "GEMINI." GEMINI was validated on experimentally verified islands in the LESB58 strain before examining its potential to decipher novel islands. Of the 6062 genes in P. aeruginosa LESB58, 596 genes were identified to be resident on 20 GIs of which 12 have not been previously reported. Comparative genomics provided evidence in support of our novel predictions. Furthermore, GEMINI unraveled the mosaic structure of islands that are composed of segments of likely different evolutionary origins, and demonstrated its ability to identify potential strain biomarkers. These newly found islands likely have contributed to the hyper-virulence and multidrug resistance of the Liverpool epidemic strain of P. aeruginosa. PMID:27536294

  4. Identification of Novel Genomic Islands in Liverpool Epidemic Strain of Pseudomonas aeruginosa Using Segmentation and Clustering

    PubMed Central

    Jani, Mehul; Mathee, Kalai; Azad, Rajeev K.

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen implicated in a myriad of infections and a leading pathogen responsible for mortality in patients with cystic fibrosis (CF). Horizontal transfers of genes among the microorganisms living within CF patients have led to highly virulent and multi-drug resistant strains such as the Liverpool epidemic strain of P. aeruginosa, namely the LESB58 strain that has the propensity to acquire virulence and antibiotic resistance genes. Often these genes are acquired in large clusters, referred to as “genomic islands (GIs).” To decipher GIs and understand their contributions to the evolution of virulence and antibiotic resistance in P. aeruginosa LESB58, we utilized a recursive segmentation and clustering procedure, presented here as a genome-mining tool, “GEMINI.” GEMINI was validated on experimentally verified islands in the LESB58 strain before examining its potential to decipher novel islands. Of the 6062 genes in P. aeruginosa LESB58, 596 genes were identified to be resident on 20 GIs of which 12 have not been previously reported. Comparative genomics provided evidence in support of our novel predictions. Furthermore, GEMINI unraveled the mosaic structure of islands that are composed of segments of likely different evolutionary origins, and demonstrated its ability to identify potential strain biomarkers. These newly found islands likely have contributed to the hyper-virulence and multidrug resistance of the Liverpool epidemic strain of P. aeruginosa. PMID:27536294

  5. Genomic Variation among Contemporary Pseudomonas aeruginosa Isolates from Chronically Infected Cystic Fibrosis Patients

    PubMed Central

    Chung, Jade C. S.; Becq, Jennifer; Fraser, Louise; Schulz-Trieglaff, Ole; Bond, Nicholas J.; Foweraker, Juliet; Bruce, Kenneth D.; Smith, Geoffrey P.

    2012-01-01

    The airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogen Pseudomonas aeruginosa. Several lines of evidence suggest that the infecting P. aeruginosa lineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain of P. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained a mutS mutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs of P. aeruginosa CF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates. PMID:22753054

  6. Genome-Wide Study of Pseudomonas aeruginosa Outer Membrane Protein Immunogenicity Using Self-Assembling Protein Microarrays▿ †

    PubMed Central

    Montor, Wagner R.; Huang, Jin; Hu, Yanhui; Hainsworth, Eugenie; Lynch, Susan; Kronish, Jeannine-Weiner; Ordonez, Claudia L.; Logvinenko, Tanya; Lory, Stephen; LaBaer, Joshua

    2009-01-01

    Pseudomonas aeruginosa is responsible for potentially life-threatening infections in individuals with compromised defense mechanisms and those with cystic fibrosis. P. aeruginosa infection is notable for the appearance of a humoral response to some known antigens, such as flagellin C, elastase, alkaline protease, and others. Although a number of immunogenic proteins are known, no effective vaccine has been approved yet. Here, we report a comprehensive study of all 262 outer membrane and exported P. aeruginosa PAO1 proteins by a modified protein microarray methodology called the nucleic acid-programmable protein array. From this study, it was possible to identify 12 proteins that trigger an adaptive immune response in cystic fibrosis and acutely infected patients, providing valuable information about which bacterial proteins are actually recognized by the immune system in vivo during the natural course of infection. The differential detections of these proteins in patients and controls proved to be statistically significant (P < 0.01). The study provides a list of potential candidates for the improvement of serological diagnostics and the development of vaccines. PMID:19737893

  7. Complete genome sequence of Pseudomonas aeruginosa lytic bacteriophage PA1O which resembles temperate bacteriophage D3112.

    PubMed

    Kim, Shukho; Rahman, Marzia; Kim, Jungmin

    2012-03-01

    A novel Pseudomonas aeruginosa lytic bacteriophage (phage), PA1Ø, was isolated, and its genome was sequenced completely. This phage is able to lyse not only P. aeruginosa but also Staphylococcus aureus. Genome analysis of PA1Ø showed that it is similar to a P. aeruginosa temperate phage, D3112, with the exception of the absence of a c repressor-encoding gene, which is known to play a critical role in the maintenance of the lysogenic state of D3112 in P. aeruginosa. PMID:22354942

  8. Draft Genome Sequence of a Pseudomonas aeruginosa Strain Able To Decompose N,N-Dimethyl Formamide

    PubMed Central

    Yan, Ming; Xu, Lin; Wei, Li; Zhang, Liting

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium, which uses a variety of organic chemicals as carbon sources. Here, we report the genome sequence of the Cu1510 isolate from wastewater containing a high concentration of N,N-dimethyl formamide. PMID:26847883

  9. Draft Genome Sequences of 63 Pseudomonas aeruginosa Isolates Recovered from Cystic Fibrosis Sputum

    PubMed Central

    Spilker, Theodore

    2016-01-01

    Here, we report the draft genome sequences of 63 Pseudomonas aeruginosa isolates, recovered in culture of sputum from 15 individuals with cystic fibrosis (CF) receiving care in a single CF care center over a 13-year period. These sequences add value to studies of within-host evolution of bacterial pathogens during chronic infection. PMID:27103710

  10. Insights into Mechanisms and Proteomic Characterisation of Pseudomonas aeruginosa Adaptation to a Novel Antimicrobial Substance

    PubMed Central

    Cierniak, Peter; Jübner, Martin; Müller, Stefan; Bender, Katja

    2013-01-01

    Antibiotic resistance has been reported since the introduction of synthetic antibiotics. Bacteria, such as one of the most common nosocomial pathogens P. aeruginosa, adapt quickly to changing environmental conditions, due to their short generation time. Thus microevolutional changes can be monitored in situ. In this study, the microevolutional process of Pseudomonas aeruginosa PAO1 resistance against a recently developed novel antibacterial zinc Schiff-base (ZSB) was investigated at the proteome level. After extended exposure to ZSB the passaged strain differed in tolerance against ZSB, with the adapted P. aeruginosa PAO1 exhibiting 1.6 times higher minimal inhibitory concentration. Using Two-dimensional Difference Gel Electrophoresis, the changes in the proteome of ZSB adapted P. aeruginosa PAO1 were examined by comparison with the non-adapted P. aeruginosa PAO1. The proteome of the adapted P. aeruginosa PAO1 strain differed significantly from the non-adapted in the abundance of two proteins when both strains were grown under stressing conditions. One protein could be identified as the outer membrane protein D that plays a role in uptake of basic amino acids as well as in carbapeneme resistance. The second protein has been identified as alkyl peroxide reductase subunit F. Our data indicated a slight increase in abundance of alkyl peroxide reductase F (AhpF) in the case of ZSB passaged P. aeruginosa PAO1. Higher abundance of Ahp has been discussed in the literature as a promoter of accelerated detoxification of benzene derivatives. The observed up-regulated AhpF thus appears to be connected to an increased tolerance against ZSB. Changes in the abundance of proteins connected to oxidative stress were also found after short-time exposure of P. aeruginosa PAO1 to the ZSB. Furthermore, adapted P. aeruginosa PAO1 showed increased tolerance against hydrogen peroxide and, in addition, showed accelerated degradation of ZSB, as determined by HPLC measurements. PMID:23869205

  11. Enhanced Clearance of Pseudomonas aeruginosa by Peroxisome Proliferator-Activated Receptor Gamma.

    PubMed

    Bedi, Brahmchetna; Yuan, Zhihong; Joo, Myungsoo; Zughaier, Susu M; Goldberg, Joanna B; Arbiser, Jack L; Hart, C Michael; Sadikot, Ruxana T

    2016-07-01

    The pathogenic profile of Pseudomonas aeruginosa is related to its ability to secrete a variety of virulence factors. Quorum sensing (QS) is a mechanism wherein small diffusible molecules, specifically acyl-homoserine lactones, are produced by P. aeruginosa to promote virulence. We show here that macrophage clearance of P. aeruginosa (PAO1) is enhanced by activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ). Macrophages treated with a PPARγ agonist (pioglitazone) showed enhanced phagocytosis and bacterial killing of PAO1. It is known that PAO1 QS molecules are inactivated by PON-2. QS molecules are also known to inhibit activation of PPARγ by competitively binding PPARγ receptors. In accord with this observation, we found that infection of macrophages with PAO1 inhibited expression of PPARγ and PON-2. Mechanistically, we show that PPARγ induces macrophage paraoxonase 2 (PON-2), an enzyme that degrades QS molecules produced by P. aeruginosa Gene silencing studies confirmed that enhanced clearance of PAO1 in macrophages by PPARγ is PON-2 dependent. Further, we show that PPARγ agonists also enhance clearance of P. aeruginosa from lungs of mice infected with PAO1. Together, these data demonstrate that P. aeruginosa impairs the ability of host cells to mount an immune response by inhibiting PPARγ through secretion of QS molecules. These studies define a novel mechanism by which PPARγ contributes to the host immunoprotective effects during bacterial infection and suggest a role for PPARγ immunotherapy for P. aeruginosa infections. PMID:27091928

  12. Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14.

    PubMed

    Fischer, Sebastian; Klockgether, Jens; Morán Losada, Patricia; Chouvarine, Philippe; Cramer, Nina; Davenport, Colin F; Dethlefsen, Sarah; Dorda, Marie; Goesmann, Alexander; Hilker, Rolf; Mielke, Samira; Schönfelder, Torben; Suerbaum, Sebastian; Türk, Oliver; Woltemate, Sabrina; Wiehlmann, Lutz; Tümmler, Burkhard

    2016-04-01

    Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant Pseudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within-clone single nucleotide sequence diversity of 8 × 10(-6) for clone C and 2 × 10(-5) for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer. PMID:26711897

  13. Intraclonal genome diversity of the major Pseudomonas aeruginosa clones C and PA14

    PubMed Central

    Fischer, Sebastian; Klockgether, Jens; Morán Losada, Patricia; Chouvarine, Philippe; Cramer, Nina; Davenport, Colin F.; Dethlefsen, Sarah; Dorda, Marie; Goesmann, Alexander; Hilker, Rolf; Mielke, Samira; Schönfelder, Torben; Suerbaum, Sebastian; Türk, Oliver; Woltemate, Sabrina; Wiehlmann, Lutz

    2016-01-01

    Summary Bacterial populations differentiate at the subspecies level into clonal complexes. Intraclonal genome diversity was studied in 100 isolates of the two dominant P seudomonas aeruginosa clones C and PA14 collected from the inanimate environment, acute and chronic infections. The core genome was highly conserved among clone members with a median pairwise within‐clone single nucleotide sequence diversity of 8 × 10−6 for clone C and 2 × 10−5 for clone PA14. The composition of the accessory genome was, on the other hand, as variable within the clone as between unrelated clones. Each strain carried a large cargo of unique genes. The two dominant worldwide distributed P. aeruginosa clones combine an almost invariant core with the flexible gain and loss of genetic elements that spread by horizontal transfer. PMID:26711897

  14. Quorum-sensing-regulated virulence factors in Pseudomonas aeruginosa are toxic to Lucilia sericata maggots

    PubMed Central

    Andersen, A. S.; Joergensen, B.; Bjarnsholt, T.; Johansen, H.; Karlsmark, T.; Givskov, M.; Krogfelt, K. A.

    2010-01-01

    Maggot debridement therapy (MDT) is widely used for debridement of chronic infected wounds; however, for wounds harbouring specific bacteria limited effect or failure of the treatment has been described. Here we studied the survival of Lucilia sericata maggots encountering Pseudomonas aeruginosa PAO1 in a simple assay with emphasis on the quorum-sensing (QS)-regulated virulence. The maggots were challenged with GFP-tagged P. aeruginosa wild-type (WT) PAO1 and a GFP-tagged P. aeruginosa ΔlasR rhlR (ΔRR) QS-deficient mutant in different concentrations. Maggots were killed in the presence of WT PAO1 whereas the challenge with the QS mutant showed a survival reduction of ∼25 % compared to negative controls. Furthermore, bacterial intake by the maggots was lower in the presence of WT PAO1 compared to the PAO1 ΔRR mutant. Maggot excretions/secretions (ES) were assayed for the presence of QS inhibitors; only high doses of ES showed inhibition of QS in P. aeruginosa. Thus P. aeruginosa was shown to be toxic to L. sericata maggots. This, coupled to the preferential feeding by the maggots and reduced ingestion of P. aeruginosa, could explain MDT failure in wounds colonized by P. aeruginosa. Wounds heavily colonized with P. aeruginosa should be a counterindication for MDT unless used in combination with a pre-treatment with other topical therapeutics targeting P. aeruginosa. PMID:19892758

  15. Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium.

    PubMed

    Freschi, Luca; Jeukens, Julie; Kukavica-Ibrulj, Irena; Boyle, Brian; Dupont, Marie-Josée; Laroche, Jérôme; Larose, Stéphane; Maaroufi, Halim; Fothergill, Joanne L; Moore, Matthew; Winsor, Geoffrey L; Aaron, Shawn D; Barbeau, Jean; Bell, Scott C; Burns, Jane L; Camara, Miguel; Cantin, André; Charette, Steve J; Dewar, Ken; Déziel, Éric; Grimwood, Keith; Hancock, Robert E W; Harrison, Joe J; Heeb, Stephan; Jelsbak, Lars; Jia, Baofeng; Kenna, Dervla T; Kidd, Timothy J; Klockgether, Jens; Lam, Joseph S; Lamont, Iain L; Lewenza, Shawn; Loman, Nick; Malouin, François; Manos, Jim; McArthur, Andrew G; McKeown, Josie; Milot, Julie; Naghra, Hardeep; Nguyen, Dao; Pereira, Sheldon K; Perron, Gabriel G; Pirnay, Jean-Paul; Rainey, Paul B; Rousseau, Simon; Santos, Pedro M; Stephenson, Anne; Taylor, Véronique; Turton, Jane F; Waglechner, Nicholas; Williams, Paul; Thrane, Sandra W; Wright, Gerard D; Brinkman, Fiona S L; Tucker, Nicholas P; Tümmler, Burkhard; Winstanley, Craig; Levesque, Roger C

    2015-01-01

    The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care. PMID:26483767

  16. Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium

    PubMed Central

    Freschi, Luca; Jeukens, Julie; Kukavica-Ibrulj, Irena; Boyle, Brian; Dupont, Marie-Josée; Laroche, Jérôme; Larose, Stéphane; Maaroufi, Halim; Fothergill, Joanne L.; Moore, Matthew; Winsor, Geoffrey L.; Aaron, Shawn D.; Barbeau, Jean; Bell, Scott C.; Burns, Jane L.; Camara, Miguel; Cantin, André; Charette, Steve J.; Dewar, Ken; Déziel, Éric; Grimwood, Keith; Hancock, Robert E. W.; Harrison, Joe J.; Heeb, Stephan; Jelsbak, Lars; Jia, Baofeng; Kenna, Dervla T.; Kidd, Timothy J.; Klockgether, Jens; Lam, Joseph S.; Lamont, Iain L.; Lewenza, Shawn; Loman, Nick; Malouin, François; Manos, Jim; McArthur, Andrew G.; McKeown, Josie; Milot, Julie; Naghra, Hardeep; Nguyen, Dao; Pereira, Sheldon K.; Perron, Gabriel G.; Pirnay, Jean-Paul; Rainey, Paul B.; Rousseau, Simon; Santos, Pedro M.; Stephenson, Anne; Taylor, Véronique; Turton, Jane F.; Waglechner, Nicholas; Williams, Paul; Thrane, Sandra W.; Wright, Gerard D.; Brinkman, Fiona S. L.; Tucker, Nicholas P.; Tümmler, Burkhard; Winstanley, Craig; Levesque, Roger C.

    2015-01-01

    The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care. PMID:26483767

  17. Description of genomic islands associated to the multidrug-resistant Pseudomonas aeruginosa clone ST277.

    PubMed

    Silveira, Melise Chaves; Albano, Rodolpho Mattos; Asensi, Marise Dutra; Carvalho-Assef, Ana Paula D'Alincourt

    2016-08-01

    Multidrug-resistant Pseudomonas aeruginosa clone ST277 is disseminated in Brazil where it is mainly associated with the presence of metallo-β-lactamase SPM-1. Furthermore, it carries the class I integron In163 and a 16S rRNA methylase rmtD that confers aminoglycoside resistance. To analyze the genetic characteristics that might be responsible for the success of this endemic clone, genomes of four P. aeruginosa strains that were isolated in distinct years and in different Brazilian states were sequenced. The strains differed regarding the presence of the genes blaSPM-1 and rmtD. Genomic comparisons that included genomes of other clones that have spread worldwide from this species were also performed. These analyses revealed a 763,863bp region in the P. aeruginosa chromosome that concentrates acquired genetic structures comprising two new genomic islands (PAGI-13 and PAGI-14), a mobile element that could be used for ST277 fingerprinting and a recently reported Integrative and Conjugative Element (ICE) associated to blaSPM-1. The genetic elements rmtD and In163 are inserted in PAGI-13 while PAGI-14 has genes encoding proteins related to type III restriction system and phages. The data reported in this study provide a basis for a clearer understanding of the genetic content of clone ST277 and illustrate the mechanisms that are responsible for the success of these endemic clones. PMID:27108807

  18. Genetic and Phenotypic Characterization of a Pseudomonas aeruginosa Population with High Frequency of Genomic Islands

    PubMed Central

    Morales-Espinosa, Rosario; Soberón-Chávez, Gloria; Delgado-Sapién, Gabriela; Sandner-Miranda, Luisa; Méndez, José L.; González-Valencia, Gerardo; Cravioto, Alejandro

    2012-01-01

    Various genomic islands, PAPI-1, PAPI-2, PAGI-1, PAGI-2, PAGI-3, and PAGI-4, and the element pKLC102 have been characterized in different P. aeruginosa strains from diverse habitats and geographical locations. Chromosomal DNA macroarray of 100 P. aeruginosa strains isolated from 85 unrelated patients hospitalized in an intensive care unit was created to assess the occurrence of these genomic islands (GEIs). The macroarray was then hybridized with labeled probes derived from each genomic island. In addition, PFGE patterns with SpeI, frequency of virulence genes, and antimicrobial resistance patterns of the strains were studied. Our results showed that almost all P. aeruginosa strains presented up to eight virulence genes. By SpeI macrorestriction fragment analysis we were able to identify 49 restriction patterns; 35 patterns correspond to single strains and the remaining 14 to strains subgroup (a–n). Most of the strains showed variation in number or composition of GEIs and a specific antimicrobial pattern indicating that each strain was an unrelated isolate. In terms of the number of genomic islands per strain, 7 GEIs were found in 34% of the strains, 6 in 18%, 5 in 12%, 4 in 14%, 3 in 10%, 2 in 7%, and 1 in 4%; only one isolate did not present any GEI. The genomic islands PAPI-1 and PAPI-2 and the element pKLC102 were the most frequently detected. The analysis of the location of each GEI in the chromosome of two strains show that the islands PAGI-3, PAPI-1, PAPI-2 and pKLC102 are present in the insertion site previously reported, but that PAGI-2 and PAGI-4 are inserted in another chromosome place in a site not characterized yet. In conclusion our data show that P. aeruginosa strains exhibited an epidemic population structure with horizontal transfer of DNA resulting in a high frequency of GEIs. PMID:22662157

  19. Genetic and phenotypic characterization of a Pseudomonas aeruginosa population with high frequency of genomic islands.

    PubMed

    Morales-Espinosa, Rosario; Soberón-Chávez, Gloria; Delgado-Sapién, Gabriela; Sandner-Miranda, Luisa; Méndez, José L; González-Valencia, Gerardo; Cravioto, Alejandro

    2012-01-01

    Various genomic islands, PAPI-1, PAPI-2, PAGI-1, PAGI-2, PAGI-3, and PAGI-4, and the element pKLC102 have been characterized in different P. aeruginosa strains from diverse habitats and geographical locations. Chromosomal DNA macroarray of 100 P. aeruginosa strains isolated from 85 unrelated patients hospitalized in an intensive care unit was created to assess the occurrence of these genomic islands (GEIs). The macroarray was then hybridized with labeled probes derived from each genomic island. In addition, PFGE patterns with SpeI, frequency of virulence genes, and antimicrobial resistance patterns of the strains were studied. Our results showed that almost all P. aeruginosa strains presented up to eight virulence genes. By SpeI macrorestriction fragment analysis we were able to identify 49 restriction patterns; 35 patterns correspond to single strains and the remaining 14 to strains subgroup (a-n). Most of the strains showed variation in number or composition of GEIs and a specific antimicrobial pattern indicating that each strain was an unrelated isolate. In terms of the number of genomic islands per strain, 7 GEIs were found in 34% of the strains, 6 in 18%, 5 in 12%, 4 in 14%, 3 in 10%, 2 in 7%, and 1 in 4%; only one isolate did not present any GEI. The genomic islands PAPI-1 and PAPI-2 and the element pKLC102 were the most frequently detected. The analysis of the location of each GEI in the chromosome of two strains show that the islands PAGI-3, PAPI-1, PAPI-2 and pKLC102 are present in the insertion site previously reported, but that PAGI-2 and PAGI-4 are inserted in another chromosome place in a site not characterized yet. In conclusion our data show that P. aeruginosa strains exhibited an epidemic population structure with horizontal transfer of DNA resulting in a high frequency of GEIs. PMID:22662157

  20. Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1

    PubMed Central

    Andrade-Domínguez, Andrés

    2016-01-01

    Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. PMID:27563032

  1. Complete Genome Sequence of Pseudomonas aeruginosa Phage AAT-1.

    PubMed

    Andrade-Domínguez, Andrés; Kolter, Roberto

    2016-01-01

    Aspects of the interaction between phages and animals are of interest and importance for medical applications. Here, we report the genome sequence of the lytic Pseudomonas phage AAT-1, isolated from mammalian serum. AAT-1 is a double-stranded DNA phage, with a genome of 57,599 bp, containing 76 predicted open reading frames. PMID:27563032

  2. Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa.

    PubMed

    Latino, Libera; Midoux, Cédric; Hauck, Yolande; Vergnaud, Gilles; Pourcel, Christine

    2016-05-01

    Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of Pseudomonas aeruginosa and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of P. aeruginosa strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10- 5 for single phage infection and 10- 6 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure. PMID:26921273

  3. Comparative genomics reveals diversified CRISPR-Cas systems of globally distributed Microcystis aeruginosa, a freshwater bloom-forming cyanobacterium

    PubMed Central

    Yang, Chen; Lin, Feibi; Li, Qi; Li, Tao; Zhao, Jindong

    2015-01-01

    Microcystis aeruginosa is one of the most common and dominant bloom-forming cyanobacteria in freshwater lakes around the world. Microcystis cells can produce toxic secondary metabolites, such as microcystins, which are harmful to human health. Two M. aeruginosa strains were isolated from two highly eutrophic lakes in China and their genomes were sequenced. Comparative genomic analysis was performed with the 12 other available M. aeruginosa genomes and closely related unicellular cyanobacterium. Each genome of M. aeruginosa containing at least one clustered regularly interspaced short palindromic repeat (CRISPR) locus and total 71 loci were identified, suggesting it is ubiquitous in M. aeruginosa genomes. In addition to the previously reported subtype I-D cas gene sets, three CAS subtypes I-A, III-A and III-B were identified and characterized in this study. Seven types of CRISPR direct repeat have close association with CAS subtype, confirming that different and specific secondary structures of CRISPR repeats are important for the recognition, binding and process of corresponding cas gene sets. Homology search of the CRISPR spacer sequences provides a history of not only resistance to bacteriophages and plasmids known to be associated with M. aeruginosa, but also the ability to target much more exogenous genetic material in the natural environment. These adaptive and heritable defense mechanisms play a vital role in keeping genomic stability and self-maintenance by restriction of horizontal gene transfer. Maintaining genomic stability and modulating genomic plasticity are both important evolutionary strategies for M. aeruginosa in adaptation and survival in various habitats. PMID:26029174

  4. Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors

    PubMed Central

    Zrieq, Rafat; Sana, Thibault G.; Vergin, Sandra; Garvis, Steve; Volfson, Irina; Bleves, Sophie; Voulhoux, Romé; Hegemann, Johannes H.

    2015-01-01

    Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions. PMID:26636043

  5. Genome Sequencing of a Mung Bean Plant Growth Promoting Strain of P. aeruginosa with Biocontrol Ability

    PubMed Central

    Illakkiam, Devaraj; Shankar, Manoharan; Ponraj, Paramasivan; Rajendhran, Jeyaprakash

    2014-01-01

    Pseudomonas aeruginosa PGPR2 is a mung bean rhizosphere strain that produces secondary metabolites and hydrolytic enzymes contributing to excellent antifungal activity against Macrophomina phaseolina, one of the prevalent fungal pathogens of mung bean. Genome sequencing was performed using the Ion Torrent Personal Genome Machine generating 1,354,732 reads (6,772,433 sequenced bases) achieving ~25-fold coverage of the genome. Reference genome assembly using MIRA 3.4.0 yielded 198 contigs. The draft genome of PGPR2 encoded 6803 open reading frames, of which 5314 were genes with predicted functions, 1489 were genes of known functions, and 80 were RNA-coding genes. Strain specific and core genes of P. aeruginosa PGPR2 that are relevant to rhizospheric habitat were identified by pangenome analysis. Genes involved in plant growth promoting function such as synthesis of ACC deaminase, indole-3-acetic acid, trehalose, mineral scavenging siderophores, hydrogen cyanide, chitinases, acyl homoserine lactones, acetoin, 2,3-butanediol, and phytases were identified. In addition, niche-specific genes such as phosphate solubilising 3-phytase, adhesins, pathway-specific transcriptional regulators, a diguanylate cyclase involved in cellulose synthesis, a receptor for ferrienterochelin, a DEAD/DEAH-box helicase involved in stress tolerance, chemotaxis/motility determinants, an HtpX protease, and enzymes involved in the production of a chromanone derivative with potent antifungal activity were identified. PMID:25184130

  6. Selection of an Antibiotic-Hypersusceptible Mutant of Pseudomonas aeruginosa: Identification of the GlmR Transcriptional Regulator

    PubMed Central

    Ramos-Aires, Julio; Plésiat, Patrick; Kocjancic-Curty, Lasta; Köhler, Thilo

    2004-01-01

    Tn501 random mutagenesis was applied to the Pseudomonas aeruginosa wild-type strain PAO1 to select for mutants hypersusceptible to aminoglycoside antimicrobial agents. One such mutant, called 19A, was found to be hypersusceptible to a wide range of antibiotics including aminoglycosides, β-lactams, fluoroquinolones, colistin, erythromycin, rifampin, and glycopeptides. Light microscopy of the mutant strain revealed abnormal morphology characterized by large, filamentous cells. The drug supersusceptibility of 19A was accompanied by loss of motility, reduced resistance to osmotic and heat shock stress, and impaired growth at low temperatures. The insertion site of the Tn501 transposon in mutant 19A has occurred in an open reading frame (PA5550 according to the PAO1 genome project), whose gene product shows amino acid sequence similarity to the DeoR family of transcriptional repressors. The gene, which we called glmR, is located between the glmS (PA5549) and glmU (PA5552) homologues of E. coli, responsible for the synthesis of UDP-N-acetylglucosamine-1-P, a precursor of both lipopolysaccharide (LPS) and peptidoglycan. We showed that GlmR represses the transcription of the adjacent glmS homologue (PA5549) in P. aeruginosa, possibly affecting the pool of precursors for peptidoglycan and LPS synthesis. To our knowledge GlmR is the first regulator in P. aeruginosa that affects susceptibility to a large variety of antibiotics and is therefore a potential target for novel anti-infective agents. PMID:14982774

  7. Genome-wide patterns of recombination in the opportunistic human pathogen Pseudomonas aeruginosa.

    PubMed

    Dettman, Jeremy R; Rodrigue, Nicolas; Kassen, Rees

    2015-01-01

    The bacterium Pseudomonas aeruginosa is a significant cause of acute nosocomial infections as well as chronic respiratory infections in patients with cystic fibrosis (CF). Recent reports of the intercontinental spread of a CF-specific epidemic strain, combined with high intrinsic levels of antibiotic resistance, have made this opportunistic pathogen an important public health concern. Strain-specific differences correlate with variation in clinical outcomes of infected CF patients, increasing the urgency to understand the evolutionary origin of genetic factors conferring important phenotypes that enable infection, virulence, or resistance. Here, we describe the genome-wide patterns of homologous and nonhomologous recombination in P. aeruginosa, and the extent to which the genomes are affected by these diversity-generating processes. Based on whole-genome sequence data from 32 clinical isolates of P. aeruginosa, we examined the rate and distribution of recombination along the genome, and its effect on the reconstruction of phylogenetic relationships. Multiple lines of evidence suggested that recombination was common and usually involves short stretches of DNA (200-300 bp). Although mutation was the main source of nucleotide diversity, the import of polymorphisms by homologous recombination contributed nearly as much. We also identified the genomic regions with frequent recombination, and the specific sequences of recombinant origin within epidemic strains. The functional characteristics of the genes contained therein were examined for potential associations with a pathogenic lifestyle or adaptation to the CF lung environment. A common link between many of the high-recombination genes was their functional affiliation with the cell wall, suggesting that the products of recombination may be maintained by selection for variation in cell-surface molecules that allows for evasion of the host immune system. PMID:25480685

  8. Complete Genome Sequences of Broad-Host-Range Pseudomonas aeruginosa Bacteriophages ΦR18 and ΦS12-1

    PubMed Central

    Furusawa, Takaaki; Higuchi, Hidetoshi; Usui, Masaru; Maruyama, Fumito; Nakagawa, Ichiro; Yokota, Hiroshi; Tamura, Yutaka

    2016-01-01

    Pseudomonas aeruginosa is an important cause of racehorse keratitis. Bacteriophage therapy has the potential to aid in the prevention and treatment of diseases caused by P. aeruginosa. We present here the complete genome sequences of two phages, ΦR18 and ΦS12-1, which exhibit infectivity for a broad range of P. aeruginosa isolates. PMID:27151780

  9. Cloning, expression and purification of penicillin-binding protein 3 from Pseudomonas aeruginosa CMCC 10104.

    PubMed

    An, Yan Dong; Du, Qi Zhen; Tong, Li Yan; Yu, Zhao Wu; Gong, Xing Wen

    2015-06-01

    Penicillin-binding protein 3 (PBP3) of Pseudomonas aeruginosa is the primary target of β-lactams used to treat pseudomonas infections. Meanwhile, structure change and overproduction of PBP3 play important roles in the drug resistance of P. aeruginosa. Therefore, studies on the gene and structure of PBP3 are urgently needed. P. aeruginosa CMCC 10104 is a type culture strain common used in China. However, there is no report on its genomic and proteomic profiles. In this study, based on ftsI of P. aeruginosa PAO1, the gene encoding PBP3 was cloned from CMCC 10104. A truncated version of the ftsI gene, omitting the bases encoding the hydrophobic leader peptide (amino acids 1-34), was amplified by PCR. The cloned DNA shared 99.76% identity with ftsI from PAO1. Only four bases were different (66 C-A, 1020 T-C, 1233 T-C, and 1527 T-C). However, there were no differences between their deduced amino acid sequences. The recombinant PBP3 (rPBP3), containing a 6-histidine tag, was expressed in Escherichia coli BL21 (DE3). Immobilized metal affinity chromatography (IMAC) with Ni(2+)-NTA agarose was used for its purification. The purified rPBP3 was identified by SDS-PAGE and western blot analysis, and showed a single band at about 60kDa with purity higher than 95%. The penicillin-binding assay indicated that the obtained rPBP3 was functional and not hindered by the presence of the C-terminal His-tag. The protocol described in this study offers a method for obtaining purified recombinant PBP3 from P. aeruginosa CMCC 10104. PMID:25514204

  10. Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

    PubMed Central

    Rakhimova, Elza; Munder, Antje; Wiehlmann, Lutz; Bredenbruch, Florian; Tümmler, Burkhard

    2008-01-01

    Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild. PMID:18301762

  11. Iron release from transferrin by pyoverdin and elastase from Pseudomonas aeruginosa.

    PubMed Central

    Wolz, C; Hohloch, K; Ocaktan, A; Poole, K; Evans, R W; Rochel, N; Albrecht-Gary, A M; Abdallah, M A; Döring, G

    1994-01-01

    Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochelin as well as receptors for siderophores in response to iron deprivation. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. aeruginosa elastase (LasB), which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagenesis to investigate whether this mutant differs in growth from the parental strain PAO1 in an iron-depleted medium supplemented with transferrin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 microM Fe2-transferrin as the only iron source. Urea gel electrophoresis indicated iron release from intact transferrin during the logarithmic growth phase of PAO1 and PAO1E. A total of 333 microM LasB was synthesized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 microM pyroverdin was produced during growth of the strains in medium supplemented with Fe2-transferrin or 10% human serum. Incubation of Fe2-transferrin and purified pyoverdin in concentrations similar to those found in the culture supernatant resulted in release iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate constant for iron acquisition of pyoverdin from transferrin. We conclude that P. aeruginosa can use transferrin as an iron source without further need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, and its LasB-negative mutant, K437E. Though K437 and K437E did not differ in growth with Fe2-transferrin as the only iron source, their growth was significantly reduced relative to that of PAO1 and PAO1E. Images PMID:8063422

  12. Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing

    PubMed Central

    Quick, Joshua; Cumley, Nicola; Wearn, Christopher M; Niebel, Marc; Constantinidou, Chrystala; Thomas, Chris M; Pallen, Mark J; Moiemen, Naiem S; Bamford, Amy; Oppenheim, Beryl; Loman, Nicholas J

    2014-01-01

    Objectives Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition. Study design An observational prospective cohort study. Setting Burns care ward and critical care ward in the UK. Participants Patients with >7% total burns by surface area were recruited into the study. Methods All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates. Results WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment. Conclusions This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a

  13. Biofilm Formation Mechanisms of Pseudomonas aeruginosa Predicted via Genome-Scale Kinetic Models of Bacterial Metabolism

    PubMed Central

    Vital-Lopez, Francisco G.; Reifman, Jaques; Wallqvist, Anders

    2015-01-01

    A hallmark of Pseudomonas aeruginosa is its ability to establish biofilm-based infections that are difficult to eradicate. Biofilms are less susceptible to host inflammatory and immune responses and have higher antibiotic tolerance than free-living planktonic cells. Developing treatments against biofilms requires an understanding of bacterial biofilm-specific physiological traits. Research efforts have started to elucidate the intricate mechanisms underlying biofilm development. However, many aspects of these mechanisms are still poorly understood. Here, we addressed questions regarding biofilm metabolism using a genome-scale kinetic model of the P. aeruginosa metabolic network and gene expression profiles. Specifically, we computed metabolite concentration differences between known mutants with altered biofilm formation and the wild-type strain to predict drug targets against P. aeruginosa biofilms. We also simulated the altered metabolism driven by gene expression changes between biofilm and stationary growth-phase planktonic cultures. Our analysis suggests that the synthesis of important biofilm-related molecules, such as the quorum-sensing molecule Pseudomonas quinolone signal and the exopolysaccharide Psl, is regulated not only through the expression of genes in their own synthesis pathway, but also through the biofilm-specific expression of genes in pathways competing for precursors to these molecules. Finally, we investigated why mutants defective in anthranilate degradation have an impaired ability to form biofilms. Alternative to a previous hypothesis that this biofilm reduction is caused by a decrease in energy production, we proposed that the dysregulation of the synthesis of secondary metabolites derived from anthranilate and chorismate is what impaired the biofilms of these mutants. Notably, these insights generated through our kinetic model-based approach are not accessible from previous constraint-based model analyses of P. aeruginosa biofilm

  14. Complete Genome Sequence of Pseudomonas aeruginosa PA1, Isolated from a Patient with a Respiratory Tract Infection

    PubMed Central

    Lu, Shuguang; Le, Shuai; Li, Gang; Shen, Mengyu; Tan, Yinling; Zhao, Xia; Wang, Jing; Shen, Wei; Guo, Keke; Yang, Yuhui; Zhu, Hongbin; Li, Shu; Li, Ming; Zhu, Junmin; Rao, Xiancai

    2015-01-01

    We report the 6,498,072-bp complete genome sequence of Pseudomonas aeruginosa PA1, which was isolated from a patient with a respiratory tract infection in Chongqing, People's Republic of China. Whole-genome sequencing was performed using single-molecule real-time (SMRT) technology, and de novo assembly revealed a single contig with 396-fold sequence coverage. PMID:26659688

  15. Complete genome sequences of three Pseudomonas aeruginosa isolates with phenotypes of polymyxin B adaptation and inducible resistance.

    PubMed

    Boyle, Brian; Fernandez, Lucia; Laroche, Jerome; Kukavica-Ibrulj, Irena; Mendes, Caio M F; Hancock, Robert W; Levesque, Roger C

    2012-01-01

    Clinical "superbug" isolates of Pseudomonas aeruginosa were previously observed to be resistant to several antibiotics, including polymyxin B, and/or to have a distinct, reproducible adaptive polymyxin resistance phenotype, identified by observing "skipped" wells (appearance of extra turbid wells) during broth microdilution testing. Here we report the complete assembled draft genome sequences of three such polymyxin resistant P. aeruginosa strains (9BR, 19BR, and 213BR). PMID:22207740

  16. Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18

    PubMed Central

    2011-01-01

    Background Our previously published reports have described an effective biocontrol agent named Pseudomonas sp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those of P. aeruginosa, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures. Results The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conserved P. aeruginosa core genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains a capB gene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with other P. aeruginosa strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that of P. aeruginosa strain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C. Conclusions The P. aeruginosa strain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche. PMID:21884571

  17. Draft genome sequence of blaVeb-1, blaoxa-10 producing multi-drug resistant (MDR) Pseudomonas aeruginosa strain VRFPA09 recovered from bloodstream infection.

    PubMed

    Murugan, Nandagopal; Malathi, Jambulingam; Umashankar, Vetrivel; Madhavan, Hajib NarahariRao

    2015-01-01

    Pseudomonas aeruginosa (P. aeruginosa) bacteremia causes significant mortality rate due to emergence of multidrug resistant (MDR) nosocomial infections. We report the draft genome sequence of P. aeruginosa strain VRFPA09, a human bloodstream isolate, phenotypically proven as MDR strain. Whole genome sequencing on VRFPA09, deciphered betalactamase encoding blav(eb-1) and bla(OXA-10) genes and multiple drug resistance, virulence factor encoding genes. PMID:26413042

  18. Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

    PubMed

    Lødeng, A G G; Ahlén, C; Lysvand, H; Mandal, L H; Iversen, O J

    2006-08-01

    This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype. PMID:16842571

  19. From the Environment to the Host: Re-Wiring of the Transcriptome of Pseudomonas aeruginosa from 22°C to 37°C

    PubMed Central

    Bielecki, Piotr; Suárez-Diez, María; Puchałka, Jacek; Albertí, Sebastian; dos Santos, Vitor Martins; Goldberg, Joanna B.

    2014-01-01

    Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe2+ extracellular concentration at 37°C and a peak in Fe3+ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature. PMID:24587139

  20. Draft Genome Sequence of Pseudomonas aeruginosa Strain N002, Isolated from Crude Oil-Contaminated Soil from Geleky, Assam, India

    PubMed Central

    Roy, Abhjit Sarma; Baruah, Reshita; Gogoi, Dhrubajyoti; Borah, Maina

    2013-01-01

    Here, we report the draft genome sequence of crude oil-degrading Pseudomonas aeruginosa strain N002, isolated from a crude oil-polluted soil sample from Geleky, Assam, India. Multiple genes potentially involved in crude oil degradation were identified. PMID:23405324

  1. Antibiotic and metal resistance in a ST395 Pseudomonas aeruginosa environmental isolate: A genomics approach.

    PubMed

    Teixeira, Pedro; Tacão, Marta; Alves, Artur; Henriques, Isabel

    2016-09-15

    We analyzed the resistome of Pseudomonas aeruginosa E67, an epiphytic isolate from a metal-contaminated estuary. The aim was to identify genetic determinants of resistance to antibiotics and metals, assessing possible co-selection mechanisms. Identification was based on phylogenetic analysis and average nucleotide identity value calculation. MLST affiliated E67 to ST395, previously described as a high-risk clone. Genome analysis allowed identifying genes probably involved in resistance to antibiotics (e.g. beta-lactams, aminoglycosides and chloramphenicol) and metals (e.g. mercury and copper), consistent with resistance phenotypes. Several genes associated with efflux systems, as well as genetic determinants contributing to gene motility, were identified. Pseudomonas aeruginosa E67 possesses an arsenal of resistance determinants, probably contributing to adaptation to a polluted ecosystem. Association to mobile structures highlights the role of these platforms in multi-drug resistance. Physical links between metal and antibiotic resistance genes were not identified, suggesting a predominance of cross-resistance associated with multidrug efflux pumps. PMID:27371958

  2. Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases

    PubMed Central

    Huang, Cheng-Yang

    2015-01-01

    Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC50 values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC50 values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver−Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the Kd value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases. PMID:25993634

  3. Elastase Deficiency Phenotype of Pseudomonas aeruginosa Canine Otitis Externa Isolates

    PubMed Central

    Petermann, Shana R.; Doetkott, Curt; Rust, Lynn

    2001-01-01

    Pseudomonas aeruginosa veterinary isolates were assayed for elastase and total matrix protease activity. The elastase activity of canine ear isolates was much less than that of strain PAO1 and that of all other veterinary isolates (P < 0.0001). The results indicate that canine ear isolates have a distinct elastase phenotype. PMID:11329471

  4. The genomic basis of adaptation to the fitness cost of rifampicin resistance in Pseudomonas aeruginosa

    PubMed Central

    Toll-Riera, Macarena; Heilbron, Karl

    2016-01-01

    Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings. PMID:26763710

  5. Genome Sequence of a Virulent Pseudomonas aeruginosa Strain, 12-4-4(59), Isolated from the Blood Culture of a Burn Patient

    PubMed Central

    Karna, S. L. Rajasekhar; Chen, Tsute; Chen, Ping; Peacock, Trent J.; Abercrombie, Johnathan J.

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that frequently infects wounds, significantly impairs wound healing, and causes morbidity and mortality in burn patients. Here, we report the genome sequence of a virulent strain of P. aeruginosa, 12-4-4(59), isolated from the blood culture of a burn patient. PMID:26941150

  6. Genomic analysis of Pseudomonas aeruginosa PA96, the host of carbapenem resistance plasmid pOZ176.

    PubMed

    Déraspe, Maxime; Alexander, David C; Xiong, Jianhui; Ma, Jennifer H; Low, Donald E; Jamieson, Frances B; Roy, Paul H

    2014-07-01

    Pseudomonas aeruginosa PA96 is a clinical isolate from Guangzhou, China, that is multiresistant to antibiotics. We previously described the 500-kb IncP-2 plasmid, pOZ176 that encodes many resistance genes including the IMP-9 carbapenemase. Whole-genome sequencing of PA96 enabled characterization of its genomic islands, virulence factors, and chromosomal resistance genes. We filled gaps using PCR and used optical mapping to confirm the correct contig order. We automatically annotated the core genome and manually annotated the genomic islands. The genome is 6 444 091 bp and encodes 5853 ORFs. From the whole-genome sequence, we constructed a physical map and constructed a phylogenetic tree for comparison with sequenced P. aeruginosa strains. Analysis of known core genome virulence factors and resistance genes revealed few differences with other strains, but the major virulence island is closer to that of DK2 than to PA14. PA96 most closely resembles the environmental strain M18, and notably shares a common serotype, pyoverdin type, flagellar operon, type IV pilin, and several genomic islands with M18. PMID:24673340

  7. ZnuA and zinc homeostasis in Pseudomonas aeruginosa

    PubMed Central

    Pederick, Victoria G.; Eijkelkamp, Bart A.; Begg, Stephanie L.; Ween, Miranda P.; McAllister, Lauren J.; Paton, James C.; McDevitt, Christopher A.

    2015-01-01

    Pseudomonas aeruginosa is a ubiquitous environmental bacterium and a clinically significant opportunistic human pathogen. Central to the ability of P. aeruginosa to colonise both environmental and host niches is the acquisition of zinc. Here we show that P. aeruginosa PAO1 acquires zinc via an ATP-binding cassette (ABC) permease in which ZnuA is the high affinity, zinc-specific binding protein. Zinc uptake in Gram-negative organisms predominantly occurs via an ABC permease, and consistent with this expectation a P. aeruginosa ΔznuA mutant strain showed an ~60% reduction in cellular zinc accumulation, while other metal ions were essentially unaffected. Despite the major reduction in zinc accumulation, minimal phenotypic differences were observed between the wild-type and ΔznuA mutant strains. However, the effect of zinc limitation on the transcriptome of P. aeruginosa PAO1 revealed significant changes in gene expression that enable adaptation to low-zinc conditions. Genes significantly up-regulated included non-zinc-requiring paralogs of zinc-dependent proteins and a number of novel import pathways associated with zinc acquisition. Collectively, this study provides new insight into the acquisition of zinc by P. aeruginosa PAO1, revealing a hitherto unrecognized complexity in zinc homeostasis that enables the bacterium to survive under zinc limitation. PMID:26290475

  8. Whole-Genome Sequence of Multidrug-Resistant Pseudomonas aeruginosa Strain BAMCPA07-48, Isolated from a Combat Injury Wound

    PubMed Central

    Sanjar, Fatemeh; Karna, S. L. Rajasekhar; Chen, Tsute; Chen, Ping; Abercrombie, Johnathan J.

    2016-01-01

    We report here the complete genome sequence of Pseudomonas aeruginosa strain BAMCPA07-48, isolated from a combat injury wound. The closed genome sequence of this isolate is a valuable resource for pathogenome characterization of P. aeruginosa associated with wounds, which will aid in the development of a higher-resolution phylogenomic framework for molecular-guided pathogen-surveillance. PMID:27389262

  9. Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa

    SciTech Connect

    Sokolova, O.S.; Shaburova, O.V.; Pechnikova, E.V.; Shaytan, A.K.; Krylov, S.V.; Kiselev, N.A.; Krylov, V.N.

    2014-11-15

    A unique feature of the Pseudomonas aeruginosa giant phage phiKZ is its way of genome packaging onto a spool-like protein structure, the inner body. Until recently, no similar structures have been detected in other phages. We have studied DNA packaging in P. aeruginosa phages EL and Lin68 using cryo-electron microscopy and revealed the presence of inner bodies. The shape and positioning of the inner body and the density of the DNA packaging in EL are different from those found in phiKZ and Lin68. This internal organization explains how the shorter EL genome is packed into a large EL capsid, which has the same external dimensions as the capsids of phiKZ and Lin68. The similarity in the structural organization in EL and other phiKZ-like phages indicates that EL is phylogenetically related to other phiKZ-like phages, and, despite the lack of detectable DNA homology, EL, phiKZ, and Lin68 descend from a common ancestor. - Highlights: • We performed a comparative structural study of giant P. aeruginosa phages: EL, Lin68 and phiKZ. • We revealed that the inner body is a common feature in giant phages. • The phage genome size correlates with the overall dimensions of the inner body.

  10. Optimization and comparative characterization of neuraminidase activities from Pseudomonas aeruginosa with Klebsiella pneumoniae, Hep-2 cell, sheep kidney and rat liver lysosome

    PubMed Central

    Ghazaei, C; Ahmadi, M; Hosseini Jazani, N

    2010-01-01

    Background and Objectives The properties of neuraminidase produced by P. aeruginosa strain PAO1 during growth in a defined medium (BHI) was examined and compared with some neuraminidase features of K. pneumoniae in this investigation. Materials and Methods The enzyme was isolated from concentrated culture supernatants of P. aeruginosa which was used in a sensitive fluorometric assay by using 2′-(4-methylumbelliferyl) α-D-N acetylneuraminic acid as substrate. Results Neuraminidase production in P. aeruginosa PAO1 paralleled bacterial growth in defined medium (BHI) and was maximal in the late logarithmic phase of growth but decreased during the stationary phase, probably owing to protease production or thermal instability. Highest production of P. aeruginosa PAO1 neuraminidase was in BHI culture media. The neuraminidase of P. aeruginosa PAO1 possessed an optimum temperature of activity at 56°C and the activity was maximal at pH 5. Heating the enzyme to 56°C for 45 min., in the presence of bovine serum albumin destroyed 33.1% of it's activity and addition of Ca+2, EDTA and NANA also decreased activity markedly. Conclusion The results revealed that the highest specific activity is for p. aeruginosa PAO1. PMID:22347548

  11. Draft Genome Sequences of Pseudomonas aeruginosa Isolates from Wounded Military Personnel

    PubMed Central

    Arivett, Brock A.; Ream, Dave C.; Fiester, Steven E.; Kidane, Destaalem

    2016-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work. PMID:27516516

  12. Draft Genome Sequences of Pseudomonas aeruginosa Isolates from Wounded Military Personnel.

    PubMed

    Arivett, Brock A; Ream, Dave C; Fiester, Steven E; Kidane, Destaalem; Actis, Luis A

    2016-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital-acquired infections, is grouped as an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogen because of its extensive drug resistance phenotypes and effects on human health worldwide. Five multidrug resistant P. aeruginosa strains isolated from wounded military personnel were sequenced and annotated in this work. PMID:27516516

  13. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

    PubMed Central

    Hmelo, Laura R.; Borlee, Bradley R.; Almblad, Henrik; Love, Michelle E.; Randall, Trevor E.; Tseng, Boo Shan; Lin, Chuyang; Irie, Yasuhiko; Storek, Kelly M.; Yang, Jaeun Jane; Siehnel, Richard J.; Howell, P. Lynne; Singh, Pradeep K.; Tolker-Nielsen, Tim; Parsek, Matthew R.; Schweizer, Herbert P.; Harrison, Joe J.

    2016-01-01

    Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knockins, as well as single nucleotide insertions, deletions and substitutions in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selection are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks. PMID:26492139

  14. Structure and fate of a Pseudomonas aeruginosa population originating from a combined sewer and colonizing a wastewater treatment lagoon.

    PubMed

    Lavenir, Raphaël; Petit, Stéphanie M-C; Alliot, Nolwenn; Ribun, Sébastien; Loiseau, Laurence; Marjolet, Laurence; Briolay, Jérôme; Nazaret, Sylvie; Cournoyer, Benoit

    2014-04-01

    The efficacy of a wastewater treatment lagoon (WWTL) at preventing the spread of Pseudomonas aeruginosa into natural aquatic habitats was investigated. A WWTL and its connected combined sewer and brook were exhaustively sampled. Physico-chemical analyses showed a stratification of the first pond according to pH, temperature and oxygen content. The P. aeruginosa counts partially matched this stratification with higher values among the bottom anaerobic waters of the first half of this pond. Genotyping of 494 WWTL P. aeruginosa strains was performed and led to the definition of 85 lineages. Dominant lineages were observed, with some being found all over the WWTL including the connected brook. IS5 was used as an indicator of genomic changes, and 1 to 12 elements were detected among 16 % of the strains. IS-driven lasR (genetic regulator) disruptions were detected among nine strains that were not part of the dominant lineages. These insertional mutants did not show significant elastase activities but showed better growth than the PAO1 reference strain in WWTL waters. Differences in growth patterns were related to a better survival of these mutants at an alkaline pH and a better ability at using some C-sources such as alanine. The opportunistic colonization of a WWTL by P. aeruginosa can involve several metabolic strategies which appeared lineage specific. Some clones appeared more successful than others at disseminating from a combined sewer toward the overflow of a WWTL. PMID:24407782

  15. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells.

    PubMed

    Choudhary, Geetika S; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  16. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells

    PubMed Central

    Choudhary, Geetika S.; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A.; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  17. Complete genome sequence analysis of Pseudomonas aeruginosa N002 reveals its genetic adaptation for crude oil degradation.

    PubMed

    Das, Dhrubajyoti; Baruah, Reshita; Sarma Roy, Abhijit; Singh, Anil Kumar; Deka Boruah, Hari Prasanna; Kalita, Jatin; Bora, Tarun Chandra

    2015-03-01

    The present research work reports the whole genome sequence analysis of Pseudomonas aeruginosa strain N002 isolated from crude oil contaminated soil of Assam, India having high crude oil degradation ability. The whole genome of the strain N002 was sequenced by shotgun sequencing using Ion Torrent method and complete genome sequence analysis was done. It was found that the strain N002 revealed versatility for degradation, emulsification and metabolizing of crude oil. Analysis of cluster of orthologous group (COG) revealed that N002 has significantly higher gene abundance for cell motility, lipid transport and metabolism, intracellular trafficking, secretion and vesicular transport, secondary metabolite biosynthesis, transport and catabolism, signal transduction mechanism and transcription than average levels found in other genome sequences of the same bacterial species. However, lower gene abundance for carbohydrate transport and metabolism, replication, recombination and repair, translation, ribosomal structure, biogenesis was observed in N002 than average levels of other bacterial species. PMID:25546474

  18. Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

    PubMed

    Doukyu, Noriyuki; Nihei, Shyou

    2015-07-01

    An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6β-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and β-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 μM and 15.9 μmol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa. PMID:25573142

  19. First Complete Genome Sequence of Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (DSM 50071T), Determined Using PacBio Single-Molecule Real-Time Technology.

    PubMed

    Nakano, Kazuma; Terabayashi, Yasunobu; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

    2015-01-01

    The first complete genome sequence of the type strain Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (DSM 50071(T)) was determined in a single contig by PacBio RS II. The genome (6,317,050 bp, G+C content of 66.52%) contained 10 sets of >1,000-bp identical sequence pairs and 183 tandem repeats. PMID:26294631

  20. First Complete Genome Sequence of Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (DSM 50071T), Determined Using PacBio Single-Molecule Real-Time Technology

    PubMed Central

    Terabayashi, Yasunobu; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

    2015-01-01

    The first complete genome sequence of the type strain Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 (DSM 50071T) was determined in a single contig by PacBio RS II. The genome (6,317,050 bp, G+C content of 66.52%) contained 10 sets of >1,000-bp identical sequence pairs and 183 tandem repeats. PMID:26294631

  1. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

    PubMed

    Danis-Wlodarczyk, Katarzyna; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Gula, Grzegorz; Briers, Yves; Jang, Ho Bin; Vandenheuvel, Dieter; Duda, Katarzyna Anna; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2015-01-01

    We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants. PMID:25996839

  2. Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

    PubMed Central

    Danis-Wlodarczyk, Katarzyna; Olszak, Tomasz; Arabski, Michal; Wasik, Slawomir; Majkowska-Skrobek, Grazyna; Augustyniak, Daria; Gula, Grzegorz; Briers, Yves; Jang, Ho Bin; Vandenheuvel, Dieter; Duda, Katarzyna Anna; Lavigne, Rob; Drulis-Kawa, Zuzanna

    2015-01-01

    We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants. PMID:25996839

  3. Evidence of a plasmid-encoded oxidative xylose-catabolic pathway in Arthrobacter nicotinovorans pAO1.

    PubMed

    Mihasan, Marius; Stefan, Marius; Hritcu, Lucian; Artenie, Vlad; Brandsch, Roderich

    2013-01-01

    Due to its high abundance, the D-xylose fraction of lignocellulose provides a promising resource for production of various chemicals. Examples of efficient utilization of d-xylose are nevertheless rare, mainly due to the lack of enzymes with suitable properties for biotechnological applications. The genus Arthrobacter, which occupies an ecological niche rich in lignocellulosic materials and containing species with high resistance and tolerance to environmental factors, is a very suitable candidate for finding D-xylose-degrading enzymes with new properties. In this work, the presence of the pAO1 megaplasmid in cells of Arthrobacter nicotinovorans was directly linked to the ability of this microorganism to ferment D-xylose and to sustain longer log growth. Three pAO1 genes (orf32, orf39, orf40) putatively involved in degradation of xylose were identified and cloned, and the corresponding proteins purified and characterized. ORF40 was shown to be a homotetrameric NADP(+)/NAD(+) sugar dehydrogenase with a strong preference for d-xylose; ORF39 is a monomeric aldehyde dehydrogenase with wide substrate specificity and ORF32 is a constitutive expressed transcription factor putatively involved in control of the entire catabolic pathway. Based on analogies with other pentose degradation pathways, a putative xylose oxidative pathway similar to the Weimberg pathway is postulated. PMID:23063486

  4. Glycerol metabolism promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Scoffield, Jessica; Silo-Suh, Laura

    2016-08-01

    Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation. PMID:27392247

  5. CLONING AND EXPRESSION OF THE CATA AND CATBC GENE CLUSTERS FROM PSEUDOMONAS AERUGINOSA PAO

    EPA Science Inventory

    A 9.9-kilobase (kb) BAMIII estriction endonuclease fragment encoding the catA and catBC gene clusters was selected from a gene bank of the Pseudomonas aeruginosa PAO1c chromosome. he catA, catB, and catC genes encode enzymes that catalyze consecutive reactions in the catechol bra...

  6. Sensitizing Pseudomonas aeruginosa to antibiotics by electrochemical disruption of membrane functions.

    PubMed

    Niepa, Tagbo H R; Snepenger, Laura M; Wang, Hao; Sivan, Shiril; Gilbert, Jeremy L; Jones, Marcus B; Ren, Dacheng

    2016-01-01

    Recently, we reported synergistic effects between 70 μA/cm(2) direct current and tobramycin in killing Pseudomonas aeruginosa PAO1 persister cells, a phenomenon we named electrochemical control of persister cells (ECCP; Niepa et al. Biomaterials 33: 7356-7365, 2012). To understand the mechanism of ECCP, the effects of electrochemical treatments mediated via stainless steel 304 and carbon electrodes on P. aeruginosa PAO1 were systematically compared using complementary approaches in this study. Electron microscopic analysis revealed that μA/cm(2) level direct current (DC) caused substantial changes in the structure and membrane integrity of P. aeruginosa PAO1 cells. DC treatments using SS304 electrodes induced cell lysis, while the same level of DC generated using carbon electrodes led to aggregation of intracellular proteins and increased permeabilization of P. aeruginosa PAO1 cells to antibiotics. The profound effects of DC on the physiology of persister cells were corroborated with DNA microarray analysis, which revealed the induction of genes associated with pyocin production and SOS response in DC-treated persister cells. Interestingly, sequential treatment using DC mediated with carbon electrodes followed by tobramycin was found more effective than concurrent treatment; and total eradication of persister cells was achieved. PMID:26461119

  7. A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus.

    PubMed

    Gi, Mia; Lee, Kang-Mu; Kim, Sang Cheol; Yoon, Joo-Heon; Yoon, Sang Sun; Choi, Jae Young

    2015-01-01

    Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 μM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 μM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection. PMID:26446565

  8. A novel siderophore system is essential for the growth of Pseudomonas aeruginosa in airway mucus

    PubMed Central

    Gi, Mia; Lee, Kang-Mu; Kim, Sang Cheol; Yoon, Joo-Heon; Yoon, Sang Sun; Choi, Jae Young

    2015-01-01

    Pseudomonas aeruginosa establishes airway infections in Cystic Fibrosis patients. Here, we investigate the molecular interactions between P. aeruginosa and airway mucus secretions (AMS) derived from the primary cultures of normal human tracheal epithelial (NHTE) cells. PAO1, a prototype strain of P. aeruginosa, was capable of proliferating during incubation with AMS, while all other tested bacterial species perished. A PAO1 mutant lacking PA4834 gene became susceptible to AMS treatment. The ΔPA4834 mutant was grown in AMS supplemented with 100 μM ferric iron, suggesting that the PA4834 gene product is involved in iron metabolism. Consistently, intracellular iron content was decreased in the mutant, but not in PAO1 after the AMS treatment. Importantly, a PAO1 mutant unable to produce both pyoverdine and pyochelin remained viable, suggesting that these two major siderophore molecules are dispensable for maintaining viability during incubation with AMS. The ΔPA4834 mutant was regrown in AMS amended with 100 μM nicotianamine, a phytosiderophore whose production is predicted to be mediated by the PA4836 gene. Infectivity of the ΔPA4834 mutant was also significantly compromised in vivo. Together, our results identify a genetic element encoding a novel iron acquisition system that plays a previously undiscovered role in P. aeruginosa airway infection. PMID:26446565

  9. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database.

    PubMed

    Winsor, Geoffrey L; Griffiths, Emma J; Lo, Raymond; Dhillon, Bhavjinder K; Shay, Julie A; Brinkman, Fiona S L

    2016-01-01

    The Pseudomonas Genome Database (http://www.pseudomonas.com) is well known for the application of community-based annotation approaches for producing a high-quality Pseudomonas aeruginosa PAO1 genome annotation, and facilitating whole-genome comparative analyses with other Pseudomonas strains. To aid analysis of potentially thousands of complete and draft genome assemblies, this database and analysis platform was upgraded to integrate curated genome annotations and isolate metadata with enhanced tools for larger scale comparative analysis and visualization. Manually curated gene annotations are supplemented with improved computational analyses that help identify putative drug targets and vaccine candidates or assist with evolutionary studies by identifying orthologs, pathogen-associated genes and genomic islands. The database schema has been updated to integrate isolate metadata that will facilitate more powerful analysis of genomes across datasets in the future. We continue to place an emphasis on providing high-quality updates to gene annotations through regular review of the scientific literature and using community-based approaches including a major new Pseudomonas community initiative for the assignment of high-quality gene ontology terms to genes. As we further expand from thousands of genomes, we plan to provide enhancements that will aid data visualization and analysis arising from whole-genome comparative studies including more pan-genome and population-based approaches. PMID:26578582

  10. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database

    PubMed Central

    Winsor, Geoffrey L.; Griffiths, Emma J.; Lo, Raymond; Dhillon, Bhavjinder K.; Shay, Julie A.; Brinkman, Fiona S. L.

    2016-01-01

    The Pseudomonas Genome Database (http://www.pseudomonas.com) is well known for the application of community-based annotation approaches for producing a high-quality Pseudomonas aeruginosa PAO1 genome annotation, and facilitating whole-genome comparative analyses with other Pseudomonas strains. To aid analysis of potentially thousands of complete and draft genome assemblies, this database and analysis platform was upgraded to integrate curated genome annotations and isolate metadata with enhanced tools for larger scale comparative analysis and visualization. Manually curated gene annotations are supplemented with improved computational analyses that help identify putative drug targets and vaccine candidates or assist with evolutionary studies by identifying orthologs, pathogen-associated genes and genomic islands. The database schema has been updated to integrate isolate metadata that will facilitate more powerful analysis of genomes across datasets in the future. We continue to place an emphasis on providing high-quality updates to gene annotations through regular review of the scientific literature and using community-based approaches including a major new Pseudomonas community initiative for the assignment of high-quality gene ontology terms to genes. As we further expand from thousands of genomes, we plan to provide enhancements that will aid data visualization and analysis arising from whole-genome comparative studies including more pan-genome and population-based approaches. PMID:26578582

  11. [Comparison of the genome for phylogenetically related bacteriophages phiKZ and EL of Pseudomonas aeruginosa: evolutionary aspects and minimal genome size].

    PubMed

    Krylov, V N; Pletneva, E A; Lavigne, R; Hertveldt, K; Volkaert, G; Sernova, N V; Georgopoulos, C; Korchevsliĭ, R V; Kurochkina, L P; Mesianzhinov, V V

    2005-04-01

    Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phiKZ and EL, which belong to a group of (phiKZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phiKZ and EL genomes. PMID:15909907

  12. A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.

    PubMed

    Malphettes, Laetitia; Weber, Cornelia C; El-Baba, Marie Daoud; Schoenmakers, Ronald G; Aubel, Dominique; Weber, Wilfried; Fussenegger, Martin

    2005-01-01

    We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its O(NIC)-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric O(NIC)-containing promoters (P(NIC); O(NIC) fused to a minimal eukaryotic promoter [P(min)]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-O(NIC) and/or O(NIC)-P(min) spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing. PMID:16002786

  13. Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    PubMed Central

    Okkotsu, Yuta; Tieku, Prince; Fitzsimmons, Liam F.; Churchill, Mair E.

    2013-01-01

    AlgR is a key Pseudomonas aeruginosa transcriptional response regulator required for virulence. AlgR activates alginate production and twitching motility but represses the Rhl quorum-sensing (QS) system, including rhamnolipid production. The role of AlgR phosphorylation is enigmatic, since phosphorylated AlgR (AlgR-P) is required for twitching motility through the fimU promoter but is not required for the activation of alginate production. In order to examine the role of AlgR phosphorylation in vivo, a PAO1 algRD54E strain (with algR encoding a D-to-E change at position 54), which constitutively activates fimU transcription and exhibits twitching motility, was created. A corresponding PAO1 algRD54N strain (with algR encoding a D-to-N change at position 54) that does not activate fimU or twitching motility was compared to PAO1, PAO1 algRD54E, PAO1 ΔalgZ (deletion of the algZ [fimS] gene, encoding a putative histidine kinase), and PAO1 ΔalgR for swarming motility, rhamnolipid production, and rhlA transcription. PAO1 and PAO1 algRD54E produced approximately 2-fold-higher levels of rhamnolipids than PAO1 algRD54N and PAO1 ΔalgZ, thereby indicating that phosphorylated AlgR is required for normal rhamnolipid production. Examination of purified AlgR, AlgR-P, AlgR D54N, and AlgR D54E showed that AlgR-P and AlgR D54E bound preferentially to the fimU and rhlA promoters. Additionally, AlgR-P bound specifically to two sites within the rhlA promoter that were not bound by unphosphorylated AlgR. Taken together, these results indicate that phosphorylated AlgR-P has increased affinity for the rhlA promoter and is required for the coordinate activation of twitching motility, rhamnolipid production, and swarming motility in P. aeruginosa. PMID:24097945

  14. Recombination is a key driver of genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis infection.

    PubMed

    Darch, Sophie E; McNally, Alan; Harrison, Freya; Corander, Jukka; Barr, Helen L; Paszkiewicz, Konrad; Holden, Stephen; Fogarty, Andrew; Crusz, Shanika A; Diggle, Stephen P

    2015-01-01

    The Cystic Fibrosis (CF) lung harbors a complex, polymicrobial ecosystem, in which Pseudomonas aeruginosa is capable of sustaining chronic infections, which are highly resistant to multiple antibiotics. Here, we investigate the phenotypic and genotypic diversity of 44 morphologically identical P. aeruginosa isolates taken from a single CF patient sputum sample. Comprehensive phenotypic analysis of isolates revealed large variances and trade-offs in growth, virulence factors and quorum sensing (QS) signals. Whole genome analysis of 22 isolates revealed high levels of intra-isolate diversity ranging from 5 to 64 SNPs and that recombination and not spontaneous mutation was the dominant driver of diversity in this population. Furthermore, phenotypic differences between isolates were not linked to mutations in known genes but were statistically associated with distinct recombination events. We also assessed antibiotic susceptibility of all isolates. Resistance to antibiotics significantly increased when multiple isolates were mixed together. Our results highlight the significant role of recombination in generating phenotypic and genetic diversification during in vivo chronic CF infection. We also discuss (i) how these findings could influence how patient-to-patient transmission studies are performed using whole genome sequencing, and (ii) the need to refine antibiotic susceptibility testing in sputum samples taken from patients with CF. PMID:25578031

  15. Recombination is a key driver of genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis infection

    PubMed Central

    Darch, Sophie E.; McNally, Alan; Harrison, Freya; Corander, Jukka; Barr, Helen L.; Paszkiewicz, Konrad; Holden, Stephen; Fogarty, Andrew; Crusz, Shanika A.; Diggle, Stephen P.

    2015-01-01

    The Cystic Fibrosis (CF) lung harbors a complex, polymicrobial ecosystem, in which Pseudomonas aeruginosa is capable of sustaining chronic infections, which are highly resistant to multiple antibiotics. Here, we investigate the phenotypic and genotypic diversity of 44 morphologically identical P. aeruginosa isolates taken from a single CF patient sputum sample. Comprehensive phenotypic analysis of isolates revealed large variances and trade-offs in growth, virulence factors and quorum sensing (QS) signals. Whole genome analysis of 22 isolates revealed high levels of intra-isolate diversity ranging from 5 to 64 SNPs and that recombination and not spontaneous mutation was the dominant driver of diversity in this population. Furthermore, phenotypic differences between isolates were not linked to mutations in known genes but were statistically associated with distinct recombination events. We also assessed antibiotic susceptibility of all isolates. Resistance to antibiotics significantly increased when multiple isolates were mixed together. Our results highlight the significant role of recombination in generating phenotypic and genetic diversification during in vivo chronic CF infection. We also discuss (i) how these findings could influence how patient-to-patient transmission studies are performed using whole genome sequencing, and (ii) the need to refine antibiotic susceptibility testing in sputum samples taken from patients with CF. PMID:25578031

  16. Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis

    PubMed Central

    Pan, Xuewei; Cui, Xiaoli; Zhang, Fenjiao; He, Yang; Li, Lingyan; Yang, Hongjiang

    2016-01-01

    Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA) synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The Lipopolysaccharide profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor. PMID:26973628

  17. A Drug-Repositioning Screening Identifies Pentetic Acid as a Potential Therapeutic Agent for Suppressing the Elastase-Mediated Virulence of Pseudomonas aeruginosa

    PubMed Central

    Gi, Mia; Jeong, Junhui; Lee, Keehoon; Lee, Kang-Mu; Toyofuku, Masanori; Yong, Dong Eun

    2014-01-01

    Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 μM DTPA. Supplementation with Zn2+ or Mn2+ ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection. PMID:25246397

  18. Chlorinated phenol-induced physiological antibiotic resistance in Pseudomonas aeruginosa.

    PubMed

    Muller, Jocelyn Fraga; Ghosh, Sudeshna; Ikuma, Kaoru; Stevens, Ann M; Love, Nancy G

    2015-11-01

    Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen with the ability to rapidly develop multidrug resistance under selective pressure. Previous work demonstrated that upon exposure to the environmental contaminant pentachlorophenol (PCP), P. aeruginosa PAO1 increases expression of multiple multidrug efflux pumps, including the MexAB-OprM pump. The current study describes increases in the antibiotic resistance of PAO1 upon exposure to PCP and other chlorinated organics, including triclosan. Only exposure to chlorinated phenols induced the mexAB-oprM-mediated antibiotic-resistant phenotype. Thus, chlorinated phenols have the potential to contribute to transient phenotypic increases of antibiotic resistance that are relevant when both compounds are present in the environment. PMID:26403431

  19. Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa

    PubMed Central

    Scott, Martin; Worden, Paul; Huntington, Peter; Hudson, Bernard; Karagiannis, Thomas; Charles, Ian G.; Djordjevic, Steven P.

    2016-01-01

    Pseudomonas aeruginosa are noscomially acquired, opportunistic pathogens that pose a major threat to the health of burns patients and the immunocompromised. We sequenced the genomes of P. aeruginosa isolates RNS_PA1, RNS_PA46 and RNS_PAE05, which displayed resistance to almost all frontline antibiotics, including gentamicin, piperacillin, timentin, meropenem, ceftazidime and colistin. We provide evidence that the isolates are representatives of P. aeruginosa sequence type (ST) 235 and carry Tn6162 and Tn6163 in genomic islands 1 (GI1) and 2 (GI2), respectively. GI1 disrupts the endA gene at precisely the same chromosomal location as in P. aeruginosa strain VR-143/97, of unknown ST, creating an identical CA direct repeat. The class 1 integron associated with Tn6163 in GI2 carries a blaGES-5–aacA4–gcuE15–aphA15 cassette array conferring resistance to carbapenems and aminoglycosides. GI2 is flanked by a 12 nt direct repeat motif, abuts a tRNA-gly gene, and encodes proteins with putative roles in integration, conjugative transfer as well as integrative conjugative element-specific proteins. This suggests that GI2 may have evolved from a novel integrative conjugative element. Our data provide further support to the hypothesis that genomic islands play an important role in de novo evolution of multiple antibiotic resistance phenotypes in P. aeruginosa. PMID:26962050

  20. Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa.

    PubMed

    Roy Chowdhury, Piklu; Scott, Martin; Worden, Paul; Huntington, Peter; Hudson, Bernard; Karagiannis, Thomas; Charles, Ian G; Djordjevic, Steven P

    2016-03-01

    Pseudomonas aeruginosa are noscomially acquired, opportunistic pathogens that pose a major threat to the health of burns patients and the immunocompromised. We sequenced the genomes of P. aeruginosa isolates RNS_PA1, RNS_PA46 and RNS_PAE05, which displayed resistance to almost all frontline antibiotics, including gentamicin, piperacillin, timentin, meropenem, ceftazidime and colistin. We provide evidence that the isolates are representatives of P. aeruginosa sequence type (ST) 235 and carry Tn6162 and Tn6163 in genomic islands 1 (GI1) and 2 (GI2), respectively. GI1 disrupts the endA gene at precisely the same chromosomal location as in P. aeruginosa strain VR-143/97, of unknown ST, creating an identical CA direct repeat. The class 1 integron associated with Tn6163 in GI2 carries a blaGES-5-aacA4-gcuE15-aphA15 cassette array conferring resistance to carbapenems and aminoglycosides. GI2 is flanked by a 12 nt direct repeat motif, abuts a tRNA-gly gene, and encodes proteins with putative roles in integration, conjugative transfer as well as integrative conjugative element-specific proteins. This suggests that GI2 may have evolved from a novel integrative conjugative element. Our data provide further support to the hypothesis that genomic islands play an important role in de novo evolution of multiple antibiotic resistance phenotypes in P. aeruginosa. PMID:26962050

  1. Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis and In Silico Serotyping of Pseudomonas aeruginosa Isolates.

    PubMed

    Thrane, Sandra Wingaard; Taylor, Véronique L; Lund, Ole; Lam, Joseph S; Jelsbak, Lars

    2016-07-01

    Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates high-throughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data. PMID:27098958

  2. Identification and functional analysis of a bacteriocin, pyocin S6, with ribonuclease activity from a Pseudomonas aeruginosa cystic fibrosis clinical isolate.

    PubMed

    Dingemans, Jozef; Ghequire, Maarten G K; Craggs, Michael; De Mot, René; Cornelis, Pierre

    2016-06-01

    S-type pyocins are bacteriocins produced by Pseudomonas aeruginosa isolates to antagonize or kill other strains of the same species. They have a modular organization comprising a receptor-binding domain recognizing a surface constituent of the target bacterium, a domain for translocation through the periplasm, and a killing or toxic domain with DNase, tRNase, or pore-forming activity. Pyocins S2, S3, S4, and S5 recognize TonB-dependent ferri-siderophore receptors in the outer membrane. We here describe a new nuclease bacteriocin, pyocin S6, encoded in the genome of a P. aeruginosa cystic fibrosis (CF) clinical isolate, CF_PA39. Similarly to pyocins S1 and S2, the S6 toxin-immunity gene tandem was recruited to the genomic region encoding exotoxin A. The pyocin S6 receptor-binding and translocation domains are identical to those of pyocin S1, whereas the killing domain is similar to the 16S ribonuclease domain of Escherichia coli colicin E3. The cytotoxic activity was abolished in pyocin S6 forms with a mutation in the colicin E3-equivalent catalytic motif. The CF_PA39 S6 immunity gene displays a higher expression level than the gene encoding the killing protein, the latter being only detected when bacteria are grown under iron-limiting conditions. In the S1-pyocinogenic strain P. aeruginosa ATCC 25324 and pyocin S2 producer P. aeruginosa PAO1, a remnant of the pyocin S6 killing domain and an intact S6-type immunity gene are located downstream of their respective pyocin operons. Strain PAO1 is insensitive for pyocin S6, and its S6-type immunity gene provides protection against pyocin S6 activity. Purified pyocin S6 inhibits one-fifth of 110 P. aeruginosa CF clinical isolates tested, showing clearer inhibition zones when the target cells are grown under iron limitation. In this panel, about half of the CF clinical isolates were found to host the S6 genes. The pyocin S6 locus is also present in the genome of some non-CF clinical isolates. PMID:26860427

  3. Involvement of Fe uptake systems and AmpC β-lactamase in susceptibility to the siderophore monosulfactam BAL30072 in Pseudomonas aeruginosa.

    PubMed

    van Delden, Christian; Page, Malcolm G P; Köhler, Thilo

    2013-05-01

    BAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosa PAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC β-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression of ampC and the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of the fecI promoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. A fecI mutant and a fecA mutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 μg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. The piuC gene, coding for a Fe(II)-dependent dioxygenase located next to the piuA iron receptor gene, was also downregulated. The MICs of BAL30072 for piuC and piuA transposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility of P. aeruginosa PAO1 to BAL30072. PMID:23422914

  4. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment.

    PubMed

    Wagner, Victoria E; Bushnell, Daniel; Passador, Luciano; Brooks, Andrew I; Iglewski, Barbara H

    2003-04-01

    Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the las and rhl systems, have been identified previously in the opportunistic pathogen Pseudomonas aeruginosa. High-density oligonucleotide microarrays for the P. aeruginosa PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying las and/or rhl QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (P

  5. From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C.

    PubMed

    Barbier, Mariette; Damron, F Heath; Bielecki, Piotr; Suárez-Diez, María; Puchałka, Jacek; Albertí, Sebastian; Dos Santos, Vitor Martins; Goldberg, Joanna B

    2014-01-01

    Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe²⁺ extracellular concentration at 37°C and a peak in Fe³⁺ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature. PMID

  6. Molecular epidemiology of Pseudomonas aeruginosa from cystic fibrosis in Sicily: genome macrorestriction analysis and rapid PCR-ribotyping.

    PubMed

    Agodi, A; Sciacca, A; Campanile, F; Messina, C; Barchitta, M; Sciacca, S; Stefani, S

    2000-07-01

    This study addresses the epidemiologic relatedness of a collection of Pseudomonas aeruginosa isolates from cystic fibrosis patients attending the Pediatric Clinic, Catania, Sicily. Genome macrorestriction analysis after pulsed field gel electrophoresis (PFGE) was used to characterise all strains. Furthermore, a rapid typing procedure, developed in this study, based on polymerase chain reaction amplified ribosomal DNA spacer polymorphisms (PCR-ribotyping), straight from bacterial cultures, was used. On the basis of macrorestriction analysis after PFGE, persistence of infection was shown in all patients; two cross-transmission episodes were identified in the nosocomial as well as in the familiar environment. PCR-ribotyping proved to be useful for a DNA-based identification test, suitable for screening purposes. The rapid amplification protocol here tested is proposed to evaluate the discriminatory power of other specific target sequences in PCR-based typing assays, for epidemiologic purposes. PMID:10939047

  7. Comparative genomics of isolates of a Pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients.

    PubMed

    Jeukens, Julie; Boyle, Brian; Kukavica-Ibrulj, Irena; Ouellet, Myriam M; Aaron, Shawn D; Charette, Steve J; Fothergill, Joanne L; Tucker, Nicholas P; Winstanley, Craig; Levesque, Roger C

    2014-01-01

    Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung. PMID:24505294

  8. Truncation of type IV pilin induces mucoidy in Pseudomonas aeruginosa strain PAO579

    PubMed Central

    Ryan Withers, T; Heath Damron, F; Yin, Yeshi; Yu, Hongwei D

    2013-01-01

    Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ22). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosa strain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA108), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA108 confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high PalgU and PalgD promoter activities of PAO579 require pilA108, algW, algU, and rpoN encoding the alternative sigma factor σ54. We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway. PMID:23533140

  9. Draft Genome Sequence of a Clinically Isolated Extensively Drug-Resistant Pseudomonas aeruginosa Strain.

    PubMed

    Manivannan, Bhavani; Mahalingam, Niranjana; Jadhao, Sudhir; Mishra, Amrita; Nilawe, Pravin; Pradeep, Bulagonda Eswarappa

    2016-01-01

    We present the draft genome assembly of an extensively drug-resistant (XDR)Pseudomonas aeruginosastrain isolated from a patient with a history of genito urinary tuberculosis. The draft genome is 7,022,546 bp with a G+C content of 65.48%. It carries 7 phage genomes, genes for quorum sensing, biofilm formation, virulence, and antibiotic resistance. PMID:27013045

  10. Reduction of PCN biosynthesis by NO in Pseudomonas aeruginosa.

    PubMed

    Gao, Lei; Zhang, Yuying; Wang, Yan; Qiao, Xinhua; Zi, Jing; Chen, Chang; Wan, Yi

    2016-08-01

    Pyocyanin (PCN), a virulence factor synthesized by Pseudomonas aeruginosa, plays an important role during clinical infections. There is no study of the effect of nitric oxide (NO) on PCN biosynthesis. Here, the effect of NO on PCN levels in Pseudomonas aeruginosa strain PAO1, a common reference strain, was tested. The results showed that the NO donor sodium nitroprusside (SNP) can significantly reduce PCN levels (82.5% reduction at 60μM SNP). Furthermore, the effect of endogenous NO on PCN was tested by constructing PAO1 nor (NO reductase gene) knockout mutants. Compared to the wild-type strain, the Δnor strain had a lower PCN (86% reduction in Δnor). To examine whether the results were universal with other P. aeruginosa strains, we collected 4 clinical strains from a hospital, tested their PCN levels after SNP treatment, and obtained similar results, i.e., PCN biosynthesis was inhibited by NO. These results suggest that NO treatment may be a new strategy to inhibit PCN biosynthesis and could provide novel insights into eliminating P. aeruginosa virulence as a clinical goal. PMID:26874276

  11. Extensively Drug-Resistant Pseudomonas aeruginosa Isolates Containing blaVIM-2 and Elements of Salmonella Genomic Island 2: a New Genetic Resistance Determinant in Northeast Ohio

    PubMed Central

    Perez, Federico; Hujer, Andrea M.; Marshall, Steven H.; Ray, Amy J.; Rather, Philip N.; Suwantarat, Nuntra; Dumford, Donald; O'Shea, Patrick; Domitrovic, T. Nicholas J.; Salata, Robert A.; Chavda, Kalyan D.; Chen, Liang; Kreiswirth, Barry N.; Vila, Alejandro J.; Haussler, Susanne; Jacobs, Michael R.

    2014-01-01

    Carbapenems are a mainstay of treatment for infections caused by Pseudomonas aeruginosa. Carbapenem resistance mediated by metallo-β-lactamases (MBLs) remains uncommon in the United States, despite the worldwide emergence of this group of enzymes. Between March 2012 and May 2013, we detected MBL-producing P. aeruginosa in a university-affiliated health care system in northeast Ohio. We examined the clinical characteristics and outcomes of patients, defined the resistance determinants and structure of the genetic element harboring the blaMBL gene through genome sequencing, and typed MBL-producing P. aeruginosa isolates using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST). Seven patients were affected that were hospitalized at three community hospitals, a long-term-care facility, and a tertiary care center; one of the patients died as a result of infection. Isolates belonged to sequence type 233 (ST233) and were extensively drug resistant (XDR), including resistance to all fluoroquinolones, aminoglycosides, and β-lactams; two isolates were nonsusceptible to colistin. The blaMBL gene was identified as blaVIM-2 contained within a class 1 integron (In559), similar to the cassette array previously detected in isolates from Norway, Russia, Taiwan, and Chicago, IL. Genomic sequencing and assembly revealed that In559 was part of a novel 35-kb region that also included a Tn501-like transposon and Salmonella genomic island 2 (SGI2)-homologous sequences. This analysis of XDR strains producing VIM-2 from northeast Ohio revealed a novel recombination event between Salmonella and P. aeruginosa, heralding a new antibiotic resistance threat in this region's health care system. PMID:25070102

  12. Cloning and nucleotide sequence of the pvdA gene encoding the pyoverdin biosynthetic enzyme L-ornithine N5-oxygenase in Pseudomonas aeruginosa.

    PubMed Central

    Visca, P; Ciervo, A; Orsi, N

    1994-01-01

    A gene was expressed in P. aeruginosa under the control of the T7 promoter. Induction of the T7 RNA polymerase system resulted in parallel increases of the L-Orn N5-oxygenase activity and of the amount of a 47.7-kDa polypeptide. We also constructed a site-specific pvdA mutant by insertion of a tetracycline-resistance cassette in the chromosomal pvdA gene of P. aeruginosa PAO1. Similarly to strain PALS124, the pvdA mutant obtained by gene disruption also disclosed no pyoverdin synthesis, lacked L-Orn N5-oxygenase activity, was complemented by the cloned pvdA gene, and produced pyoverdin at wild-type levels when fed with the biosynthetic precursor L-N5-OH-Orn. Southern blot analysis indicated that genes homologous to pvdA could be located within a 1.7-kb DNA fragment from SphI-digested genomic DNA of different hydroxamate-producing Pseudomonas spp. Our results suggest that omega-amino acid oxygenases have been conserved over a wide evolutionary range and probably evolved from a common ancestor. Images PMID:8106324

  13. Cells of Escherichia coli are protected against severe chemical stress by co-habiting cell aggregates formed by Pseudomonas aeruginosa.

    PubMed

    Jagmann, Nina; Henke, Sebastian Franz; Philipp, Bodo

    2015-10-01

    Bacterial cells within biofilms and cell aggregates show increased resistance against chemical stress compared with suspended cells. It is not known whether bacteria that co-habit biofilms formed by other bacteria also acquire such resistance. This scenario was investigated in a proof-of-principle experiment with Pseudomonas aeruginosa strain PAO1 as cell aggregate-forming bacterium and Escherichia coli strain MG1655 as potential co-habiting bacterium equipped with an inducible bioluminescence system. Cell aggregation of strain PAO1 can be induced by the toxic detergent sodium dodecyl sulfate (SDS). In single cultures of strain MG1655, bioluminescence was inhibited by the protonophor carbonylcyanide-m-chlorophenylhydrazone (CCCP) but the cells were still viable. By applying CCCP and SDS together, cells of strain MG1655 lost their bioluminescence and viability indicating the importance of energy-dependent resistance mechanisms against SDS. In co-suspensions with strain PAO1, bioluminescence of strain MG1655 was sustained in the presence of SDS and CCCP. Image analysis showed that bioluminescent cells were located in cell aggregates formed by strain PAO1. Thus, cells of strain MG1655 that co-habited cell aggregates formed by strain PAO1 were protected against a severe chemical stress that was lethal to them in single cultures. Co-habiting could lead to increased survival of pathogens in clinical settings and could be employed in biotechnological applications involving toxic milieus. PMID:26066844

  14. The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles.

    PubMed

    Qaisar, Uzma; Kruczek, Cassandra J; Azeem, Muhammed; Javaid, Nasir; Colmer-Hamood, Jane A; Hamood, Abdul N

    2016-08-01

    Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope. PMID:27480638

  15. Catabolite repression in Pseudomonas aeruginosa PAO1 regulates the uptake of C4 -dicarboxylates depending on succinate concentration.

    PubMed

    Valentini, Martina; Lapouge, Karine

    2013-06-01

    In Pseudomonas aeruginosa carbon catabolite repression (CCR) is exerted by the CbrA/B-CrcZ-Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C4 -dicarboxylates, impairs the utilization of less preferred substrates. When non-preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non-preferred substrates. The C4 -dicarboxylate transport (Dct) system in P. aeruginosa is composed of two main transporters: DctA, more efficient at mM succinate concentrations, and DctPQM, more important at μM. In this study, we demonstrate that the Dct transporters are differentially regulated by Crc, depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post-transcriptionally. The activation of dctA is explained by a Crc-mediated repression of dctR, encoding a transcriptional repressor of dctA. At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM. As a result, expression of dctA is reduced and that of dctPQM is increased. PMID:23253107

  16. Effect of plant phenolic compounds on biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Plyuta, Vladimir; Zaitseva, Julia; Lobakova, Elena; Zagoskina, Natalia; Kuznetsov, Alexander; Khmel, Inessa

    2013-11-01

    In the natural environment, bacteria predominantly exist in matrix-enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3- to 7-fold under the action of vanillin and epicatechin, and 2- to 2.5-fold in the presence of 4-hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4-hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N-3-oxo-dodecanoyl-homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N-acyl-homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs. PMID:23594262

  17. Marine-derived quorum-sensing inhibitory activities enhance the antibacterial efficacy of tobramycin against Pseudomonas aeruginosa.

    PubMed

    Busetti, Alessandro; Shaw, George; Megaw, Julianne; Gorman, Sean P; Maggs, Christine A; Gilmore, Brendan F

    2015-01-01

    Bacterial epiphytes isolated from marine eukaryotes were screened for the production of quorum sensing inhibitory compounds (QSIs). Marine isolate KS8, identified as a Pseudoalteromonas sp., was found to display strong quorum sensing inhibitory (QSI) activity against acyl homoserine lactone (AHL)-based reporter strains Chromobacterium violaceum ATCC 12472 and CV026. KS8 supernatant significantly reduced biofilm biomass during biofilm formation (-63%) and in pre-established, mature P. aeruginosa PAO1 biofilms (-33%). KS8 supernatant also caused a 0.97-log reduction (-89%) and a 2-log reduction (-99%) in PAO1 biofilm viable counts in the biofilm formation assay and the biofilm eradication assay respectively. The crude organic extract of KS8 had a minimum inhibitory concentration (MIC) of 2 mg/mL against PAO1 but no minimum bactericidal concentration (MBC) was observed over the concentration range tested (MBC > 16 mg/mL). Sub-MIC concentrations (1 mg/mL) of KS8 crude organic extract significantly reduced the quorum sensing (QS)-dependent production of both pyoverdin and pyocyanin in P. aeruginosa PAO1 without affecting growth. A combinatorial approach using tobramycin and the crude organic extract at 1 mg/mL against planktonic P. aeruginosa PAO1 was found to increase the efficacy of tobramycin ten-fold, decreasing the MIC from 0.75 to 0.075 µg/mL. These data support the validity of approaches combining conventional antibiotic therapy with non-antibiotic compounds to improve the efficacy of current treatments. PMID:25546516

  18. Marine-Derived Quorum-Sensing Inhibitory Activities Enhance the Antibacterial Efficacy of Tobramycin against Pseudomonas aeruginosa

    PubMed Central

    Busetti, Alessandro; Shaw, George; Megaw, Julianne; Gorman, Sean P.; Maggs, Christine A.; Gilmore, Brendan F.

    2014-01-01

    Bacterial epiphytes isolated from marine eukaryotes were screened for the production of quorum sensing inhibitory compounds (QSIs). Marine isolate KS8, identified as a Pseudoalteromonas sp., was found to display strong quorum sensing inhibitory (QSI) activity against acyl homoserine lactone (AHL)-based reporter strains Chromobacterium violaceum ATCC 12472 and CV026. KS8 supernatant significantly reduced biofilm biomass during biofilm formation (−63%) and in pre-established, mature P. aeruginosa PAO1 biofilms (−33%). KS8 supernatant also caused a 0.97-log reduction (−89%) and a 2-log reduction (−99%) in PAO1 biofilm viable counts in the biofilm formation assay and the biofilm eradication assay respectively. The crude organic extract of KS8 had a minimum inhibitory concentration (MIC) of 2 mg/mL against PAO1 but no minimum bactericidal concentration (MBC) was observed over the concentration range tested (MBC > 16 mg/mL). Sub-MIC concentrations (1 mg/mL) of KS8 crude organic extract significantly reduced the quorum sensing (QS)-dependent production of both pyoverdin and pyocyanin in P. aeruginosa PAO1 without affecting growth. A combinatorial approach using tobramycin and the crude organic extract at 1 mg/mL against planktonic P. aeruginosa PAO1 was found to increase the efficacy of tobramycin ten-fold, decreasing the MIC from 0.75 to 0.075 µg/mL. These data support the validity of approaches combining conventional antibiotic therapy with non-antibiotic compounds to improve the efficacy of current treatments. PMID:25546516

  19. Pleiotropic effects of temperature-regulated 2-OH-lauroytransferase (PA0011) on Pseudomonas aeruginosa antibiotic resistance, virulence and type III secretion system.

    PubMed

    Wang, Bobo; Li, Bo; Liang, Ying; Li, Jing; Gao, Lang; Chen, Lin; Duan, Kangmin; Shen, Lixin

    2016-02-01

    Pseudomonas aeruginosa is an important human pathogen which adapts to changing environment, such as temperature variations and entering host by regulating their gene expression. Here, we report that gene PA0011 in P. aeruginosa PAO1, which encodes a 2-OH-lauroytransferase participating in lipid A biosynthesis, is involved in carbapenem resistance and virulence in a temperature-regulated manner in PAO1. The expression of PA0011 was higher at an environment temperature (21 °C) than that at a body temperature (37 °C). The inactivation of PA0011 rendered increased antibiotic susceptibility and decreased virulence both in vivo and in vitro. The impaired integrity and the decreased stability of the outer membrane were the cause of the increased susceptibility of PAO1(Δ0011) to carbapenem and many other common antibiotics. The reduced endotoxic activity of lipopolysaccharide (LPS) contributed to the decreased virulence both at 21 °C and 37 °C in PAO1 (Δ0011). In addition, we have found that PA0011 repressed the expression of TTSS virulence factors both at transcriptional and translational levels, similar to the effect of O antigen of LPS but unlike any effect of its homologue reported in other bacteria. The effect of PA0011 on resistance to many antibiotics including carbapenem and virulence in P. aeruginosa makes it a target for novel antimicrobial therapies. PMID:26596709

  20. Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water

    PubMed Central

    Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

    2015-01-01

    The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. PMID:25186059

  1. Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water.

    PubMed

    Schwartz, Thomas; Armant, Olivier; Bretschneider, Nancy; Hahn, Alexander; Kirchen, Silke; Seifert, Martin; Dötsch, Andreas

    2015-01-01

    The fitness of sensitive and resistant Pseudomonas aeruginosa in different aquatic environments depends on genetic capacities and transcriptional regulation. Therefore, an antibiotic-sensitive isolate PA30 and a multi-resistant isolate PA49 originating from waste waters were compared via whole genome and transcriptome Illumina sequencing after exposure to municipal waste water and tap water. A number of different genomic islands (e.g. PAGIs, PAPIs) were identified in the two environmental isolates beside the highly conserved core genome. Exposure to tap water and waste water exhibited similar transcriptional impacts on several gene clusters (antibiotic and metal resistance, genetic mobile elements, efflux pumps) in both environmental P. aeruginosa isolates. The MexCD-OprJ efflux pump was overexpressed in PA49 in response to waste water. The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix. Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements. Thus, the exposure of both isolates to polluted waste water and oligotrophic tap water resulted in similar expression profiles of mentioned genes. However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation. PMID:25186059

  2. Genome analysis and gene nblA identification of Microcystis aeruginosa myovirus (MaMV-DC) reveal the evidence for horizontal gene transfer events between cyanomyovirus and host.

    PubMed

    Ou, Tong; Gao, Xiao-Chan; Li, San-Hua; Zhang, Qi-Ya

    2015-12-01

    The genome sequence, genetic characterization and nblA gene function of Microcystis aeruginosa myovirus isolated from Lake Dianchi in China (MaMV-DC) have been analysed. The genome DNA is 169 223 bp long, with 170 predicted protein-coding genes (001L–170L) and a tRNA gene. About one-sixth of these genes have homologues in the host cyanobacteria M. aeruginosa. The genome carries a gene homologous to host nblA, which encodes a protein involved in the degradation of cyanobacterial phycobilisome. Its expression during MaMV-DC infection was confirmed by reverse transcriptase PCR and Western blot detection and abundant expression was companied by the significant decline of phycocyanin content and massive release of progeny MaMV-DC. In addition, expressing MaMV-DC nblA reduced the phycocyanin peak and the phycocyanin to chlorophyll ratio in model cyanobacteria. These results confirm that horizontal gene transfer events have occurred between cyanobacterial host and cyanomyovirus and suggest that MaMV-DC carrying host-derived genes (such as 005L, that codes for NblA) is responsible for more efficient expression of cyanophage genes and release of progeny cyanophage. This study provides novel insight into the horizontal gene transfer in cyanophage and the interactions between cyanophage and their host. PMID:26399243

  3. Biological cost of hypermutation in Pseudomonas aeruginosa strains from patients with cystic fibrosis.

    PubMed

    Montanari, Sara; Oliver, Antonio; Salerno, Paola; Mena, Ana; Bertoni, Giovanni; Tümmler, Burkhard; Cariani, Lisa; Conese, Massimo; Döring, Gerd; Bragonzi, Alessandra

    2007-05-01

    The high prevalence of hypermutable (mismatch repair-deficient) Pseudomonas aeruginosa strains in patients with cystic fibrosis (CF) is thought to be driven by their co-selection with adaptive mutations required for long-term persistence. Whether the increased mutation rate of naturally hypermutable strains is associated with a biological benefit or cost for the colonization of secondary environments is not known. Thirty-nine P. aeruginosa strains were collected from ten patients with CF during their course of chronic lung infections and screened for hypermutability. Seven hypermutable P. aeruginosa strains (18 %) isolated from six patients with CF (60 %) were identified and assigned to five different genotypes. Complementation and sequence analysis in the mutS, mutL and uvrD genes of these hypermutable P. aeruginosa strains revealed novel mutations. To understand the consequences of hypermutation for the fitness of the organisms, five pairs of clinical wild-type/hypermutable, clonally related P. aeruginosa strains and the laboratory strains PAO1/PAO1DeltamutS were subjected to competition in vitro and in the agar-beads mouse model of chronic airway infection. When tested in competition assay in vitro, the wild-type outcompeted four clinical hypermutable strains and the PAO1DeltamutS strain. In vivo, all of the hypermutable strains were less efficient at establishing lung infection than their wild-type clones. These results suggest that P. aeruginosa hypermutation is associated with a biological cost, reducing the potential for colonization of new environments and therefore strain transmissibility. PMID:17464058

  4. A Novel Insight into Dehydroleucodine Mediated Attenuation of Pseudomonas aeruginosa Virulence Mechanism

    PubMed Central

    Mustafi, S.; Veisaga, M. L.; López, L. A.; Barbieri, M. A.

    2015-01-01

    Increasing resistance of Pseudomonas aeruginosa (P. aeruginosa) to conventional treatments demands the search for novel therapeutic strategies. In this study, the antimicrobial activity of dehydroleucodine (DhL), a sesquiterpene lactone obtained from Artemisia (A.) douglasiana, was screened against several pathogenic virulence effectors of P. aeruginosa. In vitro, minimum inhibitory concentration of DhL was determined against P. aeruginosa strains PAO1, PA103, PA14, and multidrug resistant clinical strain, CDN118. Results showed that DhL was active against each strain where PAO1 and PA103 showed higher susceptibility (MIC 0.48 mg/mL) as compared to PA14 (MIC 0.96 mg/mL) and CDN118 (MIC 0.98 mg/mL). Also, when PAO1 strain was grown in the presence of DhL (MIC50, 0.12 mg/mL), a delay in the generation time was noticed along with significant inhibition of secretory protease and elastase activities, interruption in biofilm attachment phase in a stationary culture, and a significant decline in Type III effector ExoS. At MIC50, DhL treatment increased the sensitivity of P. aeruginosa towards potent antibiotics. Furthermore, treatment of P. aeruginosa with DhL prevented toxin-induced apoptosis in macrophages. These observations suggest that DhL activity was at the bacterial transcriptional level. Hence, antimicrobial activity of DhL may serve as leads in the development of new anti-Pseudomonas pharmaceuticals. PMID:26640783

  5. Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

    PubMed Central

    Schwarzer, Christian; Fischer, Horst; Machen, Terry E.

    2016-01-01

    Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10–80 fold increase, termed “swarming”), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds. PMID:27031335

  6. Mitogenic effects of purified outer membrane proteins from Pseudomonas aeruginosa.

    PubMed Central

    Chen, Y H; Hancock, R E; Mishell, R I

    1980-01-01

    Three major outer membrane proteins from Pseudomonas aeruginosa PAO1 were purified and tested for their ability to stimulate resting murine lymphocytes to proliferate. It was demonstrated that picomole amounts of all three proteins were mitogenic for both intact and T-lymphocyte-depleted populations of spleen cells from C3H/HeJ mice. In contrast, they had no activity against either mature or immature thymocytes. Since the strain of mice used is unable to respond to lipopolysaccharide, we condlude that the three proteins are B-cell mitogens. Images Fig. 2 PMID:6769818

  7. A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

    PubMed Central

    Bijtenhoorn, Patrick; Mayerhofer, Hubert; Müller-Dieckmann, Jochen; Utpatel, Christian; Schipper, Christina; Hornung, Claudia; Szesny, Matthias; Grond, Stephanie; Thürmer, Andrea; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Dierking, Katja; Schulenburg, Hinrich; Streit, Wolfgang R.

    2011-01-01

    In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies. PMID:22046268

  8. Comparison of the transport and deposition of Pseudomonas aeruginosa under aerobic and anaerobic conditions

    NASA Astrophysics Data System (ADS)

    Zhang, Huixin; Zeng, Hongbo; Ulrich, Ania C.; Liu, Yang

    2016-02-01

    Laboratory-scale columns were employed to study the effect of oxygen and ionic strength on the transport of Pseudomonas aeruginosa PAO1 in porous media. In anaerobic experiments, cells were grown and transport experiments were conducted in a well-controlled anaerobic chamber. Cell surface electrokinetic potentials were measured and surface elemental composition was analyzed using X-ray photoelectron spectroscopy (XPS). Transport experimental results showed reduced travel distance of PAO1 with increased ionic strength under aerobic and anaerobic conditions, consistent with calculated Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The deposition rates of PAO1 were significantly higher in aerobic than in anaerobic condition at higher ionic strength (10 and 100 mM), although the electrokinetic potentials were similar throughout the tested ionic strength (1, 10, and 100 mM). No difference in PAO1 deposition rate was observed at 1 mM. XPS analysis showed that variation in cell surface composition due to different growth conditions played a primary role in determining the different deposition behaviors.

  9. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation.

    PubMed

    Müller, Christine; Birmes, Franziska S; Rückert, Christian; Kalinowski, Jörn; Fetzner, Susanne

    2015-11-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  10. Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

    PubMed Central

    Müller, Christine; Birmes, Franziska S.; Rückert, Christian; Kalinowski, Jörn

    2015-01-01

    Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases. PMID:26319870

  11. Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates.

    PubMed

    Conibear, Tim C R; Willcox, Mark D P; Flanagan, Judith L; Zhu, Hua

    2012-02-01

    Expression of protease IV by Pseudomonas aeruginosa during ocular infections contributes significantly to tissue damage. However, several P. aeruginosa strains isolated from ocular infections or inflammatory events produce very low levels of protease IV. The aim of the present study was to characterize, genetically and phenotypically, the presence and expression of the protease IV gene in a group of clinical isolates that cause adverse ocular events of varying degrees, and to elucidate the possible control mechanisms of expression associated with this virulence factor. Protease IV gene sequences from seven clinical isolates of P. aeruginosa were determined and compared to P. aeruginosa strains PAO1 and PA103-29. Production and enzyme activity of protease IV were measured in test strains and compared to that of quorum-sensing gene (lasRI) mutants and the expression of other virulence factors. Protease IV gene sequence similarities between the isolates were 97.5-99.5 %. The strains were classified into two distinct phylogenetic groups that correlated with the presence of exo-enzymes from type three secretion systems (TTSS). Protease IV concentrations produced by PAOΔlasRI mutants and the two clinical isolates with a lasRI gene deficiency were restored to levels comparable to strain PAO1 following complementation of the quorum-sensing gene deficiencies. The protease IV gene is highly conserved in P. aeruginosa clinical isolates that cause a range of adverse ocular events. Observed variations within the gene sequence appear to correlate with presence of specific TTSS genes. Protease IV expression was shown to be regulated by the Las quorum-sensing system. PMID:21921113

  12. The transcriptional regulator AlgR is essential for Pseudomonas aeruginosa pathogenesis.

    PubMed

    Lizewski, Stephen E; Lundberg, Derek S; Schurr, Michael J

    2002-11-01

    Chronic Pseudomonas aeruginosa lung infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. One P. aeruginosa virulence factor unique to CF isolates is overproduction of alginate, phenotypically termed mucoidy. Mucoidy is the result of increased transcription from the algD gene and is activated by the transcriptional regulator AlgR. Mutations in algR result in a nonmucoid phenotype and loss of twitching motility. Additionally, AlgR controls transcription of algC, encoding a dual-function enzyme necessary for both lipopolysaccharide (LPS) and alginate production. Therefore, to determine the effect of algR on P. aeruginosa virulence, an algR mutant was examined for sensitivity to reactive oxygen intermediates, killing by phagocytes, systemic virulence, and the ability to maintain a murine lung infection. We found that P. aeruginosa PAO700 (algR::Gm(r)) was less lethal than PAO1, as tested in an acute septicemia infection mouse model, and was cleared more efficiently in a mouse pneumonia model. Additionally, the algR mutant (PAO700) was more sensitive to hypochlorite. However, PAO700 was more resistant to hydrogen peroxide and killed less readily in an acellular myeloperoxidase assay than PAO1. There was little difference in killing between PAO1 and PAO700 with macrophage-like J774 cells and human polymorhonuclear leukocytes. Two-dimensional gel analysis of P. aeruginosa algR mutant and wild-type protein extracts revealed 47 differentially regulated proteins, suggesting that AlgR plays both a positive role and a negative role in gene expression. Together, these results imply that AlgR is necessary for virulence and regulates genes in addition to the genes associated with alginate and LPS production and pilus function. PMID:12379685

  13. Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa

    PubMed Central

    Suh, Sang-Jin; Silo-Suh, Laura; Woods, Donald E.; Hassett, Daniel J.; West, Susan E. H.; Ohman, Dennis E.

    1999-01-01

    The sigma factor RpoS (ςS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50°C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the

  14. Ambroxol interferes with Pseudomonas aeruginosa quorum sensing.

    PubMed

    Lu, Qi; Yu, Jialin; Yang, Xiqiang; Wang, Jiarong; Wang, Lijia; Lin, Yayin; Lin, Lihua

    2010-09-01

    The mucolytic agent ambroxol has been reported to interfere with the formation of Pseudomonas aeruginosa-derived biofilms in addition to reducing alginate production by undefined mechanisms. Since quorum sensing is a key regulator of virulence and biofilm formation, we examined the effects of ambroxol on P. aeruginosa PAO1 wild-type bacterial clearance rates, adhesion profiles and biofilm formation compared with the quorum sensing-deficient, double-mutant strains DeltalasR DeltarhlR and DeltalasI DeltarhlI. Data presented in this report demonstrated that ambroxol treatment reduced survival rates of the double-mutant strains compared with the wild-type strain in a dose-dependent manner even though the double-mutants had increased adhesion in the presence of ambroxol compared with the wild-type strain. The PAO1 wild-type strain produced a significantly thicker biofilm (21.64+/-0.57 microm) compared with the biofilms produced by the DeltalasR DeltarhlR (7.36+/-0.2 microm) and DeltalasI DeltarhlI (6.62+/-0.31 microm) isolates. Ambroxol treatment reduced biofilm thickness, increased areal porosity, and decreased the average diffusion distance and textual entropy of wild-type and double-mutant strains. However, compared with the double-mutant strains, the changes observed for the wild-type strain were more clearly defined. Finally, ambroxol exhibited significant antagonistic quorum-sensing properties, suggesting that it could be adapted for use clinically in the treatment of cystic fibrosis and to reduce biofilm formation and in the colonisation of indwelling devices. PMID:20580207

  15. C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa.

    PubMed

    Blier, Anne-Sophie; Veron, Wilfried; Bazire, Alexis; Gerault, Eloïse; Taupin, Laure; Vieillard, Julien; Rehel, Karine; Dufour, Alain; Le Derf, Franck; Orange, Nicole; Hulen, Christian; Feuilloley, Marc G J; Lesouhaitier, Olivier

    2011-07-01

    Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during

  16. C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa

    PubMed Central

    Blier, Anne-Sophie; Veron, Wilfried; Bazire, Alexis; Gerault, Eloïse; Taupin, Laure; Vieillard, Julien; Rehel, Karine; Dufour, Alain; Le Derf, Franck; Orange, Nicole; Hulen, Christian; Feuilloley, Marc G. J.

    2011-01-01

    Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during

  17. Antibiofilm activity of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 against Pseudomonas aeruginosa.

    PubMed

    Kim, Yong-Guy; Lee, Jin-Hyung; Kim, Chang-Jin; Lee, Jae-Chan; Ju, Yoon Jung; Cho, Moo Hwan; Lee, Jintae

    2012-12-01

    Members of the actinomycetes family are a rich source of bioactive compounds including diverse antibiotics. This study sought to identify novel and non-toxic biofilm inhibitors from the actinomycetes library for reducing the biofilm formation of Pseudomonas aeruginosa PAO1. After the screening of 4104 actinomycetes strains, we found that the culture spent medium (1 %, v/v) of Streptomyces sp. BFI 230 and Kribbella sp. BFI 1562 inhibited P. aeruginosa biofilm formation by 90 % without affecting the growth of planktonic P. aeruginosa cells, while the spent media enhanced the swarming motility of P. aeruginosa. Global transcriptome analyses revealed that the spent medium of Streptomyces sp. BFI 230 induced expression of phenazine, pyoverdine, pyochelin synthesis genes, and iron uptake genes in P. aeruginosa. The addition of exogenous iron restored the biofilm formation and swarming motility of P. aeruginosa in the presence of the spent medium of Streptomyces sp. BFI 230, which suggests that the Streptomyces sp. BFI 230 strain interfered iron acquisition in P. aeruginosa. Experiments on solvent extraction, heat treatment, and proteinase K treatment suggested that hydrophilic compound(s), possibly extracellular peptides or proteins from Streptomyces sp. BFI 230 cause the biofilm reduction of P. aeruginosa. Together, this study indicates that actinomycetes strains have an ability to control the biofilm of P. aeruginosa. PMID:22722911

  18. Effect of Traditional Chinese Herbal Medicine with Antiquorum Sensing Activity on Pseudomonas aeruginosa

    PubMed Central

    Zhou, Shuxin; Jiang, Yan; Zhu, Wei; Zhuang, Xiyi; Fu, Jiangyan

    2013-01-01

    Traditional Chinese herbal medicines (TCHMs) were tested for their ability of antiquorum sensing. Water extracts of Rhubarb, Fructus gardeniae, and Andrographis paniculata show antiquorumsensing activity when using Chromobacterium violaceum CV12472 as reporter; the sub-MIC concentrations of these TCHMs were tested against AHL-dependent phenotypic expressions of PAO1. Results showed significant reduction in pyocyanin pigment, protease, elastase production, and biofilm formation in PAO1 without inhibiting the bacterial growth, revealing that the QSI by the extracts is not related to static or killing effects on the bacteria. The results indicate a potential modulation of bacterial cell-cell communication, P. aeruginosa biofilm, and virulence factors by traditional Chinese herbal medicine. This study introduces not only a new mode of action for traditional Chinese herbal medicines, but also a potential new therapeutic direction for the treatment of bacterial infections, which have QSI activity and might be important in reducing virulence and pathogenicity of pathogenic bacteria. PMID:24319480

  19. Activity of Chitosans in combination with antibiotics in Pseudomonas aeruginosa

    PubMed Central

    Tin, San; Sakharkar, Kishore R.; Lim, Chu Sing; Sakharkar, Meena K.

    2009-01-01

    Chitosan and its derivative water soluble Chitosan oligosaccharide are used in a variety of applications in pharmaceutical preparations. In this study, 2 wild (ATCC 15729 and PAO1) and 2 mutant strains (PT121 and PT149) of P. aeruginosa are investigated for drug-drug interactions in vitro. 10 antimicrobial agents (antibiotics) are combined with different degree of deacetylated Chitosans and Chitosan oligosaccharide. All the chitosans show synergistic activity with sulfamethoxazole, a sulfonamide antimicrobial agent. It is interesting to observe that the MIC value for the MexEF-OprN overexpressing mutant strain of P. aeruginosa is 5 fold higher than the other strains under investigation suggesting a possible role of this efflux pump in Sulfamethoxazole efflux. The findings suggest on the use of chitosans as enhancing agent in combination with antibiotics in pharmaceutical preparations. PMID:19173037

  20. The Psl economy in early P. aeruginosa biofilm development

    NASA Astrophysics Data System (ADS)

    Zhao, Kun; Tseng, Boo Shan; Jin, Fan; Gibiansky, Max; Harrison, Joe; Parsek, Matthew; Wong, Gerard

    2012-02-01

    Psl from P. aeruginosa (PAO1) is a mannose- and galactose-rich exopolysaccharide (EPS). It has been shown that Psl plays an important role in bacterial surface adhesion. Here, we examine role of Psl in controlling motility and microcolony formation during early biofilm development, by translating video microscopy movies into searchable databases of bacterial trajectories. We use a massively-parallel cell tracking algorithm to extract the full motility history of every cell in a large community. We find that at early stages of growth, P. aeruginosa motility is guided by Psl and self-organize in a manner analogous to a capitalist economic system, resulting in a power law bacterial distribution where a small number of bacteria are extremely ``rich'' in communally produced Psl. By comparing overproducers and underproducers of Psl, we find that local Psl levels determine post-division cell fates: High local Psl levels drive the formation of sessile microcolonies that grow exponentially.

  1. Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

    PubMed Central

    Rivera, M; Bryan, L E; Hancock, R E; McGroarty, E J

    1988-01-01

    Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different. Images PMID:3123455

  2. The soluble pyocins S2 and S4 from Pseudomonas aeruginosa bind to the same FpvAI receptor.

    PubMed

    Elfarash, Ameer; Wei, Qing; Cornelis, Pierre

    2012-09-01

    Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa cells by gaining entry via a specific receptor, which, in the case of pyocin S2, is the siderophore pyoverdine receptor FpvAI, and in the case of pyocin S3, FpvAII. The nucleic acid sequence at the positions 4327697-4327359 of P. aeruginosa PAO1 genome was not annotated, but it was predicted to encode the immunity gene of the flanking pyocin S4 gene (PA3866) based on our analysis of the genome sequence. Using RT-PCR, the expression of the immunity gene was detected, confirming the existence of an immunity gene overlapping the S4 pyocin gene. The PA3866 coding for pyocin S4 and the downstream gene coding for the immunity protein were cloned and expressed in Escherichia coli and the His-tagged S4 pyocin was obtained in pure form. Forty-three P. aeruginosa strains were typed via PCR to identify their ferripyoverdine receptor gene (fpvAI-III) and were tested for their sensitivity to pyocin S4. All S4-sensitive strains had the type I ferripyoverdine receptor fpvA gene. Some S4-resistant type I fpvA-positive strains were detected, but all of them had the S4 immunity gene, and, following the deletion of the immunity gene, became S4-sensitive. The fpvAI receptor gene was deleted in a S4-sensitive strain, and, as expected, the mutant became resistant to S4. The N-terminal receptor binding domain (RBD) of pyocin S2, which also uses the FpvAI receptor to enter the cell, was cloned in the pET-15b vector, and expressed in E. coli. When the purified RBD was mixed with pyocin S4 at different ratios, an inhibition of killing was observed, indicating that S2 RBD competes with the pyocin S4 for the binding to the FpvAI receptor. The S2 RBD was also shown to enhance the expression of the pvdA pyoverdine gene, suggesting that it, like pyoverdine, works via the known siderophore-mediated signalization pathway. PMID:23170226

  3. Production of the Pseudomonas aeruginosa neuraminidase is increased under hyperosmolar conditions and is regulated by genes involved in alginate expression.

    PubMed Central

    Cacalano, G; Kays, M; Saiman, L; Prince, A

    1992-01-01

    The pathogenesis of Pseudomonas aeruginosa infection in cystic fibrosis (CF) is a complex process attributed to specific characteristics of both the host and the infecting organism. In this study, the properties of the PAO1 neuraminidase were examined to determine its potential role in facilitating Pseudomonas colonization of the respiratory epithelium. The PAO1 neuraminidase was 1000-fold more active than the Clostridium perfringens enzyme in releasing sialic acid from respiratory epithelial cells. This effect correlated with increased adherence of PAO1 to epithelial cells after exposure to PAO1 neuraminidase and was consistent with in vitro studies demonstrating Pseudomonas adherence to asialoganglioside receptors. The regulation of the neuraminidase gene nanA was examined in Pseudomonas and as cloned and expressed in Escherichia coli. In hyperosmolar conditions neuraminidase expression was increased by 50% (P less than 0.0004), an effect which was OmpR dependent in E. coli. In Pseudomonas the osmotic regulation of neuraminidase production was dependent upon algR1 and algR2, genes involved in the transcriptional activation of algD, which is responsible for the mucoid phenotype of Pseudomonas and pathognomonic for chronic infection in CF. Under the hyperosmolar conditions postulated to exist in the CF lung, nanA is likely to be expressed to facilitate the initial adherence of Pseudomonas to the respiratory tract. Images PMID:1601994

  4. Increased bactericidal activity of colistin on Pseudomonas aeruginosa biofilms in anaerobic conditions.

    PubMed

    Kolpen, Mette; Appeldorff, Cecilie F; Brandt, Sarah; Mousavi, Nabi; Kragh, Kasper N; Aydogan, Sevtap; Uppal, Haleema A; Bjarnsholt, Thomas; Ciofu, Oana; Høiby, Niels; Jensen, Peter Ø

    2016-02-01

    Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH ˙ on several strains of planktonic P. aeruginosa. Therefore, we propose that production of OH ˙ may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (ΔkatA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH⋅ formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH⋅ formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions. PMID:26458402

  5. Involvement of Pseudomonas aeruginosa rhodanese in protection from cyanide toxicity.

    PubMed

    Cipollone, Rita; Frangipani, Emanuela; Tiburzi, Federica; Imperi, Francesco; Ascenzi, Paolo; Visca, Paolo

    2007-01-01

    Cyanide is a serious environmental pollutant and a biocontrol metabolite in plant growth-promoting Pseudomonas species. Here we report on the presence of multiple sulfurtransferases in the cyanogenic bacterium Pseudomonas aeruginosa PAO1 and investigate in detail RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) which converts cyanide to less toxic thiocyanate. RhdA is a cytoplasmic enzyme acting as the principal rhodanese in P. aeruginosa. The rhdA gene forms a transcriptional unit with the PA4955 and psd genes and is controlled by two promoters located upstream of PA4955 and rhdA. Both promoters direct constitutive RhdA expression and show similar patterns of activity, involving moderate down-regulation at the stationary phase or in the presence of exogenous cyanide. We previously observed that RhdA overproduction protects Escherichia coli against cyanide toxicity, and here we show that physiological RhdA levels contribute to P. aeruginosa survival under cyanogenic conditions. The growth of a DeltarhdA mutant is impaired under cyanogenic conditions and fully restored upon complementation with rhdA. Wild-type P. aeruginosa outcompetes the DeltarhdA mutant in cyanogenic coculture assays. Hence, RhdA could be regarded as an effector of P. aeruginosa intrinsic resistance to cyanide, insofar as it provides the bacterium with a defense mechanism against endogenous cyanide toxicity, in addition to cyanide-resistant respiration. PMID:17098912

  6. Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

    PubMed Central

    Facchini, Marcella; De Fino, Ida; Riva, Camilla; Bragonzi, Alessandra

    2014-01-01

    A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host. This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease. This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies. PMID:24686327

  7. Comparative genome analysis reveals the molecular basis of nicotine degradation and survival capacities of Arthrobacter

    PubMed Central

    Yao, Yuxiang; Tang, Hongzhi; Su, Fei; Xu, Ping

    2015-01-01

    Arthrobacter is one of the most prevalent genera of nicotine-degrading bacteria; however, studies of nicotine degradation in Arthrobacter species remain at the plasmid level (plasmid pAO1). Here, we report the bioinformatic analysis of a nicotine-degrading Arthrobacter aurescens M2012083, and show that the moeB and mogA genes that are essential for nicotine degradation in Arthrobacter are absent from plasmid pAO1. Homologues of all the nicotine degradation-related genes of plasmid pAO1 were found to be located on a 68,622-bp DNA segment (nic segment-1) in the M2012083 genome, showing 98.1% nucleotide acid sequence identity to the 69,252-bp nic segment of plasmid pAO1. However, the rest sequence of plasmid pAO1 other than the nic segment shows no significant similarity to the genome sequence of strain M2012083. Taken together, our data suggest that the nicotine degradation-related genes of strain M2012083 are located on the chromosome or a plasmid other than pAO1. Based on the genomic sequence comparison of strain M2012083 and six other Arthrobacter strains, we have identified 17 σ70 transcription factors reported to be involved in stress responses and 109 genes involved in environmental adaptability of strain M2012083. These results reveal the molecular basis of nicotine degradation and survival capacities of Arthrobacter species. PMID:25721465

  8. MrkD1P from Klebsiella pneumoniae Strain IA565 Allows for Coexistence with Pseudomonas aeruginosa and Protection from Protease-Mediated Biofilm Detachment

    PubMed Central

    Childers, Brandon M.; Van Laar, Tricia A.; You, Tao; Clegg, Steven

    2013-01-01

    Biofilm formation and persistence are essential components for the continued survival of pathogens inside the host and constitute a major contributor to the development of chronic wounds with resistance to antimicrobial compounds. Understanding these processes is crucial for control of biofilm-mediated disease. Though chronic wound infections are often polymicrobial in nature, much of the research on chronic wound-related microbes has focused on single-species models. Klebsiella pneumoniae and Pseudomonas aeruginosa are microbes that are often found together in wound isolates and are able to form stable in vitro biofilms, despite the antagonistic nature of P. aeruginosa with other organisms. Mutants of the K. pneumoniae strain IA565 lacking the plasmid-borne mrkD1P gene were less competitive than the wild type in an in vitro dual-species biofilm model with P. aeruginosa (PAO1). PAO1 spent medium inhibited the formation of biofilm of mrkD1P-deficient mutants and disrupted preestablished biofilms, with no effect on IA565 and no effect on the growth of the wild type or mutants. A screen using a two-allele PAO1 transposon library identified the LasB elastase as the secreted effector involved in biofilm disruption, and a purified version of the protein produced results similar to those with PAO1 spent medium. Various other proteases had a similar effect, suggesting that the disruption of the mrkD1P gene causes sensitivity to general proteolytic effects and indicating a role for MrkD1P in protection against host antibiofilm effectors. Our results suggest that MrkD1P allows for competition of K. pneumoniae with P. aeruginosa in a mixed-species biofilm and provides defense against microbial and host-derived proteases. PMID:23980108

  9. Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response

    PubMed Central

    Bauman, Susanne J.; Kuehn, Meta J.

    2012-01-01

    Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation. PMID:16807039

  10. Pseudomonas aeruginosa inhibits the growth of Scedosporium aurantiacum, an opportunistic fungal pathogen isolated from the lungs of cystic fibrosis patients

    PubMed Central

    Kaur, Jashanpreet; Pethani, Bhavin P.; Kumar, Sheemal; Kim, Minkyoung; Sunna, Anwar; Kautto, Liisa; Penesyan, Anahit; Paulsen, Ian T.; Nevalainen, Helena

    2015-01-01

    The filamentous fungus Scedosporium aurantiacum and the bacterium Pseudomonas aeruginosa are opportunistic pathogens isolated from lungs of the cystic fibrosis (CF) patients. P. aeruginosa has been known to suppress the growth of a number of CF related fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. However, the interactions between P. aeruginosa and S. aurantiacum have not been investigated in depth. Hence we assessed the effect of P. aeruginosa reference strain PAO1 and two clinical isolates PASS1 and PASS2 on the growth of two clinical S. aurantiacum isolates WM 06.482 and WM 08.202 using solid plate assays and liquid cultures, in a synthetic medium mimicking the nutrient condition in the CF sputum. Solid plate assays showed a clear inhibition of growth of both S. aurantiacum strains when cultured with P. aeruginosa strains PASS1 and PAO1. The inhibitory effect was confirmed by confocal microscopy. In addition to using chemical fluorescent stains, strains tagged with yfp (P. aeruginosa PASS1) and mCherry (S. aurantiacum WM 06.482) were created to facilitate detailed microscopic observations on strain interaction. To our knowledge, this is the first study describing successful genetic transformation of S. aurantiacum. Inhibition of growth was observed only in co-cultures of P. aeruginosa and S. aurantiacum; the cell fractions obtained from independent bacterial monocultures failed to initiate a response against the fungus. In the liquid co-cultures, biofilm forming P. aeruginosa strains PASS1 and PAO1 displayed higher inhibition of fungal growth when compared to PASS2. No change was observed in the inhibition pattern when direct cell contact between the bacterial and fungal strains was prevented using a separation membrane suggesting the involvement of extracellular metabolites in the fungal inhibition. However, one of the most commonly described bacterial virulence factors, pyocyanin, had no effect against either of the S

  11. Evolution of metabolic divergence in Pseudomonas aeruginosa during long-term infection facilitates a proto-cooperative interspecies interaction.

    PubMed

    Frydenlund Michelsen, Charlotte; Hossein Khademi, Seyed Mohammad; Krogh Johansen, Helle; Ingmer, Hanne; Dorrestein, Pieter C; Jelsbak, Lars

    2016-06-01

    The effect of polymicrobial interactions on pathogen physiology and how it can act either to limit pathogen colonization or to potentiate pathogen expansion and virulence are not well understood. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found together in polymicrobial human infections. However, we have previously shown that the interactions between these two bacterial species are strain dependent. Whereas P. aeruginosa PAO1, a commonly used laboratory strain, effectively suppressed S. aureus growth, we observed a commensal-like interaction between the human host-adapted strain, DK2-P2M24-2003, and S. aureus. In this study, characterization by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) and mass spectral (MS) molecular networking revealed a significant metabolic divergence between P. aeruginosa PAO1 and DK2-P2M24-2003, which comprised several virulence factors and signaling 4-hydroxy-2-alkylquinoline (HAQ) molecules. Strikingly, a further modulation of the HAQ profile was observed in DK2-P2M24-2003 during interaction with S. aureus, resulting in an area with thickened colony morphology at the P. aeruginosa-S. aureus interface. In addition, we found an HAQ-mediated protection of S. aureus by DK2-P2M24-2003 from the killing effect of tobramycin. Our findings suggest a model where the metabolic divergence manifested in human host-adapted P. aeruginosa is further modulated during interaction with S. aureus and facilitate a proto-cooperative P. aeruginosa-S. aureus relationship. PMID:26684729

  12. Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume

    PubMed Central

    Pottier, Laurent; Huet, Joelle; Rabemanantsoa, Christian; Kiendrebeogo, Martin; Andriantsimahavandy, Abel; Rasamindrakotroka, Andry; Stévigny, Caroline; Duez, Pierre; El Jaziri, Mondher

    2015-01-01

    Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms. PMID:26186595

  13. Inhibition of Pseudomonas aeruginosa biofilm formation by 2,2’-bipyridyl, lipoic, kojic and picolinic acids

    PubMed Central

    Çevik, Kübra; Ulusoy, Seyhan

    2015-01-01

    Objective(s): The inhibitory effects of iron chelators, and FeCl3 chelation on biofilm formation and swarming motility were investigated against an opportunistic human pathogen Pseudomonas aeruginosa. Materials and Methods: The inhibitory activity of 2,2’-bipyridyl, lipoic acid, kojic acid and picolinic acid on biofilm formation of P. aeruginosa strain PAO1 and three clinical isolates (P. aeruginosa PAK01, P. aeruginosa PAK02 and P. aeruginosa PAK03) were investigated, based on crystal violet assay, and swarming motility test. Results: The kojic, lipoic and picolinic acid inhibited biofilm formation by 5-33% in all tested P. aeruginosa isolates. When chelated iron was added, biofilm inhibition rates were determined to be 39-57%. Among the tested chelators against P. aeruginosa, lipoic acid (84%) and kojic acid (68%) presented the highest inhibition of swarming motility. This is the first study to report the inhibitory effect of lipoic acid on biofilm formation and swarming motility of P. aeruginosa. Conclusion: It is considered that lipoic and picolinic acids can serve as alternatives for the treatment of the P. aeruginosa infections by inhibiting biofilm formation. PMID:26557964

  14. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa

    PubMed Central

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  15. Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa.

    PubMed

    Wu, Shimei; Liu, Ge; Jin, Weihua; Xiu, Pengyuan; Sun, Chaomin

    2016-01-01

    Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling. PMID:26903981

  16. Effects of Chlorine Stress on Pseudomonas aeruginosa Biofilm and Analysis of Related Gene Expressions.

    PubMed

    Kekeç, Özge; Gökalsın, Barış; Karaltı, İskender; Kayhan, Figen Esin; Sesal, Nüzhet Cenk

    2016-08-01

    Chlorine is deployed worldwide to clean waters and prevent water-originated illnesses. However, chlorine has a limited disinfection capacity against biofilms. Microorganisms form biofilms to protect themselves from biological threats such as disinfectant chemicals. Pseudomonas aeruginosa is an opportunistic pathogen and its biofilm form attaches to surfaces, living buried into exopolysaccharides, can be present in all watery environments including tap water and drinking water. This research aimed to study the biofilm trigger mechanism of the opportunistic pathogen P. aeruginosa PAO1 strain, which is known to form biofilm in water supply systems and human body, under chlorine stress levels. In addition to biofilm staining, certain genes that are relevant to the stress condition were selected for gene expression analysis. The bacteria cultures were grown under chlorine stress with concentrations of 0.5, 0.7 and 1 mg/l. Six gene regions were determined related to biofilm and stress response: rpoS, bifA, migA, katB, soxR, and algC. Biofilm formation was analyzed by basic fuchsin staining, and gene expressions were quantified by quantitative real-time PCR. According to the results, highest biofilm production was observed in P. aeruginosa PAO1 wild strain under no stress conditions. Higher biofilm amounts were observed for bacteria under 0.5 and 0.7 mg/l chlorine stress compared to 1 mg/l chlorine stress. PMID:27146505

  17. Transcriptome profiling reveals links between ParS/ParR, MexEF-OprN, and quorum sensing in the regulation of adaptation and virulence in Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    Background The ParS/ParR two component regulatory system plays critical roles for multidrug resistance in Pseudomonas aeruginosa. It was demonstrated that in the presence of antimicrobials, ParR enhances bacterial survival by distinct mechanisms including activation of the mexXY efflux genes, enhancement of lipopolysaccharide modification through the arn operon, and reduction of the expression of oprD porin. Results In this study, we report on transcriptomic analyses of P. aeruginosa PAO1 wild type and parS and parR mutants growing in a defined minimal medium. Our transcriptomic analysis provides the first estimates of transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to the known effects on drug resistance genes, transcript abundances of the quorum sensing genes (rhlIR and pqsABCDE-phnAB) were higher in both parS and parR mutants. In accordance with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of the par genes also led to increased phenazine production and swarming motility, consistent with the up-regulation of the phenazine and rhamnolipid biosynthetic genes, respectively. Conclusion Our results link the ParS/ParR two component signal transduction system to MexEF-OprN and quorum sensing systems in P. aeruginosa. These results expand our understanding of the roles of the ParS/ParR system in the regulation of gene expression in P. aeruginosa, especially in the absence of antimicrobials. PMID:24034668

  18. A Single parS Sequence from the Cluster of Four Sites Closest to oriC Is Necessary and Sufficient for Proper Chromosome Segregation in Pseudomonas aeruginosa

    PubMed Central

    Jecz, Paulina; Bartosik, Aneta A.; Glabski, Krzysztof; Jagura-Burdzy, Grazyna

    2015-01-01

    Among the mechanisms that control chromosome segregation in bacteria are highly-conserved partitioning systems comprising three components: ParA protein (a deviant Walker-type ATPase), ParB protein (a DNA-binding element) and multiple cis-acting palindromic centromere-like sequences, designated parS. Ten putative parS sites have been identified in the P. aeruginosa PAO1 genome, four localized in close proximity of oriC and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed P. aeruginosa mutants deprived of each single parS sequence and their different combinations. The analysis included evaluation of a set of phenotypic features, chromosome segregation, and ParB localization in the cells. It was found that ParB binds specifically to all ten parS sites, although with different affinities. The P. aeruginosa parS mutant with all ten parS sites modified (parSnull) is viable however it demonstrates the phenotype characteristic for parAnull or parBnull mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of parS determine its role in P. aeruginosa biology. It transpired that any one of the four parS sites proximal to oriC (parS1 to parS4), which are bound by ParB with the highest affinity, is necessary and sufficient for the parABS role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the parSnull phenotype, which indicates that none of the remaining six parS sites can substitute for these four oriC-proximal sites in this function. A single ectopic parS2 (inserted opposite oriC in the parSnull mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation. PMID:25794281

  19. Pseudomonas aeruginosa Cell Membrane Protein Expression from Phenotypically Diverse Cystic Fibrosis Isolates Demonstrates Host-Specific Adaptations.

    PubMed

    Kamath, Karthik Shantharam; Pascovici, Dana; Penesyan, Anahit; Goel, Apurv; Venkatakrishnan, Vignesh; Paulsen, Ian T; Packer, Nicolle H; Molloy, Mark P

    2016-07-01

    Pseudomonas aeruginosa is a Gram-negative, nosocomial, highly adaptable opportunistic pathogen especially prevalent in immuno-compromised cystic fibrosis (CF) patients. The bacterial cell surface proteins are important contributors to virulence, yet the membrane subproteomes of phenotypically diverse P. aeruginosa strains are poorly characterized. We carried out mass spectrometry (MS)-based proteome analysis of the membrane proteins of three novel P. aeruginosa strains isolated from the sputum of CF patients and compared protein expression to the widely used laboratory strain, PAO1. Microbes were grown in planktonic growth condition using minimal M9 media, and a defined synthetic lung nutrient mimicking medium (SCFM) limited passaging. Two-dimensional LC-MS/MS using iTRAQ labeling enabled quantitative comparisons among 3171 and 2442 proteins from the minimal M9 medium and in the SCFM, respectively. The CF isolates showed marked differences in membrane protein expression in comparison with PAO1 including up-regulation of drug resistance proteins (MexY, MexB, MexC) and down-regulation of chemotaxis and aerotaxis proteins (PA1561, PctA, PctB) and motility and adhesion proteins (FliK, FlgE, FliD, PilJ). Phenotypic analysis using adhesion, motility, and drug susceptibility assays confirmed the proteomics findings. These results provide evidence of host-specific microevolution of P. aeruginosa in the CF lung and shed light on the adaptation strategies used by CF pathogens. PMID:27246823

  20. Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody

    PubMed Central

    Behrouz, Bahador; Amirmozafari, Nour; Khoramabadi, Nima; Bahroudi, Mahboobeh; Legaee, Parisa; Mahdavi, Mehdi

    2016-01-01

    Background Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromised hosts. The single polar flagellum is an important factor in both virulence and colonization. Objectives As flagellin is the major component of the flagellar filament, the main aims of the present study are to identify, clone, express, and purify the recombinant type B flagellin (r-B-flagellin) of P. aeruginosa, as well as to evaluate the functional activity of the rabbit polyclonal antibody raised against this r-B-flagellin. Materials and Methods In the current experimental study, the r-B-flagellin gene was isolated from the P. aeruginosa PAO1 strain by PCR. It was cloned into the pET-28a vector and then transformed into the E. coli BL21 strain. Next, r-B-flagellin was overexpressed and affinity purified by Ni-NTA agarose-affinity chromatography, followed by on-column resolubilization. Polyclonal antisera against the recombinant flagellin were raised in rabbits, and the functional activity of the anti-r-B-flagellin antibody was determined by in vitro assays. Results The polyclonal antibodies raised against this r-B-flagellin inhibited the motility of the homologous PAO1 strain of P. aeruginosa, which significantly decreased the invasion of the PAO1 strain into the A549 cells and also enhanced the opsonophagocytosis of this strain. However, our polyclonal antibody showed little effect on the heterologous PAK strain. Conclusions The r-B-flagellin carried antigenic epitopes just like the native flagellin, while the polyclonal antibody raised against it exhibited functional activity. PMID:27621933

  1. Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa.

    PubMed

    Serino, L; Reimmann, C; Baur, H; Beyeler, M; Visca, P; Haas, D

    1995-11-15

    Salicylate is a precursor of pyochelin in Pseudomonas aeruginosa and both compounds display siderophore activity. To elucidate the salicylate biosynthetic pathway, we have cloned and sequenced a chromosomal region of P. aeruginosa PAO1 containing two adjacent genes, designated pchB and pchA, which are necessary for salicylate formation. The pchA gene encodes a protein of 52 kDa with extensive similarity to the chorismate-utilizing enzymes isochorismate synthase, anthranilate synthase (component I) and p-aminobenzoate synthase (component I), whereas the 11 kDa protein encoded by pchB does not show significant similarity with other proteins. The pchB stop codon overlaps the presumed pchA start codon. Expression of the pchA gene in P. aeruginosa appears to depend on the transcription and translation of the upstream pchB gene. The pchBA genes are the first salicylate biosynthetic genes to be reported. Salicylate formation was demonstrated in an Escherichia coli entC mutant lacking isochorismate synthase when this strain expressed both the pchBA genes, but not when it expressed pchB alone. By contrast, an entB mutant of E. coli blocked in the conversion of isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate formed salicylate when transformed with a pchB expression construct. Salicylate formation could also be demonstrated in vitro when chorismate was incubated with a crude extract of P. aeruginosa containing overproduced PchA and PchB proteins; salicylate and pyruvate were formed in equimolar amounts. Furthermore, salicylate-forming activity could be detected in extracts from a P. aeruginosa pyoverdin-negative mutant when grown under iron limitation, but not with iron excess. Our results are consistent with a pathway leading from chorismate to isochorismate and then to salicylate plus pyruvate, catalyzed consecutively by the iron-repressible PchA and PchB proteins in P. aeruginosa. PMID:7500944

  2. Quantification of Pseudomonas aeruginosa hydrogen cyanide production by a polarographic approach.

    PubMed

    Blier, Anne-Sophie; Vieillard, Julien; Gerault, Eloïse; Dagorn, Audrey; Varacavoudin, Tony; Le Derf, Franck; Orange, Nicole; Feuilloley, Marc; Lesouhaitier, Olivier

    2012-07-01

    Pseudomonas aeruginosa is an opportunistic pathogen responsible for numerous infections acquired in hospital especially in persons whose immune systems are weakened, such as with patient suffering from AIDS or cystic fibrosis. This bacterium produces a great diversity of virulence factors among them hydrogen cyanide (HCN) which is one of the most potent and toxic. A precise quantification of HCN or CN(-) ion is essential to understand the involvement of this toxin in the pathogenesis of P. aeruginosa. In the present study, we present a new technique based on a polarographic approach to measure the production kinetics of HCN/CN(-) by P. aeruginosa strains, in several media commonly used in microbiology labs. The method was validated using mutants (hcnB- and hcnC-) which are unable to produce detectable HCN/CN(-). The kinetics of HCN/CN(-) production by P. aeruginosa in Luria Bertani (LB) medium showed a parabolic shape with a peak observed at 4, 5 and 8h for strains PA14, PAO1 and MPAO1, respectively. When bacteria were grown in ordinary nutrient broth (ONB) 2.5% medium, a less adapted medium for bacterial growth, the general profile of the kinetics was conserved but peak production was delayed (10 and 12h for PAO1 and MPAO1, respectively). When the bacteria were cultured in minimum medium MMC, bacterial growth was particularly slow and HCN/CN(-) production was markedly reduced. Taken together, this new polarographic method appears as a useful technique to detect and quantify HCN/CN(-) in routine media where the bacteria can express and regulate high amounts of toxins. With this method, we demonstrate that HCN/CN(-) production by P. aeruginosa is maximal at the end of the exponential growth phase and depends on the richness of the growth medium used. PMID:22537820

  3. Potential effects of erythromycin on host defense systems and virulence of Pseudomonas aeruginosa.

    PubMed Central

    Hirakata, Y; Kaku, M; Mizukane, R; Ishida, K; Furuya, N; Matsumoto, T; Tateda, K; Yamaguchi, K

    1992-01-01

    We evaluated several potential effects of erythromycin (EM) on host defense systems and the virulence of Pseudomonas aeruginosa. Peritoneal macrophages obtained from mice given 250 mg of EM per kg of body weight for 7 days by the intraperitoneal, intravenous, subcutaneous, or oral route produced significantly greater amounts of thymocyte-activating factors. These data suggest that EM enhances the in vivo production of cytokines, such as interleukins 1 and 6. Treatment of P. aeruginosa D4 with subinhibitory concentrations of EM enhanced the association of bacteria with murine Kupffer cells in vitro and increased bacterial clearance from the blood in mice. EM suppressed the in vitro production of exotoxin A, total protease, elastase, and phospholipase C by P. aeruginosa D4; exotoxin A production by P. aeruginosa PA-103; and total protease production by P. aeruginosa B16 and PAO1 in a generally dose-dependent manner. These data demonstrate that EM produces various effects in addition to its direct antimicrobial activity, suggesting that it has potential as an immunomodulator or bacterial virulence-suppressing agent against P. aeruginosa and other infections. PMID:1416882

  4. Analysis of the periplasmic proteome of Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen.

    PubMed

    Imperi, Francesco; Ciccosanti, Fabiola; Perdomo, Ariel Basulto; Tiburzi, Federica; Mancone, Carmine; Alonzi, Tonino; Ascenzi, Paolo; Piacentini, Mauro; Visca, Paolo; Fimia, Gian Maria

    2009-04-01

    The Gram-negative bacterium Pseudomonas aeruginosa is a main cause of infection in hospitalized, burned, immunocompromised, and cystic fibrosis patients. Many processes essential for P. aeruginosa pathogenesis, e.g., nutrient uptake, antibiotic resistance, and virulence, take place in the cell envelope and depend on components residing in the periplasmic space. Recent high-throughput studies focused on P. aeruginosa membrane compartments. However, the composition and dynamics of its periplasm remain largely uncharacterized. Here, we report a detailed description of the periplasmic proteome of the wild-type P. aeruginosa strain PAO1 by 2-DE and MALDI-TOF/TOF analysis. Three extraction methods were compared at proteome level in order to achieve the most reliable and comprehensive periplasmic protein map. A total of 495 spots representing 395 different proteins were identified. Most of the high intensity spots corresponded to periplasmic proteins, while cytoplasmic contaminants were mainly detected among faint spots. The majority of the identified periplasmic proteins is involved in transport, cell-envelope integrity, and protein folding control. Notably, more than 30% still has an unpredicted function. This work provides the first overview of the P. aeruginosa periplasm and offers the basis for future studies on periplasmic proteome changes occurring during P. aeruginosa adaptation to different environments and/or antibiotic treatments. PMID:19333994

  5. Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa.

    PubMed

    Yang, Liang; Liu, Yang; Markussen, Trine; Høiby, Niels; Tolker-Nielsen, Tim; Molin, Søren

    2011-08-01

    Biofilm infections may not simply be the result of colonization by one bacterium, but rather the consequence of pathogenic contributions from several bacteria. Interspecies interactions of different organisms in mixed-species biofilms remain largely unexplained, but knowledge of these is very important for understanding of biofilm physiology and the treatment of biofilm-related infectious diseases. Here, we have investigated interactions of two of the major bacterial species of cystic fibrosis lung microbial communities -Pseudomonas aeruginosa and Staphylococcus aureus- when grown in co-culture biofilms. By growing co-culture biofilms of S. aureus with P. aeruginosa mutants in a flow-chamber system and observing them using confocal laser scanning microscopy, we show that wild-type P. aeruginosa PAO1 facilitates S. aureus microcolony formation. In contrast, P. aeruginosa mucA and rpoN mutants do not facilitate S. aureus microcolony formation and tend to outcompete S. aureus in co-culture biofilms. Further investigations reveal that extracellular DNA (eDNA) plays an important role in S. aureus microcolony formation and that P. aeruginosa type IV pili are required for this process, probably through their ability to bind to eDNA. Furthermore, P. aeruginosa is able to protect S. aureus against Dictyostelium discoideum phagocytosis in co-culture biofilms. PMID:21595754

  6. Gene expression and physiological role of Pseudomonas aeruginosa methionine sulfoxide reductases during oxidative stress.

    PubMed

    Romsang, Adisak; Atichartpongkul, Sopapan; Trinachartvanit, Wachareeporn; Vattanaviboon, Paiboon; Mongkolsuk, Skorn

    2013-08-01

    Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H2O2. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive. PMID:23687271

  7. Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulence.

    PubMed

    Lee, Jintae; Attila, Can; Cirillo, Suat L G; Cirillo, Jeffrey D; Wood, Thomas K

    2009-01-01

    Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7-hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)-regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI-opmD multidrug efflux pump and genes involved in the synthesis of QS-regulated virulence factors including pyocyanin (phz operon), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole-related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa. PMID:21261883

  8. High-Resolution Analysis by Whole-Genome Sequencing of an International Lineage (Sequence Type 111) of Pseudomonas aeruginosa Associated with Metallo-Carbapenemases in the United Kingdom

    PubMed Central

    Wright, Laura; Underwood, Anthony; Witney, Adam A.; Chan, Yuen-Ting; Al-Shahib, Ali; Arnold, Catherine; Doumith, Michel; Patel, Bharat; Planche, Timothy D.; Green, Jonathan; Holliman, Richard; Woodford, Neil

    2015-01-01

    Whole-genome sequencing (WGS) was carried out on 87 isolates of sequence type 111 (ST-111) of Pseudomonas aeruginosa collected between 2005 and 2014 from 65 patients and 12 environmental isolates from 24 hospital laboratories across the United Kingdom on an Illumina HiSeq instrument. Most isolates (73) carried VIM-2, but others carried IMP-1 or IMP-13 (5) or NDM-1 (1); one isolate had VIM-2 and IMP-18, and 7 carried no metallo-beta-lactamase (MBL) gene. Single nucleotide polymorphism analysis divided the isolates into distinct clusters; the NDM-1 isolate was an outlier, and the IMP isolates and 6/7 MBL-negative isolates clustered separately from the main set of 73 VIM-2 isolates. Within the VIM-2 set, there were at least 3 distinct clusters, including a tightly clustered set of isolates from 3 hospital laboratories consistent with an outbreak from a single introduction that was quickly brought under control and a much broader set dominated by isolates from a long-running outbreak in a London hospital likely seeded from an environmental source, requiring different control measures; isolates from 7 other hospital laboratories in London and southeast England were also included. Bayesian evolutionary analysis indicated that all the isolates shared a common ancestor dating back ∼50 years (1960s), with the main VIM-2 set separating approximately 20 to 30 years ago. Accessory gene profiling revealed blocks of genes associated with particular clusters, with some having high similarity (≥95%) to bacteriophage genes. WGS of widely found international lineages such as ST-111 provides the necessary resolution to inform epidemiological investigations and intervention policies. PMID:26041902

  9. Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000

    SciTech Connect

    Feil, Helene; Feil, William; Chain, Patrick S. G.; Larimer, Frank W; DiBartolo, Genevieve; Copeland, A; Lykidis, A; Trong, Stephen; Nolan, Matt; Goltsman, Eugene; Thiel, James; Malfatti, Stephanie; Loper, Joyce E.; Detter, J C; Lapidus, Alla L.; Land, Miriam L; Richardson, P M; Kyrpides, Nikos C; Ivanova, N; Lindow, Steven E.

    2005-01-01

    The complete genomic sequence of Pseudomonas syringae pv. syringae B728a (Pss B728a) has been determined and is compared with that of A syringae pv. tomato DC3000 (Pst DC3000). The two pathovars of this economically important species of plant pathogenic bacteria differ in host range and other interactions with plants, with Pss having a more pronounced epiphytic stage of growth and higher abiotic stress tolerance and Pst DC3000 having a more pronounced apoplastic growth habitat. The Pss B728a genome (6.1 Mb) contains a circular chromosome and no plasmid, whereas the Pst DC3000 genome is 6.5 mbp in size, composed of a circular chromosome and two plasmids. Although a high degree of similarity exists between the two sequenced Pseudomonads, 976 protein-encoding genes are unique to Pss B728a when compared with Pst DC3000, including large genomic islands likely to contribute to virulence and host specificity. Over 375 repetitive extragenic palindromic sequences unique to Pss B728a when compared with Pst DC3000 are widely distributed throughout the chromosome except in 14 genomic islands, which generally had lower GC content than the genome as a whole. Content of the genomic islands varies, with one containing a prophage and another the plasmid pKLC102 of Pseudomonas aeruginosa PAO1. Among the 976 genes of Pss B728a with no counterpart in Pst DC3000 are those encoding for syringopeptin, syringomycin, indole acetic acid biosynthesis, arginine degradation, and production of ice nuclei. The genomic comparison suggests that several unique genes for Pss B728a such as ectoine synthase, DNA repair, and antibiotic production may contribute to the epiphytic fitness and stress tolerance of this organism.

  10. Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000

    SciTech Connect

    Feil, H; Feil, W S; Chain, P; Larimer, F; DiBartolo, G; Copeland, A; Lykidis, A; Trong, S; Nolan, M; Goltsman, E; Thiel, J; Malfatti, S; Loper, J E; Lapidus, A; Detter, J C; Land, M; Richardson, P M; Kyrpides, N C; Ivanova, N; Lindow, S E

    2005-07-14

    The complete genomic sequence of Pseudomonas syringae pathovar syringae B728a (Pss B728a), has been determined and is compared with that of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The two pathovars of this economically important species of plant pathogenic bacteria differ in host range and other interactions with plants, with Pss having a more pronounced epiphytic stage of growth and higher abiotic stress tolerance and Pst DC3000 having a more pronounced apoplastic growth habitat. The Pss B728a genome (6.1 megabases) contains a circular chromosome and no plasmid, whereas the Pst DC3000 genome is 6.5 mbp in size, composed of a circular chromosome and two plasmids. While a high degree of similarity exists between the two sequenced Pseudomonads, 976 protein-encoding genes are unique to Pss B728a when compared to Pst DC3000, including large genomic islands likely to contribute to virulence and host specificity. Over 375 repetitive extragenic palindromic sequences (REPs) unique to Pss B728a when compared to Pst DC3000 are widely distributed throughout the chromosome except in 14 genomic islands, which generally had lower GC content than the genome as a whole. Content of the genomic islands vary, with one containing a prophage and another the plasmid pKLC102 of P. aeruginosa PAO1. Among the 976 genes of Pss B728a with no counterpart in Pst DC3000 are those encoding for syringopeptin (SP), syringomycin (SR), indole acetic acid biosynthesis, arginine degradation, and production of ice nuclei. The genomic comparison suggests that several unique genes for Pss B728a such as ectoine synthase, DNA repair, and antibiotic production may contribute to epiphytic fitness and stress tolerance of this organism.

  11. Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.

    PubMed Central

    Winteler, H V; Schneidinger, B; Jaeger, K E; Haas, D

    1996-01-01

    The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase. PMID:8795231

  12. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa.

    PubMed

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation. PMID:26263486

  13. Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa

    PubMed Central

    Kalia, Manmohit; Yadav, Vivek Kumar; Singh, Pradeep Kumar; Sharma, Deepmala; Pandey, Himanshu; Narvi, Shahid Suhail; Agarwal, Vishnu

    2015-01-01

    Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation. PMID:26263486

  14. C-di-GMP regulates Pseudomonas aeruginosa stress response to tellurite during both planktonic and biofilm modes of growth

    PubMed Central

    Chua, Song Lin; Sivakumar, Krishnakumar; Rybtke, Morten; Yuan, Mingjun; Andersen, Jens Bo; Nielsen, Thomas E.; Givskov, Michael; Tolker-Nielsen, Tim; Cao, Bin; Kjelleberg, Staffan; Yang, Liang

    2015-01-01

    Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO32–) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO32– further increased P. aeruginosa biofilm formation and resistance to TeO32–. P. aeruginosa ΔsadCΔsiaD and PAO1/plac-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO32– exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed to TeO32–. Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth. PMID:25992876

  15. Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

    PubMed Central

    Wei, Qing; Tarighi, Saeed; Dötsch, Andreas; Häussler, Susanne; Müsken, Mathias; Wright, Victoria J.; Cámara, Miguel; Williams, Paul; Haenen, Steven; Boerjan, Bart; Bogaerts, Annelies; Vierstraete, Evy; Verleyen, Peter; Schoofs, Liliane; Willaert, Ronnie; De Groote, Valérie N.; Michiels, Jan; Vercammen, Ken; Crabbé, Aurélie; Cornelis, Pierre

    2011-01-01

    Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal Findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10−5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone

  16. Ferritin and ferrihydrite nanoparticles as iron sources for Pseudomonas aeruginosa

    PubMed Central

    Dehner, Carolyn; Morales-Soto, Nydia; Behera, Rabindra K.; Shrout, Joshua; Theil, Elizabeth C.; Maurice, Patricia A.

    2013-01-01

    Metabolism of iron derived from insoluble and/ or scarce sources is essential for pathogenic and environmental microbes. The ability of Pseudomonas aeruginosa to acquire iron from exogenous ferritin was assessed; ferritin is an iron-concentrating and antioxidant protein complex composed of a catalytic protein and caged ferrihydrite nanomineral synthesized from Fe(II) and O2 or H2O2. Ferritin and free ferrihydrite supported growth of P. aeruginosa with indistinguishable kinetics and final culture densities. The P. aeruginosa PAO1 mutant (ΔpvdDΔpchEF), which is incapable of siderophore production, grew as well as the wild type when ferritin was the iron source. Such data suggest that P. aeruginosa can acquire iron by siderophore-independent mechanisms, including secretion of small-molecule reductant(s). Protease inhibitors abolished the growth of the siderophore-free strain on ferritins, with only a small effect on growth of the wild type; predictably, protease inhibitors had no effect on growth with free ferrihydrite as the iron source. Proteolytic activity was higher with the siderophore-free strain, suggesting that the role of proteases in the degradation of ferritin is particularly important for iron acquisition in the absence of siderophores. The combined results demonstrate the importance of both free ferrihydrite, a natural environmental form of iron and a model for an insoluble form of partly denatured ferritin called hemosiderin, and caged ferritin iron minerals as bacterial iron sources. Ferritin is also revealed as a growth promoter of opportunistic, pathogenic bacteria such a P. aeruginosa in diseased tissues such as the cystic fibrotic lung, where ferritin concentrations are abnormally high. PMID:23417538

  17. Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa.

    PubMed

    Kumar, Lokender; Chhibber, Sanjay; Kumar, Rajnish; Kumar, Manoj; Harjai, Kusum

    2015-04-01

    Quorum sensing in Pseudomonas aeruginosa plays an imperative role in virulence factor, biofilm formation and antimicrobial resistance. Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. Previously, our lab proved zingerone as potent anti-biofilm agent hence; further its anti-virulent and anti-quorum activities were evaluated. Zingerone, besides decreasing swimming, swarming and twitching phenotypes of P. aeruginosa PAO1, reduced biofilm forming capacity and production of virulence factors including rhamnolipid, elastase, protease, pyocyanin, cell free and cell bound hemolysin (p<0.001) indicating anti-virulent property attributing towards attenuation of virulence of P. aeruginosa. Further zingerone not only had marked effect on the production of quorum sensing signal molecules by clinical isolates of P. aeruginosa but also showed significant interference with the activation of QS reporter strains. To study the mechanism of blocking quorum sensing cascade, in silico analysis was carried out. Anti-QS activity was attributed to interference with the ligand receptor interaction of zingerone with QS receptors (TraR, LasR, RhlR and PqsR). Zingerone showed a good comparative docking score to respective autoinducer molecules which was even higher than that of vanillin, a proven anti-quorum sensing phytochemical. The results of the present study revealed the anti-quorum sensing activity of zingerone targeting ligand-receptor interaction, hence proposing zingerone as a suitable anti-virulent drug candidate against P. aeruginosa infections. PMID:25704369

  18. Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia

    PubMed Central

    Lafleur, John; Lepidi, Hubert; Papazian, Laurent; Rolain, Jean-Marc; Raoult, Didier; Elias, Mikael; Silby, Mark W.; Bzdrenga, Janek; Bregeon, Fabienne; Chabriere, Eric

    2014-01-01

    Rationale The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia. Objectives The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia. Methods To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage. Measurements and Primary Results SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality. Conclusion These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use. PMID:25350373

  19. Analysis of the swimming activity of Pseudomonas aeruginosa by using photonic force microscope

    NASA Astrophysics Data System (ADS)

    Chan, Chia-Han; Chang, Bo-Jui; Huang, Ying-Jung; Fan, Chia-Chieh; Peng, Hwei-Ling; Chi, Sien; Hsu, Long

    2005-08-01

    Swimming activity of flagella is a main factor of the motility of bacteria. Flagella expressed on the surface of bacterial species serve as a primary means of motility including swimming. We propose to use optical tweezers to analyze the swimming activity of bacteria. The sample bacteria in the work is Pseudomonas aeruginosa, and it is a gram-negative bacterium and often causes leading to burn wound infections, urinary-tract infections, and pneumonia. The single polar flagellum of P. aeruginosa has been demonstrated to be important virulence and colonization factor of this opportunistic pathogen. We demonstrate a gene to regulate the bacterial swimming activity in P. aeruginosa PAO1 by biological method. However, the change of flagellar morphology was not observed by electron microscopy analysis, suggesting that the gene regulates the flagellar rotation that could not be detected by biological method. PFM exhibits a spatial resolution of a few nanometers to detect the relative position of the probe at an acquisition rate over 1 MHz. By binding a probe such as a bead or a quantum dot on the flagella, we expect the rotation of the probe due to the flagella could be detected. It is expected that the study of the swimming activity of P. aeruginosa provide potent method for the pathogenic role of the flagella in P. aeruginosa.

  20. The ferrichrome receptor A as a new target for Pseudomonas aeruginosa virulence attenuation.

    PubMed

    Lee, Keehoon; Lee, Kang-Mu; Go, Junhyeok; Ryu, Jae-Chan; Ryu, Ji-Hwan; Yoon, Sang Sun

    2016-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ∼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen. PMID:27190289

  1. To Hit or Not to Hit, That Is the Question – Genome-wide Structure-Based Druggability Predictions for Pseudomonas aeruginosa Proteins

    PubMed Central

    Sarkar, Aurijit; Brenk, Ruth

    2015-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium known to cause opportunistic infections in immune-compromised or immunosuppressed individuals that often prove fatal. New drugs to combat this organism are therefore sought after. To this end, we subjected the gene products of predicted perturbative genes to structure-based druggability predictions using DrugPred. Making this approach suitable for large-scale predictions required the introduction of new methods for calculation of descriptors, development of a workflow to identify suitable pockets in homologous proteins and establishment of criteria to obtain valid druggability predictions based on homologs. We were able to identify 29 perturbative proteins of P. aeruginosa that may contain druggable pockets, including some of them with no or no drug-like inhibitors deposited in ChEMBL. These proteins form promising novel targets for drug discovery against P. aeruginosa. PMID:26360059

  2. To Hit or Not to Hit, That Is the Question - Genome-wide Structure-Based Druggability Predictions for Pseudomonas aeruginosa Proteins.

    PubMed

    Sarkar, Aurijit; Brenk, Ruth

    2015-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium known to cause opportunistic infections in immune-compromised or immunosuppressed individuals that often prove fatal. New drugs to combat this organism are therefore sought after. To this end, we subjected the gene products of predicted perturbative genes to structure-based druggability predictions using DrugPred. Making this approach suitable for large-scale predictions required the introduction of new methods for calculation of descriptors, development of a workflow to identify suitable pockets in homologous proteins and establishment of criteria to obtain valid druggability predictions based on homologs. We were able to identify 29 perturbative proteins of P. aeruginosa that may contain druggable pockets, including some of them with no or no drug-like inhibitors deposited in ChEMBL. These proteins form promising novel targets for drug discovery against P. aeruginosa. PMID:26360059

  3. Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa.

    PubMed

    Wilton, Mike; Wong, Megan J Q; Tang, Le; Liang, Xiaoye; Moore, Richard; Parkins, Michael D; Lewenza, Shawn; Dong, Tao G

    2016-08-01

    Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg(2+) or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities. PMID:27271742

  4. LasI/R and RhlI/R Quorum Sensing in a Strain of Pseudomonas aeruginosa Beneficial to Plants▿

    PubMed Central

    Steindler, Laura; Bertani, Iris; De Sordi, Luisa; Schwager, Stephan; Eberl, Leo; Venturi, Vittorio

    2009-01-01

    Pseudomonas aeruginosa possesses three quorum-sensing (QS) systems which are key in the expression of a large number of genes, including many virulence factors. Most studies of QS in P. aeruginosa have been performed in clinical isolates and have therefore focused on its role in pathogenicity. P. aeruginosa, however, is regarded as a ubiquitous organism capable of colonizing many different environments and also of establishing beneficial associations with plants. In this study we examined the role of the two N-acyl homoserine lactone systems known as RhlI/R and LasI/R in the environmental rice rhizosphere isolate P. aeruginosa PUPa3. Both the Rhl and Las systems are involved in the regulation of plant growth-promoting traits. The environmental P. aeruginosa PUPa3 is pathogenic in two nonmammalian infection models, and only the double las rhl mutants are attenuated for virulence. In fact it was established that the two QS systems are not hierarchically organized and that they are both important for the colonization of the rice rhizosphere. This is an in-depth genetic and molecular study of QS in an environmental P. aeruginosa strain and highlights several differences with QS regulation in the clinical isolate PAO1. PMID:19525275

  5. Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis.

    PubMed

    Scoffield, Jessica A; Wu, Hui

    2016-02-01

    Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis. PMID:26673783

  6. Mechanical Properties of Type IV Pili in P. Aeruginosa

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Touhami, Ahmed; Scheurwater, Edie; Harvey, Hanjeong; Burrows, Lori; Dutcher, John

    2009-03-01

    Type IV pili (Tfp) are thin flexible protein filaments that extend from the cell membrane of bacteria such as Pseudomonas aeruginosa and Neisseria gonorrhoeae. The mechanical properties of Tfp are of great importance since they allow bacteria to interact with and colonize various surfaces. In the present study, we have used atomic force microscopy (AFM) for both imaging and pulling on Tfp from P. aeruginosa (PAO1) and from its PilA, PilT, and FliC mutants. A single pilus filament was mechanically stretched and the resulting force-extension profiles were fitted using the worm-like-chain (WLC) model. The statistical distributions obtained for contour length, persistence length, and number of pili per bacteria pole, were used to evaluate the mechanical properties of a single pilus and the biogenesis functions of different proteins (PilA, PilT) involved in its assembly and disassembly. Importantly, the persistence length value of ˜ 1 μm measured in the present study, which is consistent with the curvature of the pili observed in our AFM images, is significantly lower than the value of 5 μm reported earlier by Skerker et al. (1). Our results shed new light on the role of mechanical forces that mediate bacteria-surface interactions and biofilm formation. 1- J.M. Skerker and H.C. Berg, Proc. Natl. Acad. Sci. USA, 98, 6901-6904 (2001).

  7. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

    PubMed Central

    Hentzer, Morten; Wu, Hong; Andersen, Jens Bo; Riedel, Kathrin; Rasmussen, Thomas B.; Bagge, Niels; Kumar, Naresh; Schembri, Mark A.; Song, Zhijun; Kristoffersen, Peter; Manefield, Mike; Costerton, John W.; Molin, Søren; Eberl, Leo; Steinberg, Peter; Kjelleberg, Staffan; Høiby, Niels; Givskov, Michael

    2003-01-01

    Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip® microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response. PMID:12881415

  8. Human Host Defense Peptide LL-37 Stimulates Virulence Factor Production and Adaptive Resistance in Pseudomonas aeruginosa

    PubMed Central

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor. PMID:24349231

  9. Human host defense peptide LL-37 stimulates virulence factor production and adaptive resistance in Pseudomonas aeruginosa.

    PubMed

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor. PMID:24349231

  10. Glucose Starvation-Induced Dispersal of Pseudomonas aeruginosa Biofilms Is cAMP and Energy Dependent

    PubMed Central

    Huynh, Tran T.; McDougald, Diane; Klebensberger, Janosch; Al Qarni, Budoor; Barraud, Nicolas; Rice, Scott A.; Kjelleberg, Staffan; Schleheck, David

    2012-01-01

    Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ). In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate) and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA). In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s) and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival. PMID:22905180

  11. The D3 bacteriophage α-polymerase inhibitor (Iap) peptide disrupts O-antigen biosynthesis through mimicry of the chain length regulator Wzz in Pseudomonas aeruginosa.

    PubMed

    Taylor, Véronique L; Udaskin, Molly L; Islam, Salim T; Lam, Joseph S

    2013-10-01

    Lysogenic bacteriophage D3 causes seroconversion of Pseudomonas aeruginosa PAO1 from serotype O5 to O16 by inverting the linkage between O-specific antigen (OSA) repeat units from α to β. The OSA units are polymerized by Wzy to modal lengths regulated by Wzz1 and Wzz2. A key component of the D3 seroconversion machinery is the inhibitor of α-polymerase (Iap) peptide, which is able to solely suppress α-linked long-chain OSA production in P. aeruginosa PAO1. To establish the target specificity of Iap for Wzyα, changes in OSA phenotypes were examined via Western immunoblotting for wzz1 and wzz2 single-knockout strains, as well as a wzz1 wzz2 double knockout, following the expression of iap from a tuneable vector. Increased induction of Iap expression completely abrogated OSA production in the wzz1 wzz2 double mutant, while background levels of OSA production were still observed in either of the single mutants. Therefore, Iap inhibition of OSA biosynthesis was most effective in the absence of both Wzz proteins. Sequence alignment analyses revealed a high degree of similarity between Iap and the first transmembrane segment (TMS) of either Wzz1 or Wzz2. Various topology prediction analyses of the Iap sequence consistently predicted the presence of a single TMS, suggesting a propensity for Iap to insert itself into the inner membrane (IM). The compromised ability of Iap to abrogate Wzyα function in the presence of Wzz1 or Wzz2 provides compelling evidence that inhibition occurs after Wzyα inserts itself into the IM and is achieved through mimicry of the first TMS from the Wzz proteins of P. aeruginosa PAO1. PMID:23955007

  12. Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection.

    PubMed

    Minandri, Fabrizia; Imperi, Francesco; Frangipani, Emanuela; Bonchi, Carlo; Visaggio, Daniela; Facchini, Marcella; Pasquali, Paolo; Bragonzi, Alessandra; Visca, Paolo

    2016-08-01

    Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe(3+) uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe(2+) acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe(3+) transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities. PMID:27271740

  13. Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

    PubMed

    Kasper, Stephen H; Bonocora, Richard P; Wade, Joseph T; Musah, Rabi Ann; Cady, Nathaniel C

    2016-04-15

    The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy. PMID:26785289

  14. Effects of Quorum Sensing Systems on Regulatory T Cells in Catheter-Related Pseudomonas aeruginosa Biofilm Infection Rat Models

    PubMed Central

    Feng, Lei; Xiang, Qingqing; Ai, Qing; Wang, Zhengli; Zhang, Yunhui; Lu, Qi

    2016-01-01

    Background. Quorum sensing (QS) systems play an important role in modulating biofilm formation. Recent studies have found that the QS molecules had complex effects on the host immune systems. In addition, regulatory T cells (Tregs), known as important negative regulators in the immune system, have been found upregulated in multiple chronic infections. Therefore, the QS systems were hypothesized to be involved in modulating Tregs in biofilm-associated infections. Object. To explore the effects of QS systems on Tregs in catheter-related Pseudomonas aeruginosa biofilm infection rat models. Method. Catheter-related Pseudomonas aeruginosa biofilm infection rat models were established; the bacterial clearance rates, total cell counts in bronchoalveolar lavage (BAL) fluid, pathological changes of lungs, and the levels of Foxp3, TGF-β1, and IL-10 in PAO1 strain group were examined and compared with the QS-mutant ΔlasRΔrhlR and ΔlasIΔrhlI groups. Results. In PAO1 group, the bacterial clearance rates were lower, total cell counts were higher, pathological changes were severer, and the levels of Foxp3, TGF-β1, and IL-10 were significantly higher compared with QS-mutant groups (p < 0.05). No significant difference was observed between the two QS-mutant groups (p > 0.05). Conclusion. QS systems can trigger host immune system, accompanied with the upregulation of Tregs. PMID:27069314

  15. Trigonella foenum-graceum (Seed) Extract Interferes with Quorum Sensing Regulated Traits and Biofilm Formation in the Strains of Pseudomonas aeruginosa and Aeromonas hydrophila

    PubMed Central

    Husain, Fohad Mabood; Ahmad, Iqbal; Khan, Mohd Shahnawaz; Al-Shabib, Nasser Abdulatif

    2015-01-01

    Trigonella foenum-graecum L. (Fenugreek) is an important plant of the Leguminosae family known to have medicinal properties. However, fraction based antiquorum sensing and antibiofilm activities have not been reported from this plant. In the present study T. foenum-graecum seed extract was sequentially fractionated and sub-MICs were tested for above activities. The methanol fraction of the extract demonstrated significant inhibition of AHL regulated virulence factors: protease, LasB elastase, pyocyanin production, chitinase, EPS, and swarming motility in Pseudomonas aeruginosa PAO1 and PAF79. Further, QS dependent virulence factor in the aquatic pathogen Aeromonas hydrophila WAF38 was also reduced. Application of T. foenum-graecum seed extract to PAO1, PAF79, and WAF38 decreased the biofilm forming abilities of the pathogens by significant levels. The extract also exhibited reduced AHL levels and subsequent downregulation of lasB gene. In vivo study showed an enhanced survival of PAO1-preinfected C. elegans after treatment with extract at 1 mg/mL. Further, the major compound detected by GC-MS, caffeine, reduced the production of QS regulated virulence factors and biofilm at 200 µg/mL concentration indicating its role in the activity of the methanol extract. The results of the present study reveal the potential anti-QS and antibiofilm property of T. foenum-graceum extract and caffeine. PMID:26000026

  16. Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms.

    PubMed

    Pezzoni, Magdalena; Pizarro, Ramón A; Costa, Cristina S

    2014-02-01

    One of the more stressful factors that Pseudomonas aeruginosa must face in nature is solar UVA radiation. In this study, the protective role of KatA catalase in both planktonic cells and biofilms of P. aeruginosa against UVA radiation was determined by using the wild-type (PAO1) and an isogenic catalase deficient strain (katA). The katA strain was more sensitive than the wild-type, especially in the case of biofilms. Moreover, the wild-type biofilm was more resistant than its planktonic counterpart, but this was not observed in the katA strain. Striking KatA activity was detected in the matrix of katA(+) strains, and to our knowledge, this is the first report of this activity in the matrix of P. aeruginosa biofilms. Provision of bovine catalase or KatA to the matrix of a katA biofilm significantly increased its UVA tolerance, demonstrating that extracellular KatA is essential to optimal defense against UVA in P. aeruginosa biofilms. Efficiency of photocatalytic treatments using TiO2 and UVA was lower in biofilms than in planktonic cells, but KatA and KatB catalases seem not to be responsible for the higher resistance of the sessile cells to this treatment. PMID:24491420

  17. Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.

    PubMed

    Kolpen, Mette; Mousavi, Nabi; Sams, Thomas; Bjarnsholt, Thomas; Ciofu, Oana; Moser, Claus; Kühl, Michael; Høiby, Niels; Jensen, Peter Østrup

    2016-02-01

    Chronic Pseudomonas aeruginosa lung infection is the most severe complication in cystic fibrosis patients. It is characterised by antibiotic-tolerant biofilms in the endobronchial mucus with zones of oxygen (O2) depletion mainly due to polymorphonuclear leucocyte activity. Whilst the exact mechanisms affecting antibiotic effectiveness on biofilms remain unclear, accumulating evidence suggests that the efficacy of several bactericidal antibiotics such as ciprofloxacin is enhanced by stimulation of the aerobic respiration of pathogens, and that lack of O2 increases their tolerance. Reoxygenation of O2-depleted biofilms may thus improve susceptibility to ciprofloxacin possibly by restoring aerobic respiration. We tested such a strategy using reoxygenation of O2-depleted P. aeruginosa strain PAO1 agarose-embedded biofilms by hyperbaric oxygen treatment (HBOT) (100% O2, 2.8bar), enhancing the diffusive supply for aerobic respiration during ciprofloxacin treatment. This proof-of-principle study demonstrates that biofilm reoxygenation by HBOT can significantly enhance the bactericidal activity of ciprofloxacin on P. aeruginosa. Combining ciprofloxacin treatment with HBOT thus clearly has potential to improve the treatment of P. aeruginosa biofilm infections. PMID:26774522

  18. Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility

    PubMed Central

    Guragain, Manita; Lenaburg, Dirk L.; Moore, Frank S.; Reutlinger, Ian; Patrauchan, Marianna A.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen causing severe acute and chronic infections. Earlier we have shown that calcium (Ca2+) induces P. aeruginosa biofilm formation and production of virulence factors. To enable further studies of the regulatory role of Ca2+, we characterized Ca2+ homeostasis in P. aeruginosa PAO1 cells. By using Ca2+-binding photoprotein aequorin, we determined that the concentration of free intracellular Ca2+ ([Ca2+]in) is 0.14±0.05 μM. In response to external Ca2+, the [Ca2+]in quickly increased at least 13 fold followed by a multi-phase decline by up to 73%. Growth at elevated Ca2+ modulated this response. Treatment with inhibitors known to affect Ca2+ channels, monovalent cations gradient, or P-type and F-type ATPases impaired [Ca2+]in response, suggesting the importance of the corresponding mechanisms in Ca2+ homeostasis. To identify Ca2+ transporters maintaining this homeostasis, bioinformatic and LC-MS/MS-based membrane proteomic analyses were used. [Ca2+]in homeostasis was monitored for seven Ca2+-affected and eleven bioinformatically predicted transporters by using transposon insertion mutants. Disruption of P-type ATPases PA2435, PA3920, and ion exchanger PA2092 significantly impaired Ca2+ homeostasis. The lack of PA3920 and vanadate treatment abolished Ca2+- induced swarming, suggesting the role of the P-type ATPase in regulating P. aeruginosa response to Ca2+. PMID:24074964

  19. N2-Succinylated intermediates in an arginine catabolic pathway of Pseudomonas aeruginosa

    PubMed Central

    Jann, Alfred; Stalon, Victor; Wauven, Corinne Vander; Leisinger, Thomas; Haas, Dieter

    1986-01-01

    Arginine-nonutilizing (aru) mutants of Pseudomonas aeruginosa strain PAO converted L-arginine to N2-succinylarginine or N-succinylglutamate, which were identified by high-voltage electrophoresis and HPLC. Addition of aminooxyacetate, an inhibitor of pyridoxal phosphate-dependent enzymes, to resting cells of the wild-type PAO1 in arginine medium led to the accumulation of N2-succinylornithine. Enzyme assays with crude P. aeruginosa extracts established the following pathway: L-arginine + succinyl-CoA → N2-succinylarginine → N2-succinylornithine → N_succinylglutamate 5-semialdehyde → N-succinylglutamate → succinate + glutamate. Succinyl-CoA may be regenerated from glutamate via 2-ketoglutarate. L-Arginine induced the enzymes of the pathway, and succinate caused catabolite repression. Purified N2-acetylornithine 5-aminotransferase (N2-acetyl-L-ornithine: 2-oxoglutarate aminotransferase, EC 2.6.1.11), an arginine biosynthetic enzyme, efficiently transaminated N2-succinylornithine; this explains the enzyme's dual role in arginine biosynthesis and catabolism. The succinylarginine pathway enables P. aeruginosa to utilize arginine efficiently as a carbon source under aerobic conditions, whereas the other three arginine catabolic pathways previously established in P. aeruginosa fulfill different functions. Images PMID:16593724

  20. Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites.

    PubMed

    Lassek, Christian; Berger, Antje; Zühlke, Daniela; Wittmann, Christoph; Riedel, Katharina

    2016-05-01

    Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites. To investigate the expression rates of P. aeruginosa proteins commonly expressed by different clinical isolates, absolute protein quantities were determined employing a gel-free and data-independent LC-IMS(E) approach. Moreover, the metabolic diversity of these isolates was investigated by (13) C-metabolic flux analyses. 812 proteins were reproducibly identified and absolutely quantified for the reference strain P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all isolates. Whilst the majority of these proteins were expressed in constant amounts, expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in burned wounds isolates compared to lung/urinary tract isolates. Moreover, proteins involved in iron/amino acids uptake were found to be highly abundant in urinary tract isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated proteome/fluxome analysis did not indicate straightforward correlation between the protein amount and flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments. All MS data have been deposited in the ProteomeXchange with identifier PXD002373 (http://proteomecentral.proteomexchange.org/dataset/PXD002373). PMID:26959854

  1. Hydnophytum formicarum Jack ethanol extract modulates quorum sensing-controlled pathogenicity in Pseudomonas aeruginosa.

    PubMed

    Hertiani, Triana; Pratiwi, Sylvia Utami Tunjung

    2015-09-01

    The discovery of new mechanism to control microbial pathogenicity by quorum sensing modulation has generated the search for quorum sensing inhibitor from natural resources. The objective of this research was to evaluate the ability of Hydnophytum formicarum Jack (Rubiaceae) ethanol extract to antagonize cell-to cell communication. Pulverized H. formicarum tuber was macerated in ethyl alcohol 96% and evaporated to yield ethanol extract. A dillution technique using Luria-Bertani (LB) medium was used to observe the capability of the extract to reduce the violacein production in Chromobacterium violaceum. Samples in two-fold dilution were prepared to obtain 2 - 0.0625 mg/mL concentration. The effects on swimming, swarming and twitching motility as well as the formation of biofilm towards Pseudomonas aeruginosa PAO1 were recorded over control. All experiments were done in triplicate. The architecture of Ps. aeruginosa biofilm treated with samples was examined by CLSM (Confocal Laser Scanning Microscopy) . Our results suggested that the ethanol extract of H. formicarum caused violacein production inhibition. Furthermore, inhibition of Ps. aeruginosa motility and biofilm formation were recorded to be significant over control in a concentration dependent manner. H. formicarum serves as a potential source for new QS-based antibacterial drugs towards Ps. aeruginosa. PMID:26408889

  2. A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions.

    PubMed

    Alves, Diana R; Perez-Esteban, P; Kot, W; Bean, J E; Arnot, T; Hansen, L H; Enright, Mark C; Jenkins, A Tobias A

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities - biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred. PMID:26347362

  3. Pseudomonas aeruginosa biofilms in disease.

    PubMed

    Mulcahy, Lawrence R; Isabella, Vincent M; Lewis, Kim

    2014-07-01

    Pseudomonas aeruginosa is a ubiquitous organism that is the focus of intense research because of its prominent role in disease. Due to its relatively large genome and flexible metabolic capabilities, this organism exploits numerous environmental niches. It is an opportunistic pathogen that sets upon the human host when the normal immune defenses are disabled. Its deadliness is most apparent in cystic fibrosis patients, but it also is a major problem in burn wounds, chronic wounds, chronic obstructive pulmonary disorder, surface growth on implanted biomaterials, and within hospital surface and water supplies, where it poses a host of threats to vulnerable patients (Peleg and Hooper, N Engl J Med 362:1804-1813, 2010; Breathnach et al., J Hosp Infect 82:19-24, 2012). Once established in the patient, P. aeruginosa can be especially difficult to treat. The genome encodes a host of resistance genes, including multidrug efflux pumps (Poole, J Mol Microbiol Biotechnol 3:255-264, 2001) and enzymes conferring resistance to beta-lactam and aminoglycoside antibotics (Vahdani et al., Annal Burns Fire Disast 25:78-81, 2012), making therapy against this gram-negative pathogen particularly challenging due to the lack of novel antimicrobial therapeutics (Lewis, Nature 485: 439-440, 2012). This challenge is compounded by the ability of P. aeruginosa to grow in a biofilm, which may enhance its ability to cause infections by protecting bacteria from host defenses and chemotherapy. Here, we review recent studies of P. aeruginosa biofilms with a focus on how this unique mode of growth contributes to its ability to cause recalcitrant infections. PMID:24096885

  4. Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.

    PubMed

    Heurlier, Karin; Dénervaud, Valérie; Haenni, Marisa; Guy, Lionel; Krishnapillai, Viji; Haas, Dieter

    2005-07-01

    In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants. PMID:15995202

  5. A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα

    PubMed Central

    Taylor, Véronique L.; Hoage, Jesse F. J.; Thrane, Sandra Wingaard; Huszczynski, Steven M.; Jelsbak, Lars; Lam, Joseph S.

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3′ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272, and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops

  6. Dynamics of Mutator and Antibiotic-Resistant Populations in a Pharmacokinetic/Pharmacodynamic Model of Pseudomonas aeruginosa Biofilm Treatment ▿

    PubMed Central

    Macià, María D.; Pérez, José L.; Molin, Soeren; Oliver, Antonio

    2011-01-01

    Biofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections by Pseudomonas aeruginosa in cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations in P. aeruginosa flow-cell biofilms, using fluorescently tagged PAO1 and PAOMS (mutator [mutS] derivative) strains. Two-day-old biofilms were treated with ciprofloxacin (CIP) for 4 days (t4) at 2 μg/ml, which correlated with the mutant prevention concentration (MPC) and provided an AUC/MIC ratio of 384 that should predict therapeutic success. Biofilms were monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development in P. aeruginosa biofilms. One-step resistant mutants (MexCD-OprJ or MexEF-OprN overexpression) were selected for both strains, while two-step resistant mutants (additional GyrA or GyrB mutation) were readily selected only from the mutator strain. CLSM analysis of competition experiments revealed that PAOMS, even when inoculated at a 0.01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting that the increased antibiotic tolerance driven by the special biofilm physiology and architecture may raise the effective MPC, favoring gradual mutational resistance development, especially in mutator strains. Moreover, the amplification of mutator populations under antibiotic treatment by coselection with resistance mutations is for the first time demonstrated in situ for P. aeruginosa biofilms. PMID:21859941

  7. Accumulation of Pyrimidine Intermediate Orotate Decreases Virulence Factor Production in Pseudomonas aeruginosa.

    PubMed

    Niazy, Abdurahman; Hughes, Lee E

    2015-08-01

    The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 μM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant's production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate. PMID:25917504

  8. Surface characteristics of Pseudomonas aeruginosa grown in a chamber implant model in mice and rats.

    PubMed Central

    Kelly, N M; Bell, A; Hancock, R E

    1989-01-01

    Pseudomonas aeruginosa PAO1 was grown in vivo in chambers implanted into the peritoneums of mice and rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of bacterial cells taken from the chambers and washed to remove loosely bound host proteins revealed the presence of the major outer membrane proteins D2, E, F, G, and H2. Western immunoblotting with specific antisera confirmed the presence of porin protein F and lipoprotein H2. However, there was no apparent induction of the phosphate starvation-inducible porin P or the divalent cation starvation-inducible protein H1. Small amounts of proteins with molecular weights similar to those of the iron-regulated outer membrane proteins were found in cells grown in vivo; however, their presence could not be confirmed immunologically. The presence of pili and flagella on the cells grown in vivo was demonstrated by electron microscopy and Western immunoblotting. A consistent alteration in the lipopolysaccharide banding pattern was observed after growth in vivo. Compared with cells of strain PAO1 grown in vitro, cells grown in vivo appeared to lack a series of high-molecular-weight O-antigen-containing lipopolysaccharide bands and gained a new series of lower-molecular-weight lipopolysaccharide bands. This alteration in the lipopolysaccharide after growth in vivo did not affect the O-antigen serotype or the resistance of the bacteria to serum. Images PMID:2492257

  9. Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

    PubMed Central

    Purevdorj, B.; Costerton, J. W.; Stoodley, P.

    2002-01-01

    Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies. PMID:12200300

  10. An investigation of Pseudomonas aeruginosa biofilm growth on novel nanocellulose fibre dressings.

    PubMed

    Powell, Lydia C; Khan, Saira; Chinga-Carrasco, Gary; Wright, Chris J; Hill, Katja E; Thomas, David W

    2016-02-10

    Nanocellulose from wood is a novel biomaterial, which is highly fibrillated at the nanoscale. This affords the material a number of advantages, including self-assembly, biodegradability and the ability to absorb and retain moisture, which highlights its potential usefulness in clinical wound-dressing applications. In these in vitro studies, the wound pathogen Pseudomonas aeruginosa PAO1 was used to assess the ability of two nanocellulose materials to impair bacterial growth (<48 h). The two nanocelluloses had a relatively small fraction of residual fibres (<4%) and thus a large fraction of nanofibrils (widths <20 nm). Scanning electron microscopy and confocal laser scanning microscopy imaging demonstrated impaired biofilm growth on the nanocellulose films and increased cell death when compared to a commercial control wound dressing, Aquacel(®). Nanocellulose suspensions inhibited bacterial growth, whilst UV-vis spectrophotometry and laser profilometry also revealed the ability of nanocellulose to form smooth, translucent films. Atomic force microscopy studies of the surface properties of nanocellulose demonstrated that PAO1 exhibited markedly contrasting morphology when grown on the nanocellulose film surfaces compared to an Aquacel(®) control dressing (p<0.05). This study highlights the potential utility of these biodegradable materials, from a renewable source, for wound dressing applications in the prevention and treatment of biofilm development. PMID:26686120

  11. Pseudomonas aeruginosa Cytotoxicity Is Attenuated at High Cell Density and Associated with the Accumulation of Phenylacetic Acid

    PubMed Central

    Wang, Jianhe; Dong, Yihu; Zhou, Tielin; Liu, Xiaoling; Deng, Yinyue; Wang, Chao; Lee, Jasmine; Zhang, Lian-Hui

    2013-01-01

    Background P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. Methodology/Findings Intriguingly, however, in this study we noticed that while a low cell density inoculum of P. aeruginosa caused severe cytotoxicity against human lung tissue cell line A549, increasing the cell density of bacterial inoculum led to decreased cytotoxicity. Addition of the supernatants from high density bacterial culture to low cell density inoculum protected the human cells from bacterial cytotoxic damage, suggesting that P. aeruginosa may produce and accumulate an inhibitory molecule(s) counteracting its pathogenic infection. The inhibitor was purified from the stationary-phase culture supernatants of P. aeruginosa strain PAO1 using bioassay-guided high performance liquid chromatography (HPLC), and characterized to be phenylacetic acid (PAA) by mass spectrometry and nuclear magnetic resonance spectroscopy. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of Type III secretion system (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Conclusions Identification of bacterial metabolite PAA as a T3SS-specific inhibitor explains this intriguing inverse cell-density-dependent-cytotoxicity phenomenon as T3SS is known to be a key virulence factor associated with cytotoxicity and acute infection. The findings may provide useful clues for design and development of new strategies to combat this formidable bacterial pathogen. PMID:23555919

  12. Surface action of gentamicin on Pseudomonas aeruginosa.

    PubMed Central

    Kadurugamuwa, J L; Clarke, A J; Beveridge, T J

    1993-01-01

    The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial protein synthesis alone appears to be insufficient to entirely explain the bactericidal effects. Bacteriolysis is also mediated through perturbation of the cell surface by gentamicin (J.L. Kadurugamuwa, J.S. Lam, and T.J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993). In order to separate the surface effect from protein synthesis in Pseudomonas aeruginosa PAO1, we chemically conjugated bovine serum albumin (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytoplasm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study the surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibiotic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro but not on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold particles were clearly seen on the cell surfaces of whole mounts and thin sections of cells, while the cytoplasm was devoid of such particles. Disruption of the cell envelope was also observed since gentamicin-BSA and gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antibiotic disrupts the packing order of lipopolysaccharide of the outer membrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially lethal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from

  13. Rapid detection of Pseudomonas aeruginosa biomarkers in biological fluids using surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Wu, Xiaomeng; Chen, Jing; Zhao, Yiping; Zughaier, Susu M.

    2014-05-01

    Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes major infection not only in Cystic Fibrosis patients but also in chronic obstructive pulmonary disease and in critically ill patients in intensive care units. Successful antibiotic treatment of the infection relies on accurate and rapid identification of the infectious agents. Conventional microbiological detection methods usually take more than 3 days to obtain accurate results. We have developed a rapid diagnostic technique based on surface-enhanced Raman scattering to directly identify PA from biological fluids. P. aeruginosa strains, PAO1 and PA14, are cultured in lysogeny broth, and the SERS spectra of the broth show the signature Raman peaks from pyocyanin and pyoverdine, two major biomarkers that P. aeruginosa secretes during its growth, as well as lipopolysaccharides. This provides the evidence that the presence of these biomarkers can be used to indicate P. aeruginosa infection. A total of 22 clinical exhaled breath condensates (EBC) samples were obtained from subjects with CF disease and from non-CF healthy donors. SERS spectra of these EBC samples were obtained and further analyzed by both principle component analysis and partial least square-discriminant analysis (PLS-DA). PLS-DA can discriminate the samples with P. aeruginosa infection and the ones without P. aeruginosa infection at 99.3% sensitivity and 99.6% specificity. In addition, this technique can also discriminate samples from subject with CF disease and healthy donor with 97.5% sensitivity and 100% specificity. These results demonstrate the potential of using SERS of EBC samples as a rapid diagnostic tool to detect PA infection.

  14. Screening of Lactobacillus spp. for the prevention of Pseudomonas aeruginosa pulmonary infections

    PubMed Central

    2014-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and cystic fibrosis patients. Its pathogenicity especially relies on the production of virulence factors or resistances to many antibiotics. Since multiplication of antibiotic resistance can lead to therapeutic impasses, it becomes necessary to develop new tools for fighting P. aeruginosa infections. The use of probiotics is one of the ways currently being explored. Probiotics are microorganisms that exert a positive effect on the host’s health and some of them are known to possess antibacterial activities. Since most of their effects have been shown in the digestive tract, experimental data compatible with the respiratory environment are strongly needed. The main goal of this study was then to test the capacity of lactobacilli to inhibit major virulence factors (elastolytic activity and biofilm formation) associated with P. aeruginosa pathogenicity. Results Sixty-seven lactobacilli were isolated from the oral cavities of healthy volunteers. These isolates together with 20 lactobacilli isolated from raw milks, were tested for their capacity to decrease biofilm formation and activity of the elastase produced by P. aeruginosa PAO1. Ten isolates, particularly efficient, were accurately identified using a polyphasic approach (API 50 CHL, mass-spectrometry and 16S/rpoA/pheS genes sequencing) and typed by pulsed-field gel electrophoresis (PFGE). The 8 remaining strains belonging to the L. fermentum (6), L. zeae (1) and L. paracasei (1) species were sensitive to all antibiotics tested with the exception of the intrinsic resistance to vancomycin. The strains were all able to grow in artificial saliva. Conclusion Eight strains belonging to L. fermentum, L. zeae and L. paracasei species harbouring anti-elastase and anti-biofilm properties are potential probiotics for fighting P. aeruginosa pulmonary infections. However, further

  15. Bactericidal effects of antimicrobial agents on epithelial cell-associated Pseudomonas aeruginosa.

    PubMed

    Hirakata, Yoichi; Yano, Hisakazu; Arai, Kazuaki; Kitagawa, Miho; Hatta, Masumitsu; Kunishima, Hiroyuki; Kaku, Mitsuo

    2012-06-01

    It is not clear whether antipseudomonal agents can kill cell-associated bacteria within a short time. Madin-Darby canine kidney (MDCK) and A549 cells were infected with Pseudomonas aeruginosa ATCC 27853 and PAO1 and the bactericidal activity of ceftazidime, imipenem, meropenem, gentamicin, and ciprofloxacin against the organisms was investigated. In both MDCK and A549 cells, β-lactams could not kill epithelial cell-associated bacteria within 2 h. Gentamicin at concentrations ≤32 μg/ml killed more than 99% of epithelial cell-associated bacteria. Ciprofloxacin at 0.5 μg/ml killed more than 99.9% of MDCK cell-associated bacteria. Ciprofloxacin has the strongest and most rapid bactericidal activity against epithelial cell-associated bacteria, which may be explained by the combination of potent in-vitro bactericidal activity and high penetration ability into epithelial cells. PMID:22116462

  16. The effects of nickel(II) complexes with imidazole derivatives on pyocyanin and pyoverdine production by Pseudomonas aeruginosa strains isolated from cystic fibrosis.

    PubMed

    Gałczyńska, Katarzyna; Kurdziel, Krystyna; Adamus-Białek, Wioletta; Wąsik, Sławomir; Szary, Karol; Drabik, Marcin; Węgierek-Ciuk, Aneta; Lankoff, Anna; Arabski, Michał

    2015-01-01

    Pseudomonas aeruginosa infection is problematic in patients with cystic fibrosis (CF). P. aeruginosa secretes a diversity of pigments, such as pyocyanin and pyoverdine. The aim of this study was to evaluate the effects of complexes of nickel(II) ([Ni(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Ni(1-allim)6](NO3)2 (1-allim = 1-allylimidazole) and NiCl2 on pyocyanin and pyoverdine production by 23 strains of P. aeruginosa isolated from cystic fibrosis under growth conditions specific for the CF respiratory system. The antibacterial effects and biophysical properties of the tested substances were measured by spectrofluorometric techniques, as well as by laser interferometry, confocal and atomic force microscopy. The cytotoxic properties of all compounds were measured by Annexin/IP assay against A549 cells. All tested compounds have no effect on pyocyanin production and decrease the pyoverdine secretion in about 40% of tested P. aeruginosa strains at non-cytotoxic range of concentrations. Imidazole-4-acetate anion and 1-allylimidazole have good diffusion properties in the mature P. aeruginosa PAO1 biofilm. In conclusion, the tested nickel(II) complexes do not have clinical implications in P. aeruginosa eradication in cystic fibrosis. The diffusion properties of 1-allylimidazole and imidazole-4-acetate and their lack of effect on A549 cells suggest that they might be considered for chemical synthesis with other transition metals. PMID:26645324

  17. Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia

    PubMed Central

    Schwarzer, Christian; Fu, Zhu; Patanwala, Maria; Hum, Lauren; Lopez-Guzman, Mirielle; Illek, Beate; Kong, Weidong; Lynch, Susan V.; Machen, Terry E.

    2014-01-01

    Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis CF) patients, a process regulated by quorum sensing molecules including N-(3-oxododecanoyl)-L-homoserine lactone, C12. C12 (10–100 μM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CFΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψmito) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca2+ and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 minutes and were complete in 1–2 hrs. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψmito and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψmito and increases in Cacyto like 10–50 μM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed. PMID:22233488

  18. Gamma-aminobutyric acid acts as a specific virulence regulator in Pseudomonas aeruginosa.

    PubMed

    Dagorn, Audrey; Hillion, Mélanie; Chapalain, Annelise; Lesouhaitier, Olivier; Duclairoir Poc, Cécile; Vieillard, Julien; Chevalier, Sylvie; Taupin, Laure; Le Derf, Franck; Feuilloley, Marc G J

    2013-02-01

    Gamma-aminobutyric acid (GABA) is widespread in the environment and can be used by animal and plants as a communication molecule. Pseudomonas species, in particular fluorescent ones, synthesize GABA and express GABA-binding proteins. In this study, we investigated the effects of GABA on the virulence of Pseudomonas aeruginosa. While exposure to GABA (10 µM) did not modify either the growth kinetics or the motility of the bacterium, its cytotoxicity and virulence were strongly increased. The Caenorhabditis elegans 'fast killing test' model revealed that GABA acts essentially through an increase in diffusible toxin(s). GABA also modulates the biofilm formation activity and adhesion properties of PAO1. GABA has no effect on cell surface polarity, biosurfactant secretion or on the lipopolysaccharide structure. The production of several exo-enzymes, pyoverdin and exotoxin A is not modified by GABA but we observed an increase in cyanogenesis which, by itself, could explain the effect of GABA on P. aeruginosa virulence. This mechanism appears to be regulated by quorum sensing. A proteomic analysis revealed that the effect of GABA on cyanogenesis is correlated with a reduction of oxygen accessibility and an over-expression of oxygen-scavenging proteins. GABA also promotes specific changes in the expression of thermostable and unstable elongation factors Tuf/Ts involved in the interaction of the bacterium with the host proteins. Taken together, these results suggest that GABA is a physiological regulator of P. aeruginosa virulence. PMID:23154974

  19. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

    PubMed Central

    Bohn, Yu-Sing Tammy; Brandes, Gudrun; Rakhimova, Elza; Horatzek, Sonja; Salunkhe, Prabhakar; Munder, Antje; van Barneveld, Andrea; Jordan, Doris; Bredenbruch, Florian; Häußler, Susanne; Riedel, Kathrin; Eberl, Leo; Jensen, Peter Østrup; Bjarnsholt, Thomas; Moser, Claus; Hoiby, Niels; Tümmler, Burkhard; Wiehlmann, Lutz

    2008-01-01

    Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population. PMID:19054330

  20. The Importance of the Pseudomonas aeruginosa Type III Secretion System in Epithelium Traversal Depends upon Conditions of Host Susceptibility

    PubMed Central

    Sullivan, Aaron B.; Tam, K. P. Connie; Metruccio, Matteo M. E.; Evans, David J.

    2015-01-01

    Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ∼16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88−/− epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88−/− mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain. PMID:25667266

  1. Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum

    PubMed Central

    Line, Laura; Alhede, Morten; Kolpen, Mette; Kühl, Michael; Ciofu, Oana; Bjarnsholt, Thomas; Moser, Claus; Toyofuku, Masanori; Nomura, Nobuhiko; Høiby, Niels; Jensen, Peter Ø.

    2014-01-01

    Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates of P. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO−3) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO−3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 μM NO−3 and 100 μM NO−3, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO−3. Activation of denitrification could be achieved by supplementation with as little as 62.5 μM of NO−3 according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts, and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO−3 followed by a transient increase of NO−2. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P

  2. Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

    PubMed

    Okamoto, K; Gotoh, N; Tsujimoto, H; Yamada, H; Yoshihara, E; Nakae, T; Nishino, T

    1999-01-01

    We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics. PMID:10338201

  3. The Pseudomonas aeruginosa pfpI gene plays an antimutator role and provides general stress protection.

    PubMed

    Rodríguez-Rojas, Alexandro; Blázquez, Jesús

    2009-02-01

    Hypermutator Pseudomonas aeruginosa strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in P. aeruginosa, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new P. aeruginosa antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described P. aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD, mutT, ung, and mutY, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the P. aeruginosa PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of pfpI resulted in a dramatic increase in the H(2)O(2)-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally, pfpI mutants are more sensitive to different types of stress and are affected in biofilm formation. PMID:19028889

  4. Efficacy of VIP as Treatment for Bacteria-Induced Keratitis Against Multiple Pseudomonas aeruginosa Strains

    PubMed Central

    Carion, Thomas W.; McWhirter, Cody R.; Grewal, Daiyajot K.; Berger, Elizabeth A.

    2015-01-01

    Purpose Previous studies have demonstrated the efficacy of vasoactive intestinal peptide (VIP) treatment in regulating inflammation following bacterial keratitis induced by the P. aeruginosa strain 19660. However, in the current study we assessed whether disease outcome is specific to 19660 or if VIP treatment is effective against multiple P. aeruginosa strains. Methods B6 mice received daily IP injections of VIP from −1 through 5 days post injection (p.i.). Control mice were similarly injected with PBS. Corneal infection was induced using PA 19660, PAO1 or KEI 1025. Disease response was documented and bacterial plate counts and myeloperoxidase assays were performed. Expression of select inflammatory mediators as well as enzymes associated with lipid mediator production was assessed after VIP treatment. KEI 1025 was characterized by cytotoxicity and invasion assays and then confirmed for ExoS/ExoU expression. Results VIP treatment converted the susceptible response to resistant for the three P. aeruginosa strains tested. Disease response was significantly reduced with no corneal perforation. Anti-inflammatory mediators were enhanced after VIP treatment, while pro-inflammatory molecules were reduced compared to controls. Furthermore, VIP reduced inflammatory cell persistence in the cornea after infection with each of the P. aeruginosa strains. Conclusions VIP treatment is effective at ameliorating disease pathogenesis for multiple P. aeruginosa strains, both cytotoxic and invasive. This study is also the first to indicate a possible role for VIP regarding lipid mediator expression in the eye. In addition, the clinical isolate, KEI 1025, was characterized as an invasive strain. Overall, this study strengthens the preclinical development of VIP as a therapeutic agent for ocular infectious disease. PMID:26513498

  5. Effect of Shear Stress on Pseudomonas aeruginosa Isolated from the Cystic Fibrosis Lung

    PubMed Central

    Dingemans, Jozef; Monsieurs, Pieter; Yu, Sung-Huan; Crabbé, Aurélie; Förstner, Konrad U.; Malfroot, Anne

    2016-01-01

    ABSTRACT Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle. PMID:27486191

  6. The effects of D-Tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa.

    PubMed

    She, Pengfei; Chen, Lihua; Liu, Hongbo; Zou, Yaru; Luo, Zhen; Koronfel, Asmaa; Wu, Yong

    2015-09-01

    The biofilm formation of microorganisms causes persistent tissue infections resistant to treatment with antimicrobial agents. Pseudomonas aeruginosa is commonly isolated from the airways of patients with chronic fibrosis (CF) and often forms biofilms, which are extremely hard to eradicate and a major cause of mortality and morbidity. Recent studies have shown that D-amino acids (D-AAs) inhibited and disrupted biofilm formation by causing the release of the protein component of the polymeric matrix. However, the effects of D-AAs combined with common antibiotics on biofilms have rarely been studied. The current study first determined whether D-AAs disrupted the biofilms of PAO1 and the clinical airway isolates of P. aeruginosa. It was then determined whether combinations of D-Tyr (the most effective one) and the antibiotic amikacin (AMK) enhanced the activity against these biofilms. The results of the current study showed that D-Tyr is the most effective among those that disassemble the D-amino acids (D-leucine, D-methionine, D-Tyrptophan, and D-tryptophan), and D-Tyr at concentrations higher than 5 mM significantly reduced the biofilm biomass of P. aeruginosa (p < 0.05) without influencing bacterial growth. It was also revealed that D-Tyr improved the efficacy of AMK to combat P. aeruginosa biofilms, as indicated by a reduction in the minimal biofilm-inhibiting concentration (MBIC50 and MBIC90) without a change in the minimal inhibitory concentration (MIC) of planktonic bacteria. Thus, the findings indicated that D-Tyr supplementation overcame the resistance of P. aeruginosa biofilms to AMK, which might be helpful for preventing AMK overuse when this specific D-Tyr is recommended for combatting these biofilms. Also, toxicity of the liver and kidney from AMK could be potentially mitigated by co-delivery with D-Tyr. PMID:26188263

  7. The synergistic effect of visible light and gentamycin on Pseudomona aeruginosa microorganisms.

    PubMed

    Reznick, Yana; Banin, Ehud; Lipovsky, Anat; Lubart, Rachel; Polak, Pazit; Zalevsky, Zeev

    2013-01-01

    Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens(1-5). The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region(4,6,7). There are also reports of biocidal effect of red and near infra red(8) as well as green light(9). In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems(10). The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's. The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light. PMID:23852319

  8. The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

    PubMed Central

    Reznick, Yana; Banin, Ehud; Lipovsky, Anat; Lubart, Rachel; Polak, Pazit; Zalevsky, Zeev

    2013-01-01

    Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9. In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10. The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's. The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light. PMID:23852319

  9. PA0148 from Pseudomonas aeruginosa Catalyzes the Deamination of Adenine

    SciTech Connect

    Goble, A.M.; Swaminathan, S.; Zhang, Z.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

    2011-08-02

    Four proteins from NCBI cog1816, previously annotated as adenosine deaminases, have been subjected to structural and functional characterization. Pa0148 (Pseudomonas aeruginosa PAO1), AAur1117 (Arthrobacter aurescens TC1), Sgx9403e, and Sgx9403g have been purified and their substrate profiles determined. Adenosine is not a substrate for any of these enzymes. All of these proteins will deaminate adenine to produce hypoxanthine with k{sub cat}/K{sub m} values that exceed 10{sup 5} M{sup -1} s{sup -1}. These enzymes will also accept 6-chloropurine, 6-methoxypurine, N-6-methyladenine, and 2,6-diaminopurine as alternate substrates. X-ray structures of Pa0148 and AAur1117 have been determined and reveal nearly identical distorted ({beta}/{alpha}){sub 8} barrels with a single zinc ion that is characteristic of members of the amidohydrolase superfamily. Structures of Pa0148 with adenine, 6-chloropurine, and hypoxanthine were also determined, thereby permitting identification of the residues responsible for coordinating the substrate and product.

  10. Pa0148 from Pseudomonas aeruginosa Catalyzes the Deamination of Adenine

    SciTech Connect

    A Goble; Z Zhang; J Sauder; S Burley; S Swaminathan; F Raushel

    2011-12-31

    Four proteins from NCBI cog1816, previously annotated as adenosine deaminases, have been subjected to structural and functional characterization. Pa0148 (Pseudomonas aeruginosa PAO1), AAur1117 (Arthrobacter aurescens TC1), Sgx9403e, and Sgx9403g have been purified and their substrate profiles determined. Adenosine is not a substrate for any of these enzymes. All of these proteins will deaminate adenine to produce hypoxanthine with k{sub cat}/K{sub m} values that exceed 10{sup 5} M{sup -1} s{sup -1}. These enzymes will also accept 6-chloropurine, 6-methoxypurine, N-6-methyladenine, and 2,6-diaminopurine as alternate substrates. X-ray structures of Pa0148 and AAur1117 have been determined and reveal nearly identical distorted ({beta}/{alpha}){sub 8} barrels with a single zinc ion that is characteristic of members of the amidohydrolase superfamily. Structures of Pa0148 with adenine, 6-chloropurine, and hypoxanthine were also determined, thereby permitting identification of the residues responsible for coordinating the substrate and product.

  11. Sodium Nitrite-Mediated Killing of the Major Cystic Fibrosis Pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia under Anaerobic Planktonic and Biofilm Conditions▿

    PubMed Central

    Major, Tiffany A.; Panmanee, Warunya; Mortensen, Joel E.; Gray, Larry D.; Hoglen, Niel; Hassett, Daniel J.

    2010-01-01

    A hallmark of airways in patients with cystic fibrosis (CF) is highly refractory, chronic infections by several opportunistic bacterial pathogens. A recent study demonstrated that acidified sodium nitrite (A-NO2−) killed the highly refractory mucoid form of Pseudomonas aeruginosa, a pathogen that significantly compromises lung function in CF patients (S. S. Yoon et al., J. Clin. Invest. 116:436-446, 2006). Therefore, the microbicidal activity of A-NO2− (pH 6.5) against the following three major CF pathogens was assessed: P. aeruginosa (a mucoid, mucA22 mutant and a sequenced nonmucoid strain, PAO1), Staphylococcus aureus USA300 (methicillin resistant), and Burkholderia cepacia, a notoriously antibiotic-resistant organism. Under planktonic, anaerobic conditions, growth of all strains except for P. aeruginosa PAO1 was inhibited by 7.24 mM (512 μg ml−1 NO2−). B. cepacia was particularly sensitive to low concentrations of A-NO2− (1.81 mM) under planktonic conditions. In antibiotic-resistant communities known as biofilms, which are reminiscent of end-stage CF airway disease, A-NO2− killed mucoid P. aeruginosa, S. aureus, and B. cepacia; 1 to 2 logs of cells were killed after a 2-day incubation with a single dose of ∼15 mM A-NO2−. Animal toxicology and phase I human trials indicate that these bactericidal levels of A-NO2− can be easily attained by aerosolization. Thus, in summary, we demonstrate that A-NO2− is very effective at killing these important CF pathogens and could be effective in other infectious settings, particularly under anaerobic conditions where bacterial defenses against the reduction product of A-NO2−, nitric oxide (NO), are dramatically reduced. PMID:20696868

  12. The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

    PubMed Central

    Darzins, A

    1993-01-01

    The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili. Images PMID:8104179

  13. Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity

    PubMed Central

    2014-01-01

    Background Pseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits. Results In this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes. Conclusions Contrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed. PMID:24773920

  14. Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells

    PubMed Central

    Stanton, Bruce A.; Coutermarsh, Bonita; Barnaby, Roxanna; Hogan, Deborah

    2015-01-01

    Background P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. Methods and Results F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. Conclusion The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials. PMID:26018799

  15. Synthesis of the A-band polysaccharide sugar D-rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa.

    PubMed

    Rocchetta, H L; Pacan, J C; Lam, J S

    1998-09-01

    Pseudomonas aeruginosa is capable of producing various cell-surface polysaccharides including alginate, A-band and B-band lipopolysaccharides (LPS). The D-mannuronic acid residues of alginate and the D-rhamnose (D-Rha) residues of A-band polysaccharide are both derived from the common sugar nucleotide precursor GDP-D-mannose (D-Man). Three genes, rmd, gmd and wbpW, which encode proteins involved in the synthesis of GDP-D-Rha, have been localized to the 5' end of the A-band gene cluster. In this study, WbpW was found to be homologous to phosphomannose isomerases (PMIs) and GDP-mannose pyrophosphorylases (GMPs) involved in GDP-D-Man biosynthesis. To confirm the enzymatic activity of WbpW, Escherichia coli PMI and GMP mutants deficient in the K30 capsule were complemented with wbpW, and restoration of K30 capsule production was observed. This indicates that WbpW, like AlgA, is a bifunctional enzyme that possesses both PMI and GMP activities for the synthesis of GDP-D-Man. No gene encoding a phosphomannose mutase (PMM) enzyme could be identified within the A-band gene cluster. This suggests that the PMM activity of AlgC may be essential for synthesis of the precursor pool of GDP-D-Man, which is converted to GDP-D-Rha for A-band synthesis. Gmd, a previously reported A-band enzyme, and Rmd are predicted to perform the two-step conversion of GDP-D-Man to GDP-D-Rha. Chromosomal mutants were generated in both rmd and wbpW. The Rmd mutants do not produce A-band LPS, while the WbpW mutants synthesize very low amounts of A band after 18 h of growth. The latter observation was thought to result from the presence of the functional homologue AlgA, which may compensate for the WbpW deficiency in these mutants. Thus, WbpW AlgA double mutants were constructed. These mutants also produced low levels of A-band LPS. A search of the PAO1 genome sequence identified a second AlgA homologue, designated ORF488, which may be responsible for the synthesis of GDP-D-Man in the absence of Wbp

  16. The Pseudomonas aeruginosa Pathogenicity Island PAPI-1 is transferred via a novel Type IV pilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments including humans is in part due to its large and diverse genomic repertoi...

  17. Pseudomonas aeruginosa d-Arabinofuranose Biosynthetic Pathway and Its Role in Type IV Pilus Assembly*

    PubMed Central

    Harvey, Hanjeong; Kus, Julianne V.; Tessier, Luc; Kelly, John; Burrows, Lori L.

    2011-01-01

    Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked d-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-d-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to d-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilAIV produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae. PMID:21676874

  18. Role of the Pseudomonas quinolone signal (PQS) in sensitising Pseudomonas aeruginosa to UVA radiation.

    PubMed

    Pezzoni, Magdalena; Meichtry, Martín; Pizarro, Ramón A; Costa, Cristina S

    2015-01-01

    One of the main stress factors that bacteria face in the environment is solar ultraviolet-A (UVA) radiation, which leads to lethal effects through oxidative damage. The aim of this work was to investigate the role of 2-heptyl-3-hydroxi-4-quinolone (the Pseudomonas quinolone signal or PQS) in the response of Pseudomonas aeruginosa to UVA radiation. PQS is an intercellular quorum sensing signal associated to membrane vesicles which, among other functions, regulates genes related to iron acquisition, forms stable complexes with iron and participates in oxidative phenomena. UVA exposure of the wild-type PAO1 strain and a pqsA mutant unable to produce PQS revealed a sensitising role for this signal. Research into the mechanism involved in this phenomenon revealed that catalase, an essential factor in the UVA defence, is not related to PQS-mediated UVA sensitivity. Absorption of UVA by PQS produced its own photo-degradation, oxidation of the probe 2',7'- dichlorodihydrofluorescein and generation of singlet oxygen and superoxide anion, suggesting that this signal could be acting as an endogenous photosensitiser. The results presented in this study could explain the high sensitivity to UVA of P. aeruginosa when compared to enteric bacteria. PMID:25535873

  19. Pel promotes symmetric, short-ranged surface attachment in P. aeruginosa

    NASA Astrophysics Data System (ADS)

    Cooley, B. J.; Thatcher, Travis; Hashmi, Sara; L'Her, Guillaume; Touhami, Ahmed; Provenzano, Daniele; Gordon, Vernita

    2013-03-01

    Bacterial biofilms are surface mounted, multicellular communities of interacting bacteria that are often associated with chronic infections that resist antibiotics and damage host tissue. Bacteria in a biofilm are bound in a matrix of polymeric materials that adhere the bacteria to the surface, give the system spatial structure, and cluster the bacteria near each other. The opportunistic human pathogen Pseudomonas aeruginosa is widely studied as a model biofilm-forming organism. The polymeric matrix of P. aeruginosa strain PAO1 biofilms is dominated by two bacteria-produced extracellular polymers, Pel and Psl. We use both optical and atomic force microscopy to examine the roles of these polymers in very early biofilm development, in the hours after initial surface attachment. In agreement with other researchers, we find that Psl mediates strong attachment to a glass surface. Unexpectedly, we find that Pel promotes symmetric attachment, in the form of the rod-shaped bacteria lying flat on the surface, independently of permanent attachment to the surface. Further, the presence of Pel makes adhesion forces more short-ranged than they are with Psl alone. We suggest that these effects may result through synergistic interactions of Pel and Psl in the polymeric matrix.

  20. Cellular organization of siderophore biosynthesis in Pseudomonas aeruginosa: Evidence for siderosomes.

    PubMed

    Gasser, Véronique; Guillon, Laurent; Cunrath, Olivier; Schalk, Isabelle J

    2015-07-01

    Pyoverdine I (PVDI) and pyochelin (PCH) are the two major siderophores produced by Pseudomonas aeruginosa PAO1 to import iron. The biochemistry of the biosynthesis of these two siderophores has been described in detail in the literature over recent years. PVDI assembly requires the coordinated action of seven cytoplasmic enzymes and is followed by a periplasmic maturation before secretion of the siderophore into the extracellular medium by the efflux system PvdRT-OpmQ. PCH biosynthesis also involves seven cytoplasmic enzymes but no periplasmic maturation. Recent findings indicate that the cytoplasmic enzymes involved in each of these two siderophore biosynthesis pathways can form siderophore-specific multi-enzymatic complexes called siderosomes associated with the inner leaflet of the cytoplasmic membrane. This organization may optimize the transfer of the siderophore precursors between the various participating enzymes and avoid the diffusion of siderophore precursors, able to chelate metals, throughout the cytoplasm. Here, we describe these recently published findings and discuss the existence of these siderosomes in P. aeruginosa. PMID:25697961

  1. X-ray Irradiated Vaccine Confers protection against Pneumonia caused by Pseudomonas Aeruginosa

    PubMed Central

    Li, Yanyan; Wang, Zhenling; Liu, Xiaoxiao; Tang, Jianying; Peng, Bin; Wei, Yuquan

    2016-01-01

    Pseudomonas aeruginosa is a gram-negative bacterium and one of the leading causes of nosocomial infection worldwide, however, no effective vaccine is currently available in the market. Here, we demonstrate that inactivation of the bacteria by X-ray irradiation inhibits its replication capability but retained antigenic expression functionally thus allowing its use as a potential vaccine. Mice immunized by this vaccine were challenged by the parental strain, the O-antigen-homologous strain PAO-1 (O2/O5) and heterologous strain PAO-6 (O6) in an acute pneumonia model. We further measured the protective effect of the vaccine, as well as host innate and cellular immunity responses. We found immunized mice could protect against both strains. Notably, the antiserum only had significant protective role against similar bacteria, while adoptive transfer of lymphocytes significantly controlled the spread of the virulent heterologous serogroup PAO-6 infection, and the protective role could be reversed by CD4 rather than CD8 antibody. We further revealed that vaccinated mice could rapidly recruit neutrophils to the airways early after intranasal challenge by PAO-6, and the irradiated vaccine was proved to be protective by the generated CD4+ IL-17+ Th17 cells. In conclusion, the generation of inactivated but metabolically active microbes is a promising strategy for safely vaccinating against Pseudomonas aeruginosa. PMID:26879055

  2. Impact of higher alginate expression on deposition of Pseudomonas aeruginosa in radial stagnation point flow and reverse osmosis systems.

    PubMed

    Herzberg, Moshe; Rezene, Tesfalem Zere; Ziemba, Christopher; Gillor, Osnat; Mathee, Kalai

    2009-10-01

    Extracellular polymeric substances (EPS) have major impact on biofouling of reverse osmosis (RO) membranes. On one hand, EPS can reduce membrane permeability and on the other, EPS production by the primary colonizers may influence their deposition and attachment rate and subsequently affect the biofouling propensity of the membrane. The role of bacterial exopolysaccharides in bacterial deposition followed by the biofouling potential of an RO membrane was evaluated using an alginate overproducing (mucoid) Pseudomonas aeruginosa. The mucoid P. aeruginosa PAOmucA22 was compared with its isogenic nonmucoid prototypic parent PAO1 microscopically in a radial stagnation point flow (RSPF) system for their bacterial deposition characteristics. Then, biofouling potential of PAO1 and PAOmucA22 was determined in a crossflow rectangular plate-and-frame membrane cell, in which the strains were cultivated on a thin-film composite, polyamide, flat RO membrane coupon (LFC-1) under laminar flow conditions. In the RSPF system, the observed deposition rate of the mucoid strain was between 5- and 10-fold lower than of the wild type using either synthetic wastewater medium (with ionic strength of 14.7 mM and pH 7.4) or 15 mM KCl solution (pH of 6.2). The slower deposition rate of the mucoid strain is explained by 5- to 25-fold increased hydrophilicity of the mucoid strain as compared to the isogenic wild type, PAO1. Corroborating with these results, a significant delay in the onset of biofouling of the RO membrane was observed when the mucoid strain was used as the membrane colonizer, in which the observed time for the induced permeate flux decline was delayed (ca. 2-fold). In conclusion, the lower initial cell attachment of the mucoid strain decelerated biofouling of the RO membrane. Bacterial deposition and attachment is a critical step in biofilm formation and governed by intimate interactions between outer membrane proteins of the bacteria and the surface. Shielding these

  3. Impaired Pulmonary Defense Against Pseudomonas aeruginosa in VEGF Gene Inactivated Mouse Lung

    PubMed Central

    Breen, Ellen C.; Malloy, Jaret L.; Tang, Kechun; Xia, Feng; Fu, Zhenxing; Hancock, Robert E. W.; Overhage, Joerg; Wagner, Peter D.; Spragg, Roger G.

    2012-01-01

    Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 hours after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection. PMID:22718316

  4. Co-Cultures of Pseudomonas aeruginosa and Roseobacter denitrificans Reveal Shifts in Gene Expression Levels Compared to Solo Cultures

    PubMed Central

    Conway, Crystal A.; Esiobu, Nwadiuto; Lopez, Jose V.

    2012-01-01

    Consistent biosynthesis of desired secondary metabolites (SMs) from pure microbial cultures is often unreliable. In a proof-of-principle study to induce SM gene expression and production, we describe mixed “co-culturing” conditions and monitoring of messages via quantitative real-time PCR (qPCR). Gene expression of model bacterial strains (Pseudomonas aeruginosa PAO1 and Roseobacter denitrificans Och114) was analyzed in pure solo and mixed cocultures to infer the effects of interspecies interactions on gene expression in vitro, Two P. aeruginosa genes (PhzH coding for portions of the phenazine antibiotic pathway leading to pyocyanin (PCN) and the RhdA gene for thiosulfate: cyanide sulfurtransferase (Rhodanese)) and two R. denitrificans genes (BetaLact for metallo-beta-lactamase and the DMSP gene for dimethylpropiothetin dethiomethylase) were assessed for differential expression. Results showed that R. denitrificans DMSP and BetaLact gene expression became elevated in a mixed culture. In contrast, P. aeruginosa co-cultures with R. denitrificans or a third species did not increase target gene expression above control levels. This paper provides insight for better control of target SM gene expression in vitro and bypass complex genetic engineering manipulations. PMID:22566756

  5. Absence of Membrane Phosphatidylcholine Does Not Affect Virulence and Stress Tolerance Phenotypes in the Opportunistic Pathogen Pseudomonas aeruginosa

    PubMed Central

    Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.

    2012-01-01

    During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ∼4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline. PMID:22363496

  6. Iminoguanidines as Allosteric Inhibitors of the Iron-Regulated Heme Oxygenase (HemO) of Pseudomonas aeruginosa.

    PubMed

    Heinzl, Geoffrey A; Huang, Weiliang; Yu, Wenbo; Giardina, Bennett J; Zhou, Yue; MacKerell, Alexander D; Wilks, Angela; Xue, Fengtian

    2016-07-28

    New therapeutic targets are required to combat multidrug resistant infections, such as the iron-regulated heme oxygenase (HemO) of Pseudomonas aeruginosa, due to links between iron and virulence and dependence on heme as an iron source during infection. Herein we report the synthesis and activity of a series of iminoguanidine-based inhibitors of HemO. Compound 23 showed a binding affinity of 5.7 μM and an MIC50 of 52.3 μg/mL against P. aeruginosa PAO1. An in cellulo activity assay was developed by coupling HemO activity to a biliverdin-IXα-dependent infrared fluorescent protein, in which compound 23 showed an EC50 of 11.3 μM. The compounds showed increased activity against clinical isolates of P. aeruginosa, further confirming the target pathway. This class of inhibitors acts by binding to an allosteric site; the novel binding site is proposed in silico and supported by saturation transfer difference (STD) NMR as well as by hydrogen exchange mass spectrometry (HXMS). PMID:27353344

  7. Pyocyanin, a virulence factor produced by Pseudomonas aeruginosa, alters root development through reactive oxygen species and ethylene signaling in Arabidopsis.

    PubMed

    Ortiz-Castro, Randy; Pelagio-Flores, Ramón; Méndez-Bravo, Alfonso; Ruiz-Herrera, León Francisco; Campos-García, Jesús; López-Bucio, José

    2014-04-01

    Pyocyanin acts as a virulence factor in Pseudomonas aeruginosa, a plant and animal pathogen. In this study, we evaluated the effect of pyocyanin on growth and development of Arabidopsis seedlings. Root inoculation with P. aeruginosa PAO1 strain inhibited primary root growth in wild-type (WT) Arabidopsis seedlings. In contrast, single lasI- and double rhlI-/lasI- mutants of P. aeruginosa defective in pyocyanin production showed decreased root growth inhibition concomitant with an increased phytostimulation. Treatment with pyocyanin modulates root system architecture, inhibiting primary root growth and promoting lateral root and root hair formation without affecting meristem viability or causing cell death. These effects correlated with altered proportions of hydrogen peroxide and superoxide in root tips and with an inhibition of cell division and elongation. Mutant analyses showed that pyocyanin modulation of root growth was likely independent of auxin, cytokinin, and abscisic acid but required ethylene signaling because the Arabidopsis etr1-1, ein2-1, and ein3-1 ethylene-related mutants were less sensitive to pyocyanin-induced root stoppage and reactive oxygen species (ROS) distribution. Our findings suggest that pyocyanin is an important factor modulating the interplay between ROS production and root system architecture by an ethylene-dependent signaling. PMID:24224532

  8. Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Hammond, John H.; Dolben, Emily F.; Smith, T. Jarrod; Bhuju, Sabin

    2015-01-01

    ABSTRACT In Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections. IMPORTANCE Pseudomonas aeruginosa causes acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients. P. aeruginosa uses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS, lasR, are frequently

  9. The Pseudomonas aeruginosa Transcriptional Landscape Is Shaped by Environmental Heterogeneity and Genetic Variation

    PubMed Central

    Schniederjans, Monika; Khaledi, Ariane; Hornischer, Klaus; Schulz, Sebastian; Bielecka, Agata; Eckweiler, Denitsa; Pohl, Sarah; Häussler, Susanne

    2015-01-01

    ABSTRACT Phenotypic variability among bacteria depends on gene expression in response to different environments, and it also reflects differences in genomic structure. In this study, we analyzed transcriptome sequencing (RNA-seq) profiles of 151 Pseudomonas aeruginosa clinical isolates under standard laboratory conditions and of one P. aeruginosa type strain under 14 different environmental conditions. Our approach allowed dissection of the impact of the genetic background versus environmental cues on P. aeruginosa gene expression profiles and revealed that phenotypic variation was larger in response to changing environments than between genomically different isolates. We demonstrate that mutations within the global regulator LasR affect more than one trait (pleiotropy) and that the interaction between mutations (epistasis) shapes the P. aeruginosa phenotypic plasticity landscape. Because of pleiotropic and epistatic effects, average genotype and phenotype measures appeared to be uncorrelated in P. aeruginosa. PMID:26126853

  10. Development of a Novel Method for Analyzing Pseudomonas aeruginosa Twitching Motility and Its Application to Define the AmrZ Regulon.

    PubMed

    Xu, Binjie; Wozniak, Daniel J

    2015-01-01

    Twitching motility is an important migration mechanism for the Gram-negative bacterium Pseudomonas aeruginosa. In the commonly used subsurface twitching assay, the sub-population of P. aeruginosa with active twitching motility is difficult to harvest for high-throughput studies. Here we describe the development of a novel method that allows efficient isolation of bacterial sub-populations conducting highly active twitching motility. The transcription factor AmrZ regulates multiple P. aeruginosa virulence factors including twitching motility, yet the mechanism of this activation remains unclear. We therefore set out to understand this mechanism by defining the AmrZ regulon using DNA microarrays in combination with the newly developed twitching motility method. We discovered 112 genes in the AmrZ regulon and many encode virulence factors. One gene of interest and the subsequent focus was lecB, which encodes a fucose-binding lectin. DNA binding assays revealed that AmrZ activates lecB transcription by directly binding to its promoter. The lecB gene was previously shown to be required for twitching motility in P. aeruginosa strain PAK; however, our lecB deletion had no effect on twitching motility in strain PAO1. Collectively, in this study a novel condition was developed for quantitative studies of twitching motility, under which the AmrZ regulon was defined. PMID:26309248

  11. Development of a Novel Method for Analyzing Pseudomonas aeruginosa Twitching Motility and Its Application to Define the AmrZ Regulon

    PubMed Central

    Xu, Binjie; Wozniak, Daniel J.

    2015-01-01

    Twitching motility is an important migration mechanism for the Gram-negative bacterium Pseudomonas aeruginosa. In the commonly used subsurface twitching assay, the sub-population of P. aeruginosa with active twitching motility is difficult to harvest for high-throughput studies. Here we describe the development of a novel method that allows efficient isolation of bacterial sub-populations conducting highly active twitching motility. The transcription factor AmrZ regulates multiple P. aeruginosa virulence factors including twitching motility, yet the mechanism of this activation remains unclear. We therefore set out to understand this mechanism by defining the AmrZ regulon using DNA microarrays in combination with the newly developed twitching motility method. We discovered 112 genes in the AmrZ regulon and many encode virulence factors. One gene of interest and the subsequent focus was lecB, which encodes a fucose-binding lectin. DNA binding assays revealed that AmrZ activates lecB transcription by directly binding to its promoter. The lecB gene was previously shown to be required for twitching motility in P. aeruginosa strain PAK; however, our lecB deletion had no effect on twitching motility in strain PAO1. Collectively, in this study a novel condition was developed for quantitative studies of twitching motility, under which the AmrZ regulon was defined. PMID:26309248

  12. Pseudomonas aeruginosa Outer Membrane Vesicles Triggered by Human Mucosal Fluid and Lysozyme Can Prime Host Tissue Surfaces for Bacterial Adhesion

    PubMed Central

    Metruccio, Matteo M. E.; Evans, David J.; Gabriel, Manal M.; Kadurugamuwa, Jagath L.; Fleiszig, Suzanne M. J.

    2016-01-01

    Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release outer membrane vesicles (OMVs) in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to phosphate buffered saline (PBS) controls (∼100-fold). Transmission electron microscopy (TEM) and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (∼4-fold, P < 0.01). Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections. PMID:27375592

  13. Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

    PubMed

    Elfarash, Ameer; Dingemans, Jozef; Ye, Lumeng; Hassan, Ahmed Amir; Craggs, Michael; Reimmann, Cornelia; Thomas, Mark S; Cornelis, Pierre

    2014-02-01

    Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain. PMID:24217175

  14. Pseudomonas aeruginosa DesB Promotes Staphylococcus aureus Growth Inhibition in Coculture by Controlling the Synthesis of HAQs

    PubMed Central

    Kim, Sejeong; Yoon, Yohan; Choi, Kyoung-Hee

    2015-01-01

    Pseudomonas aeruginosa is a pathogen that can cause serious infections and usually coexists with other pathogens, such as Staphylococcus aureus. Virulence factors are important for maintaining a presence of the organisms in these multispecies environments, and DesB plays an important role in P. aeruginosa virulence. Therefore, we investigated the effect of DesB on S. aureus reduction under competitive situation. Liquid cultures of P. aeruginosa wild type (WT) and its desB mutant were spotted on agar plates containing S. aureus, and the size of the clear zones was compared. In addition, interbacterial competition between P. aeruginosa and S. aureus was observed over time during planktonic coculture. The transcriptional profiles of the WT and desB mutant were compared by qRT-PCR and microarray to determine the role of DesB in S. aureus reduction at the molecular level. As a result, the clear zone was smaller for the desB mutant than for P. aeruginosa PAO1 (WT), and in planktonic coculture, the number of S. aureus cells was reduced in the desB mutant. qRT-PCR and microarray revealed that the expression of MvfR-controlled pqsA-E and phnAB operons was significantly decreased, but the mexEF-oprN operon was highly expressed. The results indicate that intracellular levels of 4-hydroxy-2-heptylquinoline (HHQ), a ligand of MvfR, are reduced due to MexEF-OprN-mediated efflux in desB mutant, resulting in the decrease of MvfR binding to pqsA-E promoter and the reduction of 4-hydroxy-2-alkylquinolines (HAQs) synthesis. Overexpression of mexEF-oprN operon in desB mutant was phenotypically confirmed by observing significantly increased resistance to chloramphenicol. In conclusion, these results suggest that DesB plays a role in the inhibition of S. aureus growth by controlling HAQ synthesis. PMID:26230088

  15. A Pseudomonas aeruginosa EF-Hand Protein, EfhP (PA4107), Modulates Stress Responses and Virulence at High Calcium Concentration

    PubMed Central

    Sarkisova, Svetlana A.; Lotlikar, Shalaka R.; Guragain, Manita; Kubat, Ryan; Cloud, John

    2014-01-01

    Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflamatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 ΔefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H2O2) when cultured at high [Ca2+]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca2+] and no iron. The effect of the ΔefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca2+] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca2+]. PMID

  16. Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India

    PubMed Central

    2015-01-01

    Carbapenem resistance presents severe threat to the treatment of multidrug resistant Pseudomonas aeruginosa infections. The study was undertaken to investigate the role of efflux pumps in conferring meropenem resistance and effect of single dose exposure of meropenem on transcription level of mexA gene in clinical isolates of P. aeruginosa from a tertiary referral hospital of India. Further, in this investigation an effort was made to assess whether different components of MexAB-OprM operon expresses in the same manner and the extent of contributions of those components in meropenem resistance in its natural host (P. aeruginosa) and in a heterologous host (E. coli). Out of 83 meropenem nonsusceptible isolates, 22 isolates were found to possess efflux pump activity phenotypically. Modified hodge test and multiplex PCR confirmed the absence of carbapenemase genes in those isolates. All of them were of multidrug resistant phenotype and were resistant to all the carbepenem drug tested. MexAB-OprM efflux pump was found to be overexpressed in all the study isolates. It could be observed that single dose exposure meropenem could give rise to trivial increase in transcription of mexA gene. Different constructs of MexAB-OprM (mexR-mexA-mexB-OprM; mexA-mexB-OprM; mexA-mexB) could be expressed in both its natural (P. aeruginosa PAO1) and heterologous host (E. coli JM107) but transcription level of mexA gene varied in both the hosts before and after single dose exposure of meropenem. Different components of the operon failed to enhance meropenem resistance in E. coli JM107 and P. aeruginosa PAO1. This study could prove that MexAB-OprM efflux pump can significantly contribute to meropenem resistance in hospital isolates of P. aeruginosa where an acquired resistant mechanism is absent. Thus, equal importance should be given for diagnosis of intrinsic resistance mechanism so as to minimize treatment failure. As meropenem could not enhance mexA transcriptions significantly, there

  17. Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa

    PubMed Central

    van Belkum, Alex; Soriaga, Leah B.; LaFave, Matthew C.; Akella, Srividya; Veyrieras, Jean-Baptiste; Barbu, E. Magda; Shortridge, Dee; Blanc, Bernadette; Hannum, Gregory; Zambardi, Gilles; Miller, Kristofer; Enright, Mark C.; Mugnier, Nathalie; Brami, Daniel; Schicklin, Stéphane; Felderman, Martina; Schwartz, Ariel S.; Richardson, Toby H.; Peterson, Todd C.; Hubby, Bolyn

    2015-01-01

    ABSTRACT Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. PMID:26604259

  18. Lagooning of wastewaters favors dissemination of clinically relevant Pseudomonas aeruginosa.

    PubMed

    Petit, Stéphanie M-C; Lavenir, Raphaël; Colinon-Dupuich, Céline; Boukerb, Amine M; Cholley, Pascal; Bertrand, Xavier; Freney, Jean; Doléans-Jordheim, Anne; Nazaret, Sylvie; Laurent, Frédéric; Cournoyer, Benoit

    2013-10-01

    The significance of wastewater treatment lagoons (WWTLs) as point sources of clinically relevant Pseudomonas aeruginosa that can disseminate through rural and peri-urban catchments was investigated. A panel of P. aeruginosa strains collected over three years from WWTLs and community-acquired infections was compared by pulsed field gel electrophoresis (PFGE) DNA fingerprinting and multilocus sequence typing (MLST). Forty-four distantly related PFGE profiles and four clonal complexes were found among the WWTL strains analyzed. Some genotypes were repeatedly detected from different parts of WWTLs, including the influent, suggesting an ability to migrate and persist over time. MLST showed all investigated lineages to match sequence types described in other countries and strains from major clinical clones such as PA14 of ST253 and "C" of ST17 were observed. Some of these genotypes matched isolates from community-acquired infections recorded in the WWTL geographic area. Most WWTL strains harbored the main P. aeruginosa virulence genes; 13% harbored exoU-encoded cytoxins, but on at least six different genomic islands, with some of these showing signs of genomic instability. P. aeruginosa appeared to be highly successful opportunistic colonizers of WWTLs. Lagooning of wastewaters was found to favor dissemination of clinically relevant P. aeruginosa among peri-urban watersheds. PMID:23792168

  19. Characterizing the optimal flux space of genome-scale metabolic reconstructions through modified latin-hypercube sampling.

    PubMed

    Chaudhary, Neha; Tøndel, Kristin; Bhatnagar, Rakesh; dos Santos, Vítor A P Martins; Puchałka, Jacek

    2016-03-01

    Genome-Scale Metabolic Reconstructions (GSMRs), along with optimization-based methods, predominantly Flux Balance Analysis (FBA) and its derivatives, are widely applied for assessing and predicting the behavior of metabolic networks upon perturbation, thereby enabling identification of potential novel drug targets and biotechnologically relevant pathways. The abundance of alternate flux profiles has led to the evolution of methods to explore the complete solution space aiming to increase the accuracy of predictions. Herein we present a novel, generic algorithm to characterize the entire flux space of GSMR upon application of FBA, leading to the optimal value of the objective (the optimal flux space). Our method employs Modified Latin-Hypercube Sampling (LHS) to effectively border the optimal space, followed by Principal Component Analysis (PCA) to identify and explain the major sources of variability within it. The approach was validated with the elementary mode analysis of a smaller network of Saccharomyces cerevisiae and applied to the GSMR of Pseudomonas aeruginosa PAO1 (iMO1086). It is shown to surpass the commonly used Monte Carlo Sampling (MCS) in providing a more uniform coverage for a much larger network in less number of samples. Results show that although many fluxes are identified as variable upon fixing the objective value, majority of the variability can be reduced to several main patterns arising from a few alternative pathways. In iMO1086, initial variability of 211 reactions could almost entirely be explained by 7 alternative pathway groups. These findings imply that the possibilities to reroute greater portions of flux may be limited within metabolic networks of bacteria. Furthermore, the optimal flux space is subject to change with environmental conditions. Our method may be a useful device to validate the predictions made by FBA-based tools, by describing the optimal flux space associated with these predictions, thus to improve them. PMID

  20. The Pseudomonas aeruginosa PA01 Gene Collection

    PubMed Central

    LaBaer, Joshua; Qiu, QingQing; Anumanthan, Anukanth; Mar, Wenhong; Zuo, Dongmei; Murthy, T.V.S.; Taycher, Helen; Halleck, Allison; Hainsworth, Eugenie; Lory, Stephen; Brizuela, Leonardo

    2004-01-01

    Pseudomonas aeruginosa, a common inhabitant of soil and water, is an opportunistic pathogen of growing clinical relevance. Its genome, one of the largest among bacteria [5570 open reading frames (ORFs)] approaches that of simple eukaryotes. We have constructed a comprehensive gene collection for this organism utilizing the annotated genome of P. aeruginosa PA01 and a highly automated and laboratory information management system (LIMS)-supported production line. All the individual ORFs have been successfully PCR-amplified and cloned into a recombination-based cloning system. We have isolated and archived four independent isolates of each individual ORF. Full sequence analysis of the first isolate for one-third of the ORFs in the collection has been completed. We used two sets of genes from this repository for high-throughput expression and purification of recombinant proteins in different systems. The purified proteins have been used to set up biochemical and immunological assays directed towards characterization of histidine kinases and identification of bacterial proteins involved in the immune response of cystic fibrosis patients. This gene repository provides a powerful tool for proteome- and genome-scale research of this organism, and the strategies adopted to generate this repository serve as a model for building clone sets for other bacteria. PMID:15489342

  1. The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

    PubMed

    Orgad, Oded; Oren, Yoram; Walker, Sharon L; Herzberg, Moshe

    2011-08-01

    Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment. PMID:21797737

  2. Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

    PubMed

    Beeton, M L; Alves, D R; Enright, M C; Jenkins, A T A

    2015-08-01

    The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses. PMID:26100212

  3. Development of a Pseudomonas aeruginosa Agmatine Biosensor.

    PubMed

    Gilbertsen, Adam; Williams, Bryan

    2014-12-01

    Agmatine, decarboxylated arginine, is an important intermediary in polyamine production for many prokaryotes, but serves higher functions in eukaryotes such as nitric oxide inhibition and roles in neurotransmission. Pseudomonas aeruginosa relies on the arginine decarboxylase and agmatine deiminase pathways to convert arginine into putrescine. One of the two known agmatine deiminase operons, aguBA, contains an agmatine sensitive TetR promoter controlled by AguR. We have discovered that this promoter element can produce a titratable induction of its gene products in response to agmatine, and utilized this discovery to make a luminescent agmatine biosensor in P. aeruginosa. The genome of the P. aeruginosa lab strain UCBPP-PA14 was altered to remove both its ability to synthesize or destroy agmatine, and insertion of the luminescent reporter construct allows it to produce light in proportion to the amount of exogenous agmatine applied from ~100 nM to 1mM. Furthermore it does not respond to related compounds including arginine or putrescine. To demonstrate potential applications the biosensor was used to detect agmatine in spent supernatants, to monitor the development of arginine decarboxylase over time, and to detect agmatine in the spinal cords of live mice. PMID:25587430

  4. Synergistic effect of membrane-active peptides polymyxin B and gramicidin S on multidrug-resistant strains and biofilms of Pseudomonas aeruginosa.

    PubMed

    Berditsch, Marina; Jäger, Thomas; Strempel, Nikola; Schwartz, Thomas; Overhage, Jörg; Ulrich, Anne S

    2015-09-01

    Multidrug-resistant Pseudomonas aeruginosa is a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against different P. aeruginosa isolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of ≤0.5. Remarkably, several clinical P. aeruginosa isolates that grew as small-colony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and ≤0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistant P. aeruginosa and biofilms of P. aeruginosa strain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa. PMID:26077259

  5. Pharmacokinetic/Pharmacodynamic Investigation of Colistin against Pseudomonas aeruginosa Using an In Vitro Model ▿

    PubMed Central

    Bergen, Phillip J.; Bulitta, Jurgen B.; Forrest, Alan; Tsuji, Brian T.; Li, Jian; Nation, Roger L.

    2010-01-01

    Colistin plays a key role in treatment of serious infections by Pseudomonas aeruginosa. The aims of this study were to (i) identify the pharmacokinetic/pharmacodynamic (PK/PD) index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒCmax/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT>MIC]) that best predicts colistin efficacy and (ii) determine the values for the predictive PK/PD index required to achieve various magnitudes of killing effect. Studies were conducted in a one-compartment in vitro PK/PD model for 24 h using P. aeruginosa ATCC 27853, PAO1, and the multidrug-resistant mucoid clinical isolate 19056 muc. Six intermittent dosing intervals, with a range of ƒCmax colistin concentrations, and two continuous infusion regimens were examined. PK/PD indices varied from 0.06 to 18 for targeted ƒCmax/MIC, 0.36 to 312 for ƒAUC/MIC, and 0 to 100% for ƒT>MIC. A Hill-type model was fit to killing effect data, which were expressed as the log10 ratio of the area under the CFU/ml curve for treated regimens versus control. With ƒCmax values equal to or above the MIC, rapid killing was observed following the first dose; substantial regrowth occurred by 24 h with most regimens. The overall killing effect was best correlated with ƒAUC/MIC (R2 = 0.931) compared to ƒCmax/MIC (R2 = 0.868) and ƒT>MIC (R2 = 0.785). The magnitudes of ƒAUC/MIC required for 1- and 2-log10 reductions in the area under the CFU/ml curve relative to growth control were 22.6 and 30.4, 27.1 and 35.7, and 5.04 and 6.81 for ATCC 27853, PAO1, and 19056 muc, respectively. The PK/PD targets identified will assist in designing optimal dosing strategies for colistin. PMID:20585118

  6. Structure and inhibition studies of a type II beta-carbonic anhydrase psCA3 from Pseudomonas aeruginosa.

    PubMed

    Pinard, Melissa A; Lotlikar, Shalaka R; Boone, Christopher D; Vullo, Daniela; Supuran, Claudiu T; Patrauchan, Marianna A; McKenna, Robert

    2015-08-01

    Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The β-class CAs (β-CAs) are expressed in prokaryotes, fungi, plants, and more recently have been isolated in some animals. The β-CA class is divided into two subclasses, termed type I and II, defined by pH catalytic activity profile and active site structural configuration. Type I β-CAs display catalytic activity over a broad pH range (6.5-9.0) with the active site zinc tetrahedrally coordinated by three amino acids and a hydroxide/water. In contrast, type II β-CAs are catalytically active only at a pH 8 and higher where they adopt a functional active site configuration like that of type I. However, below pH 8 they are conformationally self-inactivated by the addition of a fourth amino acid coordinating the zinc and thereby displacing the zinc bound solvent. We have determined the structure of psCA3, a type II β-CA, isolated from Pseudomonas aeruginosa (P. aeruginosa) PAO1 at pH 8.3, in its open active state to a resolution of 1.9 Å. The active site zinc is coordinated by Cys42, His98, Cys101 and a water/hydroxide molecule. P. aeruginosa is a multi-drug resistant bacterium and displays intrinsic resistance to most of the currently used antibiotics; therefore, there is a need for new antibacterial targets. Kinetic data confirm that psCA3 belongs to the type II subclass and that sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. In vivo studies identified that among six tested inhibitors representing sulfonamides, inorganic anions, and small molecules, acetazolamide has the most significant dose-dependent inhibitory effect on P. aeruginosa growth. PMID:26068018

  7. BqsR/BqsS Constitute a Two-Component System That Senses Extracellular Fe(II) in Pseudomonas aeruginosa

    PubMed Central

    Kreamer, Naomi N. K.; Wilks, Jessica C.; Marlow, Jeffrey J.; Coleman, Maureen L.

    2012-01-01

    Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium best known as the predominant opportunistic pathogen infecting the lungs of cystic fibrosis patients. In this context, it is thought to form biofilms, within which locally reducing and acidic conditions can develop that favor the stability of ferrous iron [Fe(II)]. Because iron is a signal that stimulates biofilm formation, we performed a microarray study to determine whether P. aeruginosa strain PA14 exhibits a specific transcriptional response to extracellular Fe(II). Among the genes that were most upregulated in response to Fe(II) were those encoding the two-component system BqsR/BqsS, previously identified for its role in P. aeruginosa strain PAO1 biofilm decay (13); here, we demonstrate its role in extracellular Fe(II) sensing. bqsS and bqsR form an operon together with two small upstream genes, bqsP and bqsQ, and one downstream gene, bqsT. BqsR/BqsS sense extracellular Fe(II) at physiologically relevant concentrations (>10 μM) and elicit a specific transcriptional response, including its autoregulation. The sensor distinguishes between Fe(II), Fe(III), and other dipositive cations [Ca(II), Cu(II), Mg(II), Mn(II), Zn(II)] under aerobic or anaerobic conditions. The gene that is most upregulated by BqsR/BqsS, as measured by quantitative reverse transcription-PCR (qRT-PCR), is PA14_04180, which is predicted to encode a periplasmic oligonucleotide/oligosaccharide-binding domain (OB-fold) protein. Coincident with phenazine production during batch culture growth, Fe(II) becomes the majority of the total iron pool and bqsS is upregulated. The existence of a two-component system that senses Fe(II) indicates that extracellular Fe(II) is an important environmental signal for P. aeruginosa. PMID:22194456

  8. A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis

    PubMed Central

    Silo-Suh, Laura; Suh, Sang-Jin; Sokol, Pamela A.; Ohman, Dennis E.

    2002-01-01

    A sensitive plant infection model was developed to identify virulence factors in nontypeable, alginate overproducing (mucoid) Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients with chronic pulmonary disease. Nontypeable strains with defects in lipopolysaccharide O-side chains are common to CF and often exhibit low virulence in animal models of infection. However, 1,000 such bacteria were enough to show disease symptoms in the alfalfa infection. A typical mucoid CF isolate, FRD1, and its isogenic mutants were tested for alfalfa seedling infection. Although defects in the global regulators Vfr, RpoS, PvdS, or LasR had no discernable effect on virulence, a defect in RhlR reduced the infection frequency by >50%. A defect in alginate biosynthesis resulted in plant disease with >3-fold more bacteria per plant, suggesting that alginate overproduction attenuated bacterial growth in planta. FRD1 derivatives lacking AlgT, a sigma factor required for alginate production, were reduced >50% in the frequency of infection. Thus, AlgT apparently regulates factors in FRD1, besides alginate, important for pathogenesis. In contrast, in a non-CF strain, PAO1, an algT mutation did not affect its virulence on alfalfa. Conversely, PAO1 virulence was reduced in a mucA mutant that overproduced alginate. These observations suggested that mucoid conversion in CF may be driven by a selection for organisms with attenuated virulence or growth in the lung, which promotes a chronic infection. These studies also demonstrated that the wounded alfalfa seedling infection model is a useful tool to identify factors contributing to the persistence of P. aeruginosa in CF. PMID:12426404

  9. Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

    PubMed Central

    Wong, S C; Abdelal, A T

    1990-01-01

    The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated. Images FIG. 2 FIG. 3 FIG. 7 FIG. 8 PMID:2153657

  10. Alterations of OprD in Carbapenem-Intermediate and -Susceptible Strains of Pseudomonas aeruginosa Isolated from Patients with Bacteremia in a Spanish Multicenter Study

    PubMed Central

    Cabot, Gabriel; Rodríguez, Cristina; Roman, Elena; Tubau, Fe; Macia, María D.; Moya, Bartolomé; Zamorano, Laura; Suárez, Cristina; Peña, Carmen; Domínguez, María A.; Moncalián, Gabriel; Oliver, Antonio; Martínez-Martínez, Luis

    2012-01-01

    We investigated the presence of OprD mutations in 60 strains of metallo-ß-lactamase-negative Pseudomonas aeruginosa intermediately susceptible (IS [n = 12]; MIC = 8 μg/ml) or susceptible (S [n = 48]; MICs ≤ 1 to 4 μg/ml) to imipenem and/or meropenem that were isolated from patients with bacteremia in order to evaluate their impact on carbapenem susceptibility profiles. The presence of mutations in oprD was detected by sequencing analysis. OprD expression was assessed by both outer membrane protein (OMP) analysis and real-time PCR (RT-PCR). Fourteen (23%) isolates had an OprD identical to that of PAO1, and OprD modifications were detected in 46 isolates (77%). Isolates were classified as OprD “full-length types” (T1 [n = 40, including both wild-type OprD and variants showing several polymorphisms]) and OprD “deficient types” (T2 [n = 3 for OprD frameshift mutations] and T3 [n = 17 for premature stop codons in oprD]). RT-PCR showed that 5 OprD type T1 isolates presented reduced transcription of oprD (0.1- to 0.4-fold compared to PAO1), while oprD levels increased more than 2-fold over that seen with PAO1 in 4 OprD type T1 isolates. A total of 50% of the isolates belonging to OprD “deficient types” were susceptible to both carbapenems, and 40% were susceptible to meropenem and intermediately susceptible to imipenem. Only one isolate (5%) within this group was intermediately susceptible to both carbapenems, and one (5%) was susceptible to imipenem and intermediately susceptible to meropenem. We concluded that OprD inactivating mutations in clinical isolates of P. aeruginosa are not restricted only to carbapenem-resistant isolates but are also found in isolates with imipenem or meropenem MICs of only 0.06 to 4 μg/ml. PMID:22290967

  11. Proteomics of Pseudomonas aeruginosa Australian epidemic strain 1 (AES-1) cultured under conditions mimicking the cystic fibrosis lung reveals increased iron acquisition via the siderophore pyochelin.

    PubMed

    Hare, Nathan J; Soe, Cho Zin; Rose, Barbara; Harbour, Colin; Codd, Rachel; Manos, Jim; Cordwell, Stuart J

    2012-02-01

    Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further

  12. C-Terminal Region of Pseudomonas aeruginosa Outer Membrane Porin OprD Modulates Susceptibility to Meropenem

    PubMed Central

    Epp, Simone F.; Köhler, Thilo; Plésiat, Patrick; Michéa-Hamzehpour, Mehri; Frey, Joachim; Pechère, Jean-Claude

    2001-01-01

    We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 μg/ml) but susceptible to meropenem (<1 μg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-type oprD gene were cloned and expressed in a defined oprD mutant. The meropenem MICs for strains carrying the oprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprD gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid l-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem. PMID:11353625

  13. The MerR-Like Regulator BrlR Impairs Pseudomonas aeruginosa Biofilm Tolerance to Colistin by Repressing PhoPQ

    PubMed Central

    Chambers, Jacob R.

    2013-01-01

    While the MerR-like transcriptional regulator BrlR has been demonstrated to contribute to Pseudomonas aeruginosa biofilm tolerance to antimicrobial agents known as multidrug efflux pump substrates, the role of BrlR in resistance to cationic antimicrobial peptides (CAP), which is based on reduced outer membrane susceptibility, is not known. Here, we demonstrate that inactivation of brlR coincided with increased resistance of P. aeruginosa to colistin, while overexpression of brlR resulted in increased susceptibility. brlR expression correlated with reduced transcript abundances of phoP, phoQ, pmrA, pmrB, and arnC. Inactivation of pmrA and pmrB had no effect on the susceptibility of P. aeruginosa biofilms to colistin, while inactivation of phoP and phoQ rendered biofilms more susceptible than the wild type. The susceptibility phenotype of ΔphoP biofilms to colistin was comparable to that of P. aeruginosa biofilms overexpressing brlR. BrlR was found to directly bind to oprH promoter DNA of the oprH-phoPQ operon. BrlR reciprocally contributed to colistin and tobramycin resistance in P. aeruginosa PAO1 and CF clinical isolates, with overexpression of brlR resulting in increased tobramycin MICs and increased tobramycin resistance but decreased colistin MICs and increased colistin susceptibility. The opposite trend was observed upon brlR inactivation. The difference in susceptibility to colistin and tobramycin was eliminated by combination treatment of biofilms with both antibiotics. Our findings establish BrlR as an unusual member of the MerR family, as it not only functions as a multidrug transport activator, but also acts as a repressor of phoPQ expression, thus suppressing colistin resistance. PMID:23935054

  14. The ferrichrome uptake pathway in Pseudomonas aeruginosa involves an iron release mechanism with acylation of the siderophore and recycling of the modified desferrichrome.

    PubMed

    Hannauer, Mélissa; Barda, Yaniv; Mislin, Gaëtan L A; Shanzer, Abraham; Schalk, Isabelle J

    2010-03-01

    The uptake of iron into Pseudomonas aeruginosa is mediated by two major siderophores produced by the bacterium, pyoverdine and pyochelin. The bacterium is also able of utilize several heterologous siderophores of bacterial or fungal origin. In this work, we have investigated the iron uptake in P. aeruginosa PAO1 by the heterologous ferrichrome siderophore. (55)Fe uptake assays showed that ferrichrome is transported across the outer membrane primarily (80%) by the FiuA receptor and to a lesser extent (20%) by a secondary transporter. Moreover, we demonstrate that like in the uptake of ferripyoverdine and ferripyochelin, the energy required for both pathways of ferrichrome uptake is provided by the inner membrane protein TonB1. Desferrichrome-(55)Fe uptake in P. aeruginosa was also dependent on the expression of the permease FiuB, suggesting that this protein is the inner membrane transporter of the ferrisiderophore. A biomimetic fluorescent analogue of ferrichrome, RL1194, was used in vivo to monitor the kinetics of iron release from ferrichrome in P. aeruginosa in real time. This dissociation involves acylation of ferrichrome and its biomimetic analogue RL1194 and recycling of both modified siderophores into the extracellular medium. FiuC, an N-acetyltransferase, is certainly involved in this mechanism of iron release, since its mutation abolished desferrichrome-(55)Fe uptake. The acetylated derivative reacts with iron in the extracellular medium and is able to be taken up again by the cells. All these observations are discussed in light of the current knowledge concerning ferrichrome uptake in P. aeruginosa and in Escherichia coli. PMID:20047910

  15. Chemotaxis by Pseudomonas aeruginosa.

    PubMed Central

    Moulton, R C; Montie, T C

    1979-01-01

    Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa. PMID:104961

  16. Cryo-Transmission Electron Microscopy of Frozen-Hydrated Sections of Escherichia coli and Pseudomonas aeruginosa

    PubMed Central

    Matias, Valério R. F.; Al-Amoudi, Ashraf; Dubochet, Jacques; Beveridge, Terry J.

    2003-01-01

    High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing ∼2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the periplasmic space was compressed during cryosectioning, a finding which provided supporting evidence that this space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the space between them (although still part of the periplasmic space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the periplasmic space. It is possible that the protoplast has some capacity to float freely within the periplasm. PMID:14526023

  17. Bifunctional silica nanoparticles for the exploration of biofilms of Pseudomonas aeruginosa.

    PubMed

    Mauline, L; Gressier, M; Roques, C; Hammer, P; Ribeiro, S J L; Caiut, J M A; Menu, M-J

    2013-01-01

    Luminescent silica nanoparticles are frequently employed for biotechnology applications mainly because of their easy functionalization, photo-stability, and biocompatibility. Bifunctional silica nanoparticles (BSNPs) are described here as new efficient tools for investigating complex biological systems such as biofilms. Photoluminescence is brought about by the incorporation of a silylated ruthenium(II) complex. The surface properties of the silica particles were designed by reaction with amino-organosilanes, quaternary ammonium-organosilanes, carboxylate-organosilanes and hexamethyldisilazane. BSNPs were characterized extensively by DRIFT, (13)C and (29)Si solid state NMR, XPS, and photoluminescence. Zeta potential and contact angle measurements exhibited various surface properties (hydrophilic/hydrophobic balance and electric charge) according to the functional groups. Confocal laser scanning microscopy (CLSM) measurements showed that the spatial distribution of these nanoparticles inside a biofilm of Pseudomonas aeruginosa PAO1 depends more on their hydrophilic/hydrophobic characteristics than on their size. CLSM observations using two nanosized particles (25 and 68 nm) suggest that narrow diffusion paths exist through the extracellular polymeric substances matrix. PMID:23805884

  18. Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO.

    PubMed Central

    Kropinski, A M; Lewis, V; Berry, D

    1987-01-01

    Growth of Pseudomonas aeruginosa PAO1 at 15 to 45 degrees C in tryptic soy broth resulted in changes in the lipids, lipopolysaccharides (LPSs), and outer membrane proteins of the cells. Cells grown at 15 degrees C contained, relative to those cultivated at 45 degrees C, increased levels of the phospholipid fatty acids hexadecenoate and octadecenoate and reduced levels of the corresponding saturated fatty acids. Furthermore, the lipid A fatty acids also showed thermoadaptation with decreases in dodecanoic and hexadecanoic acids and increases in the level of 3-hydroxydecanoate and 2-hydroxdodecanoate as the growth temperature decreased. In addition, LPS extracted from cells cultivated at the lower temperatures contained a higher content of long-chain S-form molecules than that isolated from cells grown at higher temperatures. On the other hand, the percentage of LPS cores substituted with side-chain material decreased from 37.6 mol% at 45 degrees C to 19.3 mol% at 15 degrees C. The outer membrane protein profiles indicated that at low growth temperatures there was an increase in a polypeptide with an apparent molecular weight of 43,000 and decreases in the content of 21,000 (protein H1)- and 27,500-molecular-weight proteins. Images PMID:3106325

  19. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation.

    PubMed

    Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tiantian; Zhu, Deyu; Peng, Jin; Xia, Zhijie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

    2016-01-01

    C-di-GMP (3',5' -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity. PMID:27388857

  20. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

    PubMed Central

    Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tiantian; Zhu, Deyu; Peng, Jin; Xia, Zhijie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

    2016-01-01

    C-di-GMP (3’,5’ -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity. PMID:27388857

  1. Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

    PubMed Central

    Jackson, Angelyca A.; Gross, Maegan J.; Daniels, Emily F.; Hampton, Thomas H.; Hammond, John H.; Vallet-Gely, Isabelle; Dove, Simon L.; Stanton, Bruce A.

    2013-01-01

    Pseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ΔplcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The Δanr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence. PMID:23667230

  2. The Pseudomonas aeruginosa pyochelin-iron uptake pathway and its metal specificity.

    PubMed

    Braud, Armelle; Hannauer, Mélissa; Mislin, Gaëtan L A; Schalk, Isabelle J

    2009-06-01

    Pyochelin (Pch) is one of the two major siderophores produced and secreted by Pseudomonas aeruginosa PAO1 to assimilate iron. It chelates iron in the extracellular medium and transports it into the cell via a specific outer membrane transporter, FptA. We used the fluorescent properties of Pch to show that this siderophore chelates, in addition to Fe(3+) albeit with substantially lower affinities, Ag(+), Al(3+), Cd(2+), Co(2+), Cr(2+), Cu(2+), Eu(3+), Ga(3+), Hg(2+), Mn(2+), Ni(2+), Pb(2+), Sn(2+), Tb(3+), Tl(+), and Zn(2+). Surprisingly, the Pch complexes with all these metals bound to FptA with affinities in the range of 10 nM to 4.8 microM (the affinity of Pch-Fe is 10 nM) and were able to inhibit, with various efficiencies, Pch-(55)Fe uptake in vivo. We used inductively coupled plasma atomic emission spectrometry to follow metal uptake by P. aeruginosa. Energy-dependent metal uptake, in the presence of Pch, was efficient only for Fe(3+). Co(2+), Ga(3+), and Ni(2+) were also transported, but the uptake rates were 23- to 35-fold lower than that for Fe(3+). No uptake was seen for all the other metals. Thus, cell surface FptA has broad metal specificity at the binding stage but is much more selective for the metal uptake process. This uptake pathway does not appear to efficiently assimilate any metal other than Fe(3+). PMID:19329644

  3. Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

    PubMed Central

    Berrazeg, M.; Jeannot, K.; Ntsogo Enguéné, Véronique Yvette; Broutin, I.; Loeffert, S.; Fournier, D.

    2015-01-01

    Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting. PMID:26248364

  4. Identification of Five Structurally Unrelated Quorum-Sensing Inhibitors of Pseudomonas aeruginosa from a Natural-Derivative Database

    PubMed Central

    Tan, Sean Yang-Yi; Chua, Song-Lin; Chen, Yicai; Rice, Scott A.; Kjelleberg, Staffan; Nielsen, Thomas E.; Givskov, Michael

    2013-01-01

    Bacteria communicate by means of small signal molecules in a process termed quorum sensing (QS). QS enables bacteria to organize their activities at the population level, including the coordinated secretion of virulence factors. Certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), have been shown to effectively block QS and subsequently attenuate the virulence of Pseudomonas aeruginosa, as well as increasing its susceptibility to both antibiotics and the immune system. In this study, a structure-based virtual screening (SB-VS) approach was used for the discovery of novel QSI candidates. Three-dimensional structures of 3,040 natural compounds and their derivatives were obtained, after which molecular docking was performed using the QS receptor LasR as a target. Based on docking scores and molecular masses, 22 compounds were purchased to determine their efficacies as quorum-sensing inhibitors. Using a live reporter assay for quorum sensing, 5 compounds were found to be able to inhibit QS-regulated gene expression in P. aeruginosa in a dose-dependent manner. The most promising compound, G1, was evaluated by isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis, and it was found to significantly affect the abundance of 46 proteins (19 were upregulated; 27 were downregulated) in P. aeruginosa PAO1. It specifically reduced the expression of several quorum-sensing-regulated virulence factors, such as protease IV, chitinase, and pyoverdine synthetases. G1 was also able to reduce extracellular DNA release and inhibited the secretion of the virulence factor, elastase, whose expression is regulated by LasR. These results demonstrate the utility of SB-VS for the discovery of target-specific QSIs. PMID:24002091

  5. Identification of five structurally unrelated quorum-sensing inhibitors of Pseudomonas aeruginosa from a natural-derivative database.

    PubMed

    Tan, Sean Yang-Yi; Chua, Song-Lin; Chen, Yicai; Rice, Scott A; Kjelleberg, Staffan; Nielsen, Thomas E; Yang, Liang; Givskov, Michael

    2013-11-01

    Bacteria communicate by means of small signal molecules in a process termed quorum sensing (QS). QS enables bacteria to organize their activities at the population level, including the coordinated secretion of virulence factors. Certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), have been shown to effectively block QS and subsequently attenuate the virulence of Pseudomonas aeruginosa, as well as increasing its susceptibility to both antibiotics and the immune system. In this study, a structure-based virtual screening (SB-VS) approach was used for the discovery of novel QSI candidates. Three-dimensional structures of 3,040 natural compounds and their derivatives were obtained, after which molecular docking was performed using the QS receptor LasR as a target. Based on docking scores and molecular masses, 22 compounds were purchased to determine their efficacies as quorum-sensing inhibitors. Using a live reporter assay for quorum sensing, 5 compounds were found to be able to inhibit QS-regulated gene expression in P. aeruginosa in a dose-dependent manner. The most promising compound, G1, was evaluated by isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis, and it was found to significantly affect the abundance of 46 proteins (19 were upregulated; 27 were downregulated) in P. aeruginosa PAO1. It specifically reduced the expression of several quorum-sensing-regulated virulence factors, such as protease IV, chitinase, and pyoverdine synthetases. G1 was also able to reduce extracellular DNA release and inhibited the secretion of the virulence factor, elastase, whose expression is regulated by LasR. These results demonstrate the utility of SB-VS for the discovery of target-specific QSIs. PMID:24002091

  6. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

    PubMed

    van 't Wout, Emily F A; van Schadewijk, Annemarie; van Boxtel, Ria; Dalton, Lucy E; Clarke, Hanna J; Tommassen, Jan; Marciniak, Stefan J; Hiemstra, Pieter S

    2015-06-01

    Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346

  7. Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis.

    PubMed

    Wright, Elli A; Di Lorenzo, Valeria; Trappetti, Claudia; Liciardi, Manuele; Orru, Germano; Viti, Carlo; Bronowski, Christina; Hall, Amanda J; Darby, Alistair C; Oggioni, Marco R; Winstanley, Craig

    2015-01-30

    Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections. PMID:25475851

  8. Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

    PubMed Central

    2010-01-01

    Background In Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link. Results Pseudomonas aeruginosa PAO1 has two ampG paralogs, PA4218 (ampP) and PA4393 (ampG). Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates ampP and ampG encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both ampP and ampG are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either ampP or ampG. Both ampG and ampP are the second members of two independent two-gene operons. Analysis of the ampG and ampP operon expression using β-galactosidase transcriptional fusions showed that in PAO1, ampG operon expression is β-lactam and ampR-independent, while ampP operon expression is β-lactam and ampR-dependent. β-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP. Conclusions In P. aeruginosa, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an ampP and ampG dependent pathway, and at high concentrations where although both ampP and ampG play a role, ampG may be of greater importance. Both ampP and amp

  9. Capsule production by Pseudomonas aeruginosa

    SciTech Connect

    Lynn, A.R.

    1984-01-01

    Mucoid strains of Pseudomonas aeruginosa, associated almost exclusively with chronic respiratory infections in patients with cystic fibrosis, possess a capsule composed of alginic acid similar to one produced by Azotobacter vinelandii. Recent reports have provided evidence that the biosynthetic pathway for alginate in P. aeruginosa may differ from the pathway proposed for A. vinelandii in that synthesis in P. aeruginosa may occur by way of the Entner-Doudoroff pathway. Incorporation of isotope from (6-/sup 14/C)glucose into alginate by both P. aueroginosa and A. vinelandii was 10-fold greater than that from either (1-/sup 14/C)/sup -/ or (2-/sup 14/C)glucose, indicating preferential utilization of the bottom half of the glucose molecule for alginate biosynthesis. These data strongly suggest that the Entner-Doudoroff pathway plays a major role in alginate synthesis in both P. aeruginosa and A. vinelandii. The enzymes of carbohydrate metabolism in mucoid strains of P. aeruginosa appear to be unchanged whether alignate is actively produced or not and activities do not differ significantly from nonmucoid strain PAO.

  10. A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance

    PubMed Central

    Moyano, Alejandro J.; Krapp, Adriana R.; Mondotte, Juan A.; Bocco, José L.; Saleh, Maria-Carla; Carrillo, Néstor; Smania, Andrea M.

    2014-01-01

    Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P . aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments. PMID:24550745

  11. The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants.

    PubMed

    Oliver, Antonio; Baquero, Fernando; Blázquez, Jesús

    2002-03-01

    We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype. PMID:11952911

  12. Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

    PubMed Central

    Tanaka, E; Kawamoto, S; Fukushima, J; Hamajima, K; Onishi, H; Miyagi, Y; Inami, S; Morihara, K; Okuda, K

    1991-01-01

    The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain. Images PMID

  13. Derivatives of Plant Phenolic Compound Affect the Type III Secretion System of Pseudomonas aeruginosa via a GacS-GacA Two-Component Signal Transduction System

    PubMed Central

    Yamazaki, Akihiro; Li, Jin; Zeng, Quan; Khokhani, Devanshi; Hutchins, William C.; Yost, Angela C.; Biddle, Eulandria; Toone, Eric J.

    2012-01-01

    Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening of exoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers of P. aeruginosa PAO1. These compounds alter exoS transcription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression of exoS through the GacSA-RsmYZ-RsmA-ExsA regulatory pathway. PMID:21968370

  14. The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase.

    PubMed

    Reimmann, C; Beyeler, M; Latifi, A; Winteler, H; Foglino, M; Lazdunski, A; Haas, D

    1997-04-01

    The global activator GacA, a highly conserved response regulator in Gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. The gacA gene of Pseudomonas aeruginosa PAO1 was isolated and its role in cell-density-dependent gene expression was characterized. Mutational inactivation of gacA resulted in delayed and reduced formation of the cell-density signal N-butyryl-L-homoserine lactone (BHL), of the cognate transcriptional activator RhIR (VsmR), and of the transcriptional activator LasR, which is known to positively regulate RhIR expression. Amplification of gacA on a multicopy plasmid caused precocious and enhanced production of BHL, RhIR and LasR. In parallel, the gacA gene dosage markedly influenced the BHL/RhIR-dependent formation of the cytotoxic compounds pyocyanin and cyanide and the exoenzyme lipase. However, the concentrations of another known cell-density signal of P. aeruginosa, N-oxododecanoyl-L-homoserine lactone, did not always match BHL concentrations. A model accounting for these observations places GacA function upstream of LasR and RhIR in the complex, cell-density-dependent signal-transduction pathway regulating several exoproducts and virulence factors of P. aeruginosa via BHL. PMID:9159518

  15. Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics.

    PubMed

    Arevalo-Ferro, Catalina; Hentzer, Morten; Reil, Gerold; Görg, Angelika; Kjelleberg, Staffan; Givskov, Michael; Riedel, Kathrin; Eberl, Leo

    2003-12-01

    The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the

  16. Dissecting the Machinery That Introduces Disulfide Bonds in Pseudomonas aeruginosa

    PubMed Central

    Arts, Isabelle S.; Ball, Geneviève; Leverrier, Pauline; Garvis, Steven; Nicolaes, Valérie; Vertommen, Didier; Ize, Bérengère; Tamu Dufe, Veronica; Messens, Joris; Voulhoux, Romé; Collet, Jean-François

    2013-01-01

    ABSTRACT Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. PMID:24327342

  17. Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation

    PubMed Central

    Dueholm, Morten S; Søndergaard, Mads T; Nilsson, Martin; Christiansen, Gunna; Stensballe, Allan; Overgaard, Michael T; Givskov, Michael; Tolker-Nielsen, Tim; Otzen, Daniel E; Nielsen, Per H

    2013-01-01

    The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation. PMID:23504942

  18. Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa

    PubMed Central

    Lee, Ji-Young; Park, Young Kyoung; Chung, Eun Seon; Na, In Young; Ko, Kwan Soo

    2016-01-01

    The increased reliance on colistin for treating multidrug-resistant Gram-negative bacterial infections has resulted in the emergence of colistin-resistant Pseudomonas aeruginosa. We attempted to identify genetic contributors to colistin resistance in vitro evolved isogenic colistin-resistant and -susceptible strains of two P. aeruginosa lineages (P5 and P155). Their evolutionary paths to acquisition and loss of colistin resistance were also tracked. Comparative genomic analysis revealed 13 and five colistin resistance determinants in the P5 and P155 lineages, respectively. Lipid A in colistin-resistant mutants was modified through the addition of 4-amino-L-arabinose; this modification was absent in colistin-susceptible revertant strains. Many amino acid substitutions that emerged during the acquisition of colistin resistance were reversed in colistin-susceptible revertants. We demonstrated that evolved colistin resistance in P. aeruginosa was mediated by a complicated regulatory network that likely emerges through diverse genetic alterations. Colistin-resistant P. aeruginosa became susceptible to the colistin upon its withdrawal because of genetic reversion. The mechanisms through which P. aeruginosa acquires and loses colistin resistance have implications on the treatment options that can be applied against P. aeruginosa infections, with respect to improving bactericidal efficacy and preventing further resistance to antibiotics. PMID:27150578

  19. Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa.

    PubMed

    Lee, Ji-Young; Park, Young Kyoung; Chung, Eun Seon; Na, In Young; Ko, Kwan Soo

    2016-01-01

    The increased reliance on colistin for treating multidrug-resistant Gram-negative bacterial infections has resulted in the emergence of colistin-resistant Pseudomonas aeruginosa. We attempted to identify genetic contributors to colistin resistance in vitro evolved isogenic colistin-resistant and -susceptible strains of two P. aeruginosa lineages (P5 and P155). Their evolutionary paths to acquisition and loss of colistin resistance were also tracked. Comparative genomic analysis revealed 13 and five colistin resistance determinants in the P5 and P155 lineages, respectively. Lipid A in colistin-resistant mutants was modified through the addition of 4-amino-L-arabinose; this modification was absent in colistin-susceptible revertant strains. Many amino acid substitutions that emerged during the acquisition of colistin resistance were reversed in colistin-susceptible revertants. We demonstrated that evolved colistin resistance in P. aeruginosa was mediated by a complicated regulatory network that likely emerges through diverse genetic alterations. Colistin-resistant P. aeruginosa became susceptible to the colistin upon its withdrawal because of genetic reversion. The mechanisms through which P. aeruginosa acquires and loses colistin resistance have implications on the treatment options that can be applied against P. aeruginosa infections, with respect to improving bactericidal efficacy and preventing further resistance to antibiotics. PMID:27150578

  20. Diverse Mobilized Class 1 Integrons Are Common in the Chromosomes of Pathogenic Pseudomonas aeruginosa Clinical Isolates

    PubMed Central

    Martinez, Elena; Marquez, Carolina; Ingold, Ana; Merlino, John; Djordjevic, Steven P.; Roy Chowdhury, Piklu

    2012-01-01

    Eleven clinical class 1 integron-containing Pseudomonas aeruginosa isolates from Australia and Uruguay were investigated for the genomic locations of these elements. Several novel class 1 integrons/transposons were found in at least four distinct locations in the chromosome, including genomic islands. These elements seem to be undergoing successful dispersal by lateral gene transfer since integrons were identified across several lineages and more than one clonal line. PMID:22271862

  1. The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

    PubMed Central

    Essoh, Christiane; Blouin, Yann; Loukou, Guillaume; Cablanmian, Arsher; Lathro, Serge; Kutter, Elizabeth; Thien, Hoang Vu; Vergnaud, Gilles; Pourcel, Christine

    2013-01-01

    Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages. PMID:23637754

  2. Impact of multidrug resistance on the pathogenicity of Pseudomonas aeruginosa: in vitro and in vivo studies.

    PubMed

    Gómez-Zorrilla, Silvia; Juan, Carlos; Cabot, Gabriel; Camoez, Mariana; Tubau, Fe; Oliver, Antonio; Dominguez, M Angeles; Ariza, Javier; Peña, Carmen

    2016-05-01

    The biological cost of multidrug resistance in Pseudomonas aeruginosa (PA) remains unclear. This study aimed to evaluate the relationship between pathogenicity and the resistance profile of different PA strains, including the most common epidemic high-risk clones. Nine PA strains were studied, including two reference strains, PAO1 and PA14 [both susceptible to all antipseudomonals (multiS)], and seven clinical strains comprising three clinical multiS strains, a non-clonal multidrug-resistant (MDR) strain and the high-risk MDR clones ST111, ST235 and ST175. In vitro studies were performed to investigate growth rate, type III secretion system (TTSS) genotype, cytotoxicity and invasiveness. Additionally, a peritonitis/sepsis model was used in C57BL/6 mice. The in vitro bacterial duplication time was shorter in clinical multiS strains than in MDR-PA (0.42±0.08h vs. 0.55±0.14h; P=0.023). Among the clinical strains, exoU(+) genotype was observed only in the epidemic clone ST235. In the animal model, the probability of mortality at 48h was 70% for clinical multiS strains vs. 7.5% for clinical MDR-PA (P<0.001, log-rank). The high-risk clone ST235 was the only MDR strain that was able to cause mortality. Bacterial concentrations in peritoneal fluid were higher in mice inoculated with multiS strains compared with MDR-PA [log CFU/mL, 8.95 (IQR 3.42-9.32) vs. 1.98 (IQR 1.08-2.80); P<0.001]. These data indicate that MDR profiles are associated with a reduction in virulence of PA in a murine model. Further studies are needed to elucidate the clinical implications of these results. PMID:27079153

  3. Transcriptome Analysis of the ArgR Regulon in Pseudomonas aeruginosa

    PubMed Central

    Lu, Chung-Dar; Yang, Zhe; Li, Wei

    2004-01-01

    Arginine metabolism in pseudomonads with multiple catabolic pathways for its utilization as carbon and nitrogen sources is of particular interest as the model system to study control of metabolic integration. We performed transcriptome analyses to identify genes controlled by the arginine regulatory protein ArgR and to better understand arginine metabolic pathways of P. aeruginosa. We compared gene expression in wild-type strain PAO1 with that in argR mutant strain PAO501 grown in glutamate minimal medium in the presence and absence of arginine. Ten putative transcriptional units of 28 genes were inducible by ArgR and arginine, including all known ArgR-regulated operons under aerobic conditions. The newly identified genes include the putative adcAB operon, which encodes a catabolic arginine decarboxylase and an antiporter protein, and PA0328, which encodes a hypothetical fusion protein of a peptidase and a type IV autotransporter. Also identified as members of the arginine network are the following solute transport systems: PA1971 (braZ) for branched-chain amino acids permease; PA2042 for a putative sodium:serine symporter; PA3934, which belongs to the family of small oligopeptide transporters; and PA5152-5155, which encodes components of an ABC transporter for a putative opine uptake system. The effect of arginine on the expression of these genes was confirmed by lacZ fusion studies and by DNA binding studies with purified ArgR. Only five transcriptional units of nine genes were qualified as repressible by ArgR and arginine, with three operons (argF, carAB, and argG) in arginine biosynthesis and two operons (gltBD and gdhA) in glutamate biosynthesis. These results indicate that ArgR is important in control of arginine and glutamate metabolism and that arginine and ArgR may have a redundant effect in inducing the uptake systems of certain compounds. PMID:15175299

  4. Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung

    PubMed Central

    Harmer, Christopher J.; Wynn, Matthew; Pinto, Rachel; Cordwell, Stuart; Rose, Barbara R.; Harbour, Colin; Triccas, James A.; Manos, Jim

    2015-01-01

    Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1. PMID:26252386

  5. Presence of the siderophores pyoverdine and pyochelin in the extracellular medium reduces toxic metal accumulation in Pseudomonas aeruginosa and increases bacterial metal tolerance.

    PubMed

    Braud, Armelle; Geoffroy, Valérie; Hoegy, Françoise; Mislin, Gaëtan L A; Schalk, Isabelle J

    2010-06-01

    In order to get access to iron, Pseudomonas aeruginosa strain PAO1 produces two major siderophores pyoverdine (PVD) and pyochelin (PCH). Both siderophores are able to chelate many other metals in addition to iron. However, despite this property, only iron is transported efficiently into the bacteria by the PVD and PCH uptake pathways. Growth studies with P. aeruginosa strains showed a lower sensitivity to toxic metals for the siderophore-producing strain than for the mutants unable to produce siderophores. Moreover, addition of PVD or PCH to the growth medium of a siderophore-deficient strain considerably reduced the toxicity of toxic metals present at concentrations of 100 µM in iron-limited and iron-supplemented growth conditions. Measurement by Inductively Coupled Plasma-Atomic Emission Spectrometry of the concentration of metals present in bacteria incubated with metals in the presence or absence of PVD or PCH indicated that both siderophores were able to sequester metals from the extracellular medium of the bacteria, decreasing metal diffusion into the bacteria. Pyoverdine was able to sequester Al(3+) , Co(2+) , Cu(2+) , Eu(3+) , Ni(2+) , Pb(2+) , Tb(3+) and Zn(2+) from the extracellular medium, and PCH, Al(3+) , Co(2+) , Cu(2+) , Ni(2+) , Pb(2+) and Zn(2+) . Moreover, the presence of 100 µM Cu(2+) and Ni(2+) increased PVD production by 290% and 380%, respectively, in a medium supplemented with iron. All these data suggest that PVD and PCH may contribute to P. aeruginosa resistance to heavy metals. PMID:23766115

  6. Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung.

    PubMed

    Harmer, Christopher J; Wynn, Matthew; Pinto, Rachel; Cordwell, Stuart; Rose, Barbara R; Harbour, Colin; Triccas, James A; Manos, Jim

    2015-01-01

    Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1. PMID:26252386

  7. Immunization with 3-oxododecanoyl-L-homoserine lactone-r-PcrV conjugate enhances survival of mice against lethal burn infections caused by Pseudomonas aeruginosa.

    PubMed

    Golpasha, Isar Dejban; Mousavi, Seyed Fazlollah; Owlia, Parviz; Siadat, Seyed Davar; Irani, Shiva

    2015-01-01

    Quorum Sensing and type III secretion system play an important role in the virulence of Pseudomonas (P.) aeruginosa in burn wound infections. We aimed to explore the feasibility of using 3-oxo-C₁₂-HSL-r-PcrV conjugate as a candidate vaccine against P. aeruginosa caused infections. 3-oxo-C₁₂-HSL-r-PcrV conjugate was prepared and used for immunization of mice (10 μg, subcutaneous, three times, at 2-week intervals). Mice were divided into five groups: I: PcrV; II: 3-oxo-C₁₂-HSL-r-PcrV (10 μg); III: 3-oxo-C₁₂-HSL-r-PcrV (20 μg); IV: 3-oxo-C₁₂-HSL; and V: PBS receiving groups. After each shot of immunization, total and isotype antibody responses against corresponding antigen were measured to determine the immunization efficacy. One month after the last immunization, all groups were burned and challenged subeschar with P. aeruginosa PAO1. Survival rate and bacterial quantity in the skin and internal organs (liver and spleen) were evaluated 25-hr after burn infection. Immunization with 3-oxo-C₁₂-HSL-r-PcrV significantly increased total IgG and specific subclass antibodies (IgG₁, IgG₂a, IgG₂b, and IgM) in the serum of the groups II and III compared to the control group (p<0.001). While all the control mice (PBS injected group) died within 2 days after bacterial challenge, 64% of the group I, 78% of group II, and 86% of group III, survived within 14 days after challenge. Interestingly, bacterial burden in the liver and spleen of 3-oxo-C₁₂-HSL-r-PcrV injected group (III) was significantly lower than the control group (p<0.001). The present study proposed two-component vaccine to inhibit Pseudomonas infections in burned mouse. PMID:26042508

  8. Nosocomial outbreak of OXA-18-producing Pseudomonas aeruginosa in Tunisia.

    PubMed

    Kalai Blagui, S; Achour, W; Abbassi, M S; Bejaoui, M; Abdeladhim, A; Ben Hassen, A

    2007-08-01

    Following systematic screening for ceftazidime-resistant (CAZ-R) Pseudomonas aeruginosa, 24 isolates producing extended-spectrum beta-lactamase (ESBL) were recovered during a 24-month period at the National Bone Marrow Transplant Centre of Tunisia. These isolates were from seven immunocompromised patients and from environmental swabs. ESBLs inhibited by clavulanic acid were detected by double-disk diffusion tests. Isoelectric focusing revealed that these isolates produced two to four beta-lactamases with pIs of 5.5, 6.1, 6.4, 7.6 or 8.2, and PCR detected the presence of bla(OXA-18), bla(SHV) and bla(TEM) genes in 24, 21 and two isolates, respectively. Pulsed-field gel electrophoresis defined two dominant genotypic groups: group A (16 isolates) and group B (four isolates). Sequencing of PCR products from representative isolates identified the bla(OXA-18) gene and revealed nucleotide sequences belonging to the bla(SHV-1) and bla(TEM-1) genes. Isolates producing OXA-18 belonged to genomic group A and were isolated from four immunocompromised patients in the haematology and graft units, and from two wash-basins in the graft unit. No immunocompromised patient harboured the clonal epidemic strain upon admission. This is the first report of the OXA-18-type ESBL in P. aeruginosa in Tunisia, and the first description of an outbreak caused by an OXA-18-producing strain of P. aeruginosa. PMID:17610599

  9. Tryptophan Inhibits Biofilm Formation by Pseudomonas aeruginosa

    PubMed Central

    Brandenburg, Kenneth S.; Rodriguez, Karien J.; McAnulty, Jonathan F.; Murphy, Christopher J.; Abbott, Nicholas L.; Schurr, Michael J.

    2013-01-01

    Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the d and l isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of d and l isoforms producing the greatest inhibitory effect. Addition of d-/l-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa. PMID:23318791

  10. Pseudomonas aeruginosa Population Structure Revisited

    PubMed Central

    Pirnay, Jean-Paul; Bilocq, Florence; Pot, Bruno; Cornelis, Pierre; Zizi, Martin; Van Eldere, Johan; Deschaght, Pieter; Vaneechoutte, Mario; Jennes, Serge; Pitt, Tyrone; De Vos, Daniel

    2009-01-01

    At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P

  11. Protective Role of Catalase in Pseudomonas aeruginosa Biofilm Resistance to Hydrogen Peroxide

    PubMed Central

    Elkins, James G.; Hassett, Daniel J.; Stewart, Philip S.; Schweizer, Herbert P.; McDermott, Timothy R.

    1999-01-01

    The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H2O2) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H2O2. Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H2O2, whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H2O2. The katB mutant, lacking the H2O2-inducible catalase KatB, was similar to the wild-type strain with respect to H2O2 resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H2O2, while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H2O2. Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H2O2, suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H2O2, while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of β-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H2O2, particularly at high H2O2

  12. Genetic Markers of Widespread Extensively Drug-Resistant Pseudomonas aeruginosa High-Risk Clones

    PubMed Central

    Cabot, Gabriel; Ocampo-Sosa, Alain A.; Domínguez, M. Angeles; Gago, Juan F.; Juan, Carlos; Tubau, Fe; Rodríguez, Cristina; Moyà, Bartolomé; Peña, Carmen; Martínez-Martínez, Luis

    2012-01-01

    Recent reports have revealed the existence of widespread extensively drug-resistant (XDR) P. aeruginosa high-risk clones in health care settings, but there is still scarce information on their specific chromosomal (mutational) and acquired resistance mechanisms. Up to 20 (10.5%) of 190 bloodstream isolates collected from 10 Spanish hospitals met the XDR criteria. A representative number (15 per group) of isolates classified as multidrug-resistant (MDR) (22.6%), resistant to 1 to 2 classes (moderately resistant [modR]) (23.7%), or susceptible to all antibiotics (multiS) (43.2%) were investigated in parallel. Multilocus sequence typing (MLST) analysis revealed that all XDR isolates belonged to sequence type 175 (ST175) (n = 19) or ST111 (n = 1), both recognized as international high-risk clones. Clonal diversity was higher among the 15 MDR isolates (4 ST175, 2 ST111, and 8 additional STs) and especially high among the 15 modR (13 different STs) and multiS (14 STs) isolates. The XDR/MDR pattern in ST111 isolates correlated with the production of VIM-2, but none of the ST175 isolates produced acquired β-lactamases. In contrast, the analysis of resistance markers in 12 representative isolates (from 7 hospitals) of ST175 revealed that the XDR pattern was driven by the combination of AmpC hyperproduction, OprD inactivation (Q142X), 3 mutations conferring high-level fluoroquinolone resistance (GyrA T83I and D87N and ParC S87W), a G195E mutation in MexZ (involved in MexXY-OprM overexpression), and the production of a class 1 integron harboring the aadB gene (gentamicin and tobramycin resistance). Of particular interest, in nearly all the ST175 isolates, AmpC hyperproduction was driven by a novel AmpR-activating mutation (G154R), as demonstrated by complementation studies using an ampR mutant of PAO1. This work is the first to describe the specific resistance markers of widespread P. aeruginosa XDR high-risk clones producing invasive infections. PMID:23045355

  13. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections

    PubMed Central

    Winstanley, Craig; O’Brien, Siobhan; Brockhurst, Michael A.

    2016-01-01

    Pseudomonas aeruginosa populations undergo a characteristic evolutionary adaptation during chronic infection of the cystic fibrosis (CF) lung, including reduced production of virulence factors, transition to a biofilm-associated lifestyle, and evolution of high-level antibiotic resistance. Populations of P. aeruginosa in chronic CF lung infections typically exhibit high phenotypic diversity, including for clinically important traits such as antibiotic resistance and toxin production, and this diversity is dynamic over time, making accurate diagnosis and treatment challenging. Population genomics studies reveal extensive genetic diversity within patients, including for transmissible strains the coexistence of highly divergent lineages acquired by patient-to-patient transmission. The inherent spatial structure and spatial heterogeneity of selection in the CF lung appears to play a key role in driving P. aeruginosa diversification. PMID:26946977

  14. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections.

    PubMed

    Winstanley, Craig; O'Brien, Siobhan; Brockhurst, Michael A

    2016-05-01

    Pseudomonas aeruginosa populations undergo a characteristic evolutionary adaptation during chronic infection of the cystic fibrosis (CF) lung, including reduced production of virulence factors, transition to a biofilm-associated lifestyle, and evolution of high-level antibiotic resistance. Populations of P. aeruginosa in chronic CF lung infections typically exhibit high phenotypic diversity, including for clinically important traits such as antibiotic resistance and toxin production, and this diversity is dynamic over time, making accurate diagnosis and treatment challenging. Population genomics studies reveal extensive genetic diversity within patients, including for transmissible strains the coexistence of highly divergent lineages acquired by patient-to-patient transmission. The inherent spatial structure and spatial heterogeneity of selection in the CF lung appears to play a key role in driving P. aeruginosa diversification. PMID:26946977

  15. Within-Host Evolution of Pseudomonas aeruginosa Reveals Adaptation toward Iron Acquisition from Hemoglobin

    PubMed Central

    Marvig, Rasmus Lykke; Damkiær, Søren; Khademi, S. M. Hossein; Markussen, Trine M.; Molin, Søren

    2014-01-01

    ABSTRACT Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients. PMID:24803516

  16. Within-host evolution of Pseudomonas aeruginosa reveals adaptation toward iron acquisition from hemoglobin.

    PubMed

    Marvig, Rasmus Lykke; Damkiær, Søren; Khademi, S M Hossein; Markussen, Trine M; Molin, Søren; Jelsbak, Lars

    2014-01-01

    ABSTRACT Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients. IMPORTANCE Most bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While

  17. A comparative study of coastal and clinical isolates of Pseudomonas aeruginosa

    PubMed Central

    Nair, Anusree V.; Joseph, Neetha; Krishna, Kiran; Sneha, K. G.; Tom, Neenu; Jangid, Kamlesh; Nair, Shanta

    2015-01-01

    Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium having a versatile metabolic potential and great ecological and clinical significance. The geographical distribution of P. aeruginosahas revealed the existence of an unbiased genetic arrangement in terrestrial isolates. In contrast, there are very few reports about P. aeruginosa strains from marine environments. The present work was aimed at studying the distribution of P. aeruginosa in coastal waters along the Indian Peninsula and understanding the environmental influence on genotypic, metabolic and phenotypic characteristics by comparing marine and clinical isolates. Of the 785 marine isolates obtained on selective media, only 32 (~4.1%) were identified as P. aeruginosa, based on their fatty acid methyl ester profiles. A low Euclidian distance value (< 2.5) obtained from chemotaxonomic analysis suggested that all the environmental (coastal and marine) isolates originated from a single species. While UPGMA analyses of AP-PCR and phenotypic profiles separated the environmental and clinical isolates, fatty acid biotyping showed overlapping between most clinical and environmental isolates. Our study revealed the genetic diversity among different environmental isolates of P. aeruginosa. While biogeographical separation was not evident based solely on phenotypic and metabolic typing, genomic and metatranscriptomic studies are more likely to show differences between these isolates. Thus, newer and more insightful methods are required to understand the ecological distribution of this complex group of bacteria. PMID:26413053

  18. Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for α-Ketoglutarate Utilization

    PubMed Central

    Tatke, Gorakh; Kumari, Hansi; Silva-Herzog, Eugenia; Ramirez, Lourdes; Mathee, Kalai

    2015-01-01

    Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved

  19. Physiological responses of Microcystis aeruginosa against the algicidal bacterium Pseudomonas aeruginosa.

    PubMed

    Zhou, Su; Yin, Hua; Tang, Shaoyu; Peng, Hui; Yin, Donggao; Yang, Yixuan; Liu, Zehua; Dang, Zhi

    2016-05-01

    Proliferation of cyanobacteria in aquatic ecosystems has caused water security problems throughout the world. Our preliminary study has showed that Pseudomonas aeruginosa can inhibit the growth of cyanobacterium, Microcystis aeruginosa. In order to explore the inhibitory mechanism of P. aeruginosa on the cell growth and synthesis of intracellular substances of M. aeruginosa, concentrations of Chlorophyll-a, intracellular protein, carbohydrate, enzyme activities and ion metabolism of M. aeruginosa, were investigated. The results indicated that 83.84% algicidal efficiency of P. aeruginosa was achieved after treatment for 7 days. The strain inhibited the reproduction of M. aeruginosa by impeding the synthesis of intracellular protein and carbohydrate of cyanobacterium, and only a very small part of intracellular protein and carbohydrate was detected after exposure to P. aeruginosa for 5 days. P. aeruginosa caused the alteration of intracellular antioxidant enzyme activity of M. aeruginosa, such as catalase, peroxidase. The accumulation of malondialdehyde aggravated membrane injury after treatment for 3 days. P. aeruginosa also affected the ion metabolism of cyanobacteria. The release of Na(+) and Cl(-) was significantly enhanced while the uptake of K(+), Ca(2+), Mg(2+), NO3(-) and SO4(2)(-) decreased. Surface morphology and intracellular structure of cyanobacteria and bacterial cells changed dramatically over time as evidenced by electron microscope (SEM) and transmission electron microscope (TEM) analysis. These results revealed that the algicidal activity of P. aeruginosa was primarily due to the fermentation liquid of P. aeruginosa that impeded the synthesis of intracellular protein and carbohydrate, and damaged the cell membrane through membrane lipid peroxidation. PMID:26866757

  20. Draft genome sequence of blaVeb-1, blaoxa-10producing multi-drug resistant (MDR) Pseudomonas aeruginosastrain VRFPA09 recovered from bloodstream infection

    PubMed Central

    Murugan, Nandagopal; Malathi, Jambulingam; Umashankar, Vetrivel; Madhavan, Hajib NarahariRao

    2015-01-01

    Pseudomonas aeruginosa (P. aeruginosa) bacteremia causes significant mortality rate due to emergence of multidrug resistant (MDR) nosocomial infections. We report the draft genome sequence of P. aeruginosa strain VRFPA09, a human bloodstream isolate, phenotypically proven as MDR strain. Whole genome sequencing on VRFPA09, deciphered betalactamase encoding blaveb-1 and blaOXA-10genes and multiple drug resistance, virulence factor encoding genes. PMID:26413042

  1. Spectroscopic Determination of Distinct Heme Ligands in Outer-Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa

    PubMed Central

    Smith, Aaron D.; Modi, Anuja R.; Sun, Shengfang; Dawson, John H.; Wilks, Angela

    2015-01-01

    Pseudomonas aeruginosa PAO1 encodes two outer membrane receptors, PhuR (Pseudomonas heme uptake) and HasR (heme assimilation system). The HasR receptor acquires heme through interaction with a secreted hemophore, HasAp. The non-hemophore-dependent PhuR is encoded along with proteins required for heme translocation into the cytoplasm. Herein, we report the isolation and characterization of the HasR and PhuR receptors. Absorption and MCD spectroscopy confirmed that, similar to other Gram-negative OM receptors, HasR coordinates heme through the conserved N-terminal plug His-221 and His-624 of the surface-exposed FRAP-loop. In contrast, PhuR showed distinct absorption and MCD spectra consistent with coordination through a Tyr residue. Sequence alignment of PhuR with all known Gram-negative OM heme receptors revealed a lack of a conserved His within the FRAP loop but two Tyr residues at positions 519 and 529. Site-directed mutagenesis and spectroscopic characterization confirmed Tyr-519 and the N-terminal plug His-124 provide the heme ligands in PhuR. We propose that PhuR and HasR represent nonredundant heme receptors capable of sensing and accessing heme across a wide range of physiological conditions on colonization and infection of the host. PMID:25849630

  2. Delayed Wound Healing in Diabetic (db/db) Mice with Pseudomonas aeruginosa Biofilm Challenge – A Model for the Study of Chronic Wounds

    PubMed Central

    Zhao, Ge; Hochwalt, Phillip C.; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.

    2010-01-01

    Chronic wounds are a major clinical problem that leads to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models. The goal of this study was to create a reproducible chronic wound model in diabetic mice by application of bacterial biofilm. Six millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms two days post-wounding, and covered with semi-occlusive dressings for two weeks. Most of the control wounds were epithelialized by 28 days post-wounding. In contrast, none of biofilm challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis and epidermal hyperplasia adjacent to challenged wounds- all indicators of an inflammatory non-healing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality and demonstrated delayed wound healing following biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing. PMID:20731798

  3. Effect of lipid and fatty acid composition of phospholipid vesicles on long-term stability and their response to Staphylococcus aureus and Pseudomonas aeruginosa supernatants.

    PubMed

    Marshall, Serena E; Hong, Sung-Ha; Thet, N T; Jenkins, A Toby A

    2013-06-11

    Phospholipid vesicles have been the focus of attention as potential vehicles for drug delivery, as they are biomimetic, easy to produce, and contain an aqueous compartment which can be used to carry hydrophilic material, such as drugs or dyes. Lipid vesicles used for this purpose present a particular challenge, as they are not especially stable and can rapidly break down and release their contents away from the target area, especially at physiological temperatures/environments. This study aims to investigate optimum methods for vesicle stabilization where the vesicles are employed as part of a system or technology that signals the presence of pathogenic bacteria via the effect of secreted cytolytic virulence factors on a sensor interface. A number of approaches have been investigated and are presented here as a systematic study of the long-term (14 day) stability at 37 °C, and at various pHs. The response of vesicles, both in suspension and within hydrogels, to Staphylococcus aureus (RN 4282) and Pseudomonas aeruginosa (PAO1) whole bacteria, and supernatants from overnight cultures of both (containing secreted proteins but free of cells), was measured via a sensitive encapsulated carboxyfluorescein release assay. The results showed that lipid chain length, cholesterol concentration, and stabilization via photopolymer stable components were critical in achieving stability. Finally, dispersion of the optimum vesicle formulation in hydrogel matrixes was investigated, culminating in the in vivo demonstration of a simple prototype wound dressing. PMID:23668367

  4. Burn sepsis: bacterial interference with Pseudomonas aeruginosa.

    PubMed

    Levenson, S M; Gruber, D K; Gruber, C; Watford, A; Seifter, E

    1981-05-01

    The pathogenicity of several strains of Pseudomonas aeruginosa for burned rats (3 degrees scald burns, 20% body surface) following topical application of the bacteria to the burn within 1 hour after burning was established. Following this, it was demonstrated that purposeful infection of such 3 degrees scald burns of rats by a strain of Ps. aeruginosa of low virulence (JB-77) protects the rats from the lethal effect of subsequent (48-hour) topical contamination of the burn by a highly virulent strain of Ps. aeruginosa (VA-134) (p less than 0.001). This finding was confirmed in a similar experiment beginning with germfree rats. When the challenge with the highly virulent Ps. aeruginosa strain was 24 hours (rather than 48 hours) after the burning and topical contamination of the burn with the low virulence strain of Ps. aeruginosa, there was little protection (p N.S.). When burned rats were given the low virulence strain of Ps. aeruginosa by gavage right after burning, there was not protection to subsequent (48 hours) challenge by topical application of the highly virulent strain of Ps. aeruginosa to the burn (11/12 vs 12/12 dying). Our finding that purposeful infection of a 3 degrees burn of rats (conventional and also germfree) by a strain of Ps. aeruginosa of low virulence protects from the lethal effect of subsequent (48-hour) topical contamination of the burn by a highly virulent strain of Ps. aeruginosa is due, we believe, to direct bacterial interference between the two strains of pseudomonas. PMID:6785444

  5. Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes.

    PubMed Central

    Stapleton, M J; Jagger, K S; Warren, R L

    1984-01-01

    Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease. Images PMID:6317657

  6. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway.

    PubMed Central

    Vander Wauven, C; Piérard, A; Kley-Raymann, M; Haas, D

    1984-01-01

    Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (O2 at less than 1 ppm), growth could be measured as an increase in protein and proceeded in a non-exponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAO1 grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arcB, and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbamate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mutant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce. PMID:6438064

  7. Carbapenem-resistant Pseudomonas aeruginosa strains from a Spanish hospital: characterization of metallo-beta-lactamases, porin OprD and integrons.

    PubMed

    Rojo-Bezares, Beatriz; Estepa, Vanesa; Cebollada, Rocío; de Toro, María; Somalo, Sergio; Seral, Cristina; Castillo, Francisco Javier; Torres, Carmen; Sáenz, Yolanda

    2014-05-01

    Molecular typing and mechanisms of carbapenem resistance such as alterations in porin OprD and presence of metallo-beta-lactamases (MBLs), as well as integrons have been studied in a collection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Spanish hospital. One hundred and twenty-three CRPA isolates were recovered from different samples of 80 patients. Clonal relationship among CRPA was analyzed by SpeI-PFGE. Susceptibility testing to 11 antibiotics and MBL phenotype was determined by microdilution, IP/IPI E-test and double disc method. The oprD gene was studied by PCR and sequencing, and mutations were determined comparing with P. aeruginosa PAO1 sequence. Characterization of MBLs, and class 1 and 2 integrons were studied by PCR and sequencing. SDS-PAGE analysis of outer membrane proteins of selected strains was performed. Seventy-four-per-cent of patients with CRPA were hospitalised in the ICU setting and 50% had long hospitalization stays. Sixty-four different PFGE patterns were detected, and 87 CRPA strains were further analyzed. MBL phenotype was detected in 43 of 87 strains (49.4%), which contained blaVIM-2 gene inside class 1 integrons. VIM-2-producing strains belonged to lineages ST175, ST235, and ST973. A great diversity of nucleotide insertions, deletions, and mutations in oprD gene, and the presence of a new insertion sequence (ISPa45) truncating oprD were identified among CRPA strains. Class 1 integrons were detected in 75% of CRPA strains, blaVIM-2 and the new arrangement aac(3)-Ia+ISPa34+aadA1 (named as In661) being the most frequent gene-cassette arrays detected. Other gene cassettes detected in integrons were: aadB, aadA6, aadA7, aac(6')-Ib', and blaOXA-46. PMID:24594145

  8. Pseudomonas aeruginosa and Achromobacter sp. Clonal Selection Leads to Successive Waves of Contamination of Water in Dental Care Units

    PubMed Central

    Abdouchakour, Fatima; Dupont, Chloé; Grau, Delphine; Aujoulat, Fabien; Mournetas, Patricia; Marchandin, Hélène; Parer, Sylvie; Gibert, Philippe; Valcarcel, Jean

    2015-01-01

    Dental care unit waterlines (DCUWs) consist of complex networks of thin tubes that facilitate the formation of microbial biofilms. Due to the predilection toward a wet environment, strong adhesion, biofilm formation, and resistance to biocides, Pseudomonas aeruginosa, a major human opportunistic pathogen, is adapted to DCUW colonization. Other nonfermentative Gram-negative bacilli, such as members of the genus Achromobacter, are emerging pathogens found in water networks. We reported the 6.5-year dynamics of bacterial contamination of waterlines in a dental health care center with 61 dental care units (DCUs) connected to the same water supply system. The conditions allowed the selection and the emergence of clones of Achromobacter sp. and P. aeruginosa characterized by multilocus sequence typing, multiplex repetitive elements-based PCR, and restriction fragment length polymorphism in pulsed-field gel electrophoresis, biofilm formation, and antimicrobial susceptibility. One clone of P. aeruginosa and 2 clones of Achromobacter sp. colonized successively all of the DCUWs: the last colonization by P. aeruginosa ST309 led to the closing of the dental care center. Successive dominance of species and clones was linked to biocide treatments. Achromobacter strains were weak biofilm producers compared to P. aeruginosa ST309, but the coculture of P. aeruginosa and Achromobacter enhanced P. aeruginosa ST309 biofilm formation. Intraclonal genomic microevolution was observed in the isolates of P. aeruginosa ST309 collected chronologically and in Achromobacter sp. clone A. The contamination control was achieved by a complete reorganization of the dental health care center by removing the connecting tubes between DCUs. PMID:26296724

  9. Pseudomonas aeruginosa and Achromobacter sp. clonal selection leads to successive waves of contamination of water in dental care units.

    PubMed

    Abdouchakour, Fatima; Dupont, Chloé; Grau, Delphine; Aujoulat, Fabien; Mournetas, Patricia; Marchandin, Hélène; Parer, Sylvie; Gibert, Philippe; Valcarcel, Jean; Jumas-Bilak, Estelle

    2015-11-01

    Dental care unit waterlines (DCUWs) consist of complex networks of thin tubes that facilitate the formation of microbial biofilms. Due to the predilection toward a wet environment, strong adhesion, biofilm formation, and resistance to biocides, Pseudomonas aeruginosa, a major human opportunistic pathogen, is adapted to DCUW colonization. Other nonfermentative Gram-negative bacilli, such as members of the genus Achromobacter, are emerging pathogens found in water networks. We reported the 6.5-year dynamics of bacterial contamination of waterlines in a dental health care center with 61 dental care units (DCUs) connected to the same water supply system. The conditions allowed the selection and the emergence of clones of Achromobacter sp. and P. aeruginosa characterized by multilocus sequence typing, multiplex repetitive elements-based PCR, and restriction fragment length polymorphism in pulsed-field gel electrophoresis, biofilm formation, and antimicrobial susceptibility. One clone of P. aeruginosa and 2 clones of Achromobacter sp. colonized successively all of the DCUWs: the last colonization by P. aeruginosa ST309 led to the closing of the dental care center. Successive dominance of species and clones was linked to biocide treatments. Achromobacter strains were weak biofilm producers compared to P. aeruginosa ST309, but the coculture of P. aeruginosa and Achromobacter enhanced P. aeruginosa ST309 biofilm formation. Intraclonal genomic microevolution was observed in the isolates of P. aeruginosa ST309 collected chronologically and in Achromobacter sp. clone A. The contamination control was achieved by a complete reorganization of the dental health care center by removing the connecting tubes between DCUs. PMID:26296724

  10. Pseudomonas aeruginosa wound infection involves activation of its iron acquisition system in response to fascial contact

    PubMed Central

    Kim, M.; Christley, S.; Khodarev, N. N.; Fleming, I.; Huang, Y.; Chang, E.; Zaborina, O.; Alverdy, J.

    2015-01-01

    Background Wound infections are traditionally thought to occur when microbial burden exceeds the innate clearance capacity of host immune system. Here we introduce the idea that the wound environment itself plays a significant contributory role to wound infection. Methods We developed a clinically relevant murine model of soft tissue infection to explore the role of activation of microbial virulence in response to tissue factors as a mechanism by which pathogenic bacteria cause wound infections. Mice underwent abdominal skin incision and light muscle injury with a crushing forceps versus skin incision alone followed by topical inoculation of P. aeruginosa. Mice were sacrificed on postoperative day 6 and abdominal tissues analyzed for clinical signs of wound infection. To determine if specific wound tissues components induce bacterial virulence, P. aeruginosa was exposed to skin, fascia, and muscle. Results Gross wound infection due to P. aeruginosa was observed to be significantly increased in injured tissues vs non-injured (80% vs 10%) tissues (n=20/group, p<0.0001). Exposure of P. aeruginosa to individual tissue components demonstrated that fascia significantly induced bacterial virulence as judged by the production of pyocyanin, a redox-active phenazine compound known to kill immune cells. Whole genome transcriptional profiling of P. aeruginosa exposed to fascia demonstrated activation of multiple genes responsible for the synthesis of the iron scavenging molecule pyochelin. Conclusion We conclude that wound elements, in particular fascia, may play a significant role in enhancing the virulence of P. aeruginosa and may contribute to the pathogenesis of clinical wound infection. PMID:25807409

  11. Proteomics of the oxidative stress response induced by hydrogen peroxide and paraquat reveals a novel AhpC-like protein in Pseudomonas aeruginosa.

    PubMed

    Hare, Nathan J; Scott, Nichollas E; Shin, Eun Hye H; Connolly, Angela M; Larsen, Martin R; Palmisano, Giuseppe; Cordwell, Stuart J

    2011-08-01

    Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P. aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide and superoxide-inducing paraquat. 2-DE and MS allowed for the identification of 59 and 58 protein spots that were statistically significantly altered following H(2) O(2) and paraquat treatment, respectively. We observed a unique mass and pI pattern for alkylhydroperoxide reductase C (AhpC) that was replicated by hypothetical protein PA3529 following treatment with 10 mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous to that known for AhpC, and by cysteine overoxidation to Cys-sulfonic acid (SO(3) H) postoxidative stress. PA3529 is therefore a functional AhpC paralog expressed under H(2) O(2) stress. Following paraquat-induced oxidative stress, we also observed the overabundance and likely oxidative modification of a second hypothetical antioxidant protein (PA3450) that shares sequence similarity with 1-Cys peroxiredoxins. Other induced proteins included known oxidative stress proteins (superoxide dismutase and catalase), as well as those involved in iron acquisition (siderophore biosynthesis and receptor proteins FpvA and FptA) and hypothetical proteins, including others predicted to be antioxidants (PA0848). These data suggest that P. aeruginosa contains a plethora of novel

  12. Occurrence of Pseudomonas aeruginosa in Kuwait soil.

    PubMed

    Al-Saleh, Esmaeil; Akbar, Abrar

    2015-02-01

    Environmentally ubiquitous bacteria such as Pseudomonas aeruginosa evolved mechanisms to adapt and prevail under diverse conditions. In the current investigation, strains of P. aeruginosa demonstrating high rates of crude oil utilization and tolerance to high concentrations of heavy metals were found in both crude oil-contaminated and uncontaminated sites in Kuwait, and were dominant in the contaminated sites. The incidence of P. aeruginosa in tested soils implies the definitive pattern of crude oil contamination in the selection of the bacterial population in petroleum-contaminated sites in Kuwait. Surprisingly, the unculturable P. aeruginosa in different soil samples showed significant high similarity coefficients based on 16S-RFLP analyses, implying that the unculturable fraction of existing bacterial population in environmental samples is more stable and, hence, reliable for phylogenetic studies compared to the culturable bacteria. PMID:25014900

  13. Antibiotic Conditioned Growth Medium of Pseudomonas Aeruginosa

    ERIC Educational Resources Information Center

    Benathen, Isaiah A.; Cazeau, Barbara; Joseph, Njeri

    2004-01-01

    A simple method to study the consequences of bacterial antibiosis after interspecific competition between microorganisms is presented. Common microorganisms are used as the test organisms and Pseudomonas aeruginosa are used as the source of the inhibitor agents.

  14. The Widespread Multidrug-Resistant Serotype O12 Pseudomonas aeruginosa Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters

    PubMed Central

    Thrane, Sandra Wingaard; Taylor, Véronique L.; Freschi, Luca; Kukavica-Ibrulj, Irena; Boyle, Brian; Laroche, Jérôme; Pirnay, Jean-Paul; Lévesque, Roger C.

    2015-01-01

    ABSTRACT The O-specific antigen (OSA) in Pseudomonas aeruginosa lipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classified P. aeruginosa into 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how the P. aeruginosa OSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83 P. aeruginosa strains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinct P. aeruginosa strains. Specifically, we identified a “serotype island” ranging from 62 kb to 185 kb containing the P. aeruginosa O12 OSA gene cluster, an antibiotic resistance determinant (gyrAC248T), and other genes that have been transferred between P. aeruginosa strains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related to P. aeruginosa PA7. Acquisition and recombination of the “serotype island” resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings. PMID:26396243

  15. Two mechanisms of killing of Pseudomonas aeruginosa by tobramycin assessed at multiple inocula via mechanism-based modeling.

    PubMed

    Bulitta, Jürgen B; Ly, Neang S; Landersdorfer, Cornelia B; Wanigaratne, Nicholin A; Velkov, Tony; Yadav, Rajbharan; Oliver, Antonio; Martin, Lisandra; Shin, Beom Soo; Forrest, Alan; Tsuji, Brian T

    2015-04-01

    Bacterial resistance is among the most serious threats to human health globally, and many bacterial isolates have emerged that are resistant to all antibiotics in monotherapy. Aminoglycosides are often used in combination therapies against severe infections by multidrug-resistant bacteria. However, models quantifying different antibacterial effects of aminoglycosides are lacking. While the mode of aminoglycoside action on protein synthesis has often been studied, their disruptive action on the outer membrane of Gram-negative bacteria remains poorly characterized. Here, we developed a novel quantitative model for these two mechanisms of aminoglycoside action, phenotypic tolerance at high bacterial densities, and adaptive bacterial resistance in response to an aminoglycoside (tobramycin) against three Pseudomonas aeruginosa strains. At low-intermediate tobramycin concentrations (<4 mg/liter), bacterial killing due to the effect on protein synthesis was most important, whereas disruption of the outer membrane was the predominant killing mechanism at higher tobramycin concentrations (≥8 mg/liter). The extent of killing was comparable across all inocula; however, the rate of bacterial killing and growth was substantially lower at the 10(8.9) CFU/ml inoculum than that at the lower inocula. At 1 to 4 mg/liter tobramycin for strain PAO1-RH, there was a 0.5- to 6-h lag time of killing that was modeled via the time to synthesize hypothetical lethal protein(s). Disruption of the outer bacterial membrane by tobramycin may be critical to enhance the target site penetration of antibiotics used in synergistic combinations with aminoglycosides and thereby combat multidrug-resistant bacteria. The two mechanisms of aminoglycoside action and the new quantitative model hold great promise to rationally design novel, synergistic aminoglycoside combination dosage regimens. PMID:25645838

  16. In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients.

    PubMed

    Olszak, Tomasz; Zarnowiec, Paulina; Kaca, Wieslaw; Danis-Wlodarczyk, Katarzyna; Augustyniak, Daria; Drevinek, Pavel; de Soyza, Anthony; McClean, Siobhán; Drulis-Kawa, Zuzanna

    2015-07-01

    The goal of the study was to determine the relationship between in vitro/in vivo efficacy of environmental Pseudomonas phages and certain phenotypical properties of Pseudomonas aeruginosa (PA) strains. We studied the diversity between particular isolates and determined phage sensitivity in vitro and in vivo in the Galleria mellonella insect model. Twenty-eight lytic bacteriophages specific for PA were tested against 121 CF PA isolates including 29 mucoid PA strains. Most strains from cystic fibrosis (CF) patients were lysed by at least three phages (93.6 %), but completely insensitive strains were also present (6.4 %). Two phages PA5oct and KT28 exhibited high rates of lytic potency on 55-68 % of PA strains (72-86 % of mucoid isolates). We further explored phage activity against six PA strains (CF and non-CF) in vitro, comparing clonal differences in phage susceptibility with bacterial properties such as the ability to form biofilms, mucosity, twitching motility, and biochemical profiles. We observed the relationship between variation in phage susceptibility and Fourier transform infrared spectroscopy (FTIR) analysis in the spectra window of carbohydrates. The protective efficacy of two selected phages against PA PAO1 and 0038 infection was confirmed in vivo in G. mellonella larvae. Generally, the wax moth model results confirmed the data from in vitro assays, but in massive infection of CF isolates, the application of lytic phages probably led to the release of toxic compound causing an increase in larvae mortality. We assumed that apart of in vitro phage activity testing, a simple and convenient wax moth larvae model should be applied for the evaluation of in vivo effectiveness of particular phage preparations. PMID:25758956

  17. Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections.

    PubMed

    Rojo-Molinero, Estrella; Macià, María D; Rubio, Rosa; Moyà, Bartolomé; Cabot, Gabriel; López-Causapé, Carla; Pérez, José L; Cantón, Rafael; Oliver, Antonio

    2016-05-01

    Traditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance in Pseudomonas aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t = 2), 4 (t = 4), and 6 days (t = 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one

  18. Vaccination against respiratory Pseudomonas aeruginosa infection

    PubMed Central

    Grimwood, Keith; Kyd, Jennelle M; Owen, Suzzanne J; Massa, Helen M; Cripps, Allan W

    2014-01-01

    Respiratory infections caused by Pseudomonas aeruginosa are a major clinical problem globally, particularly for patients with chronic pulmonary disorders, such as those with cystic fibrosis (CF), non-CF bronchiectasis (nCFB) and severe chronic obstructive pulmonary disease (COPD). In addition, critically ill and immunocompromised patients are also at significant risk of P. aeruginosa infection. For almost half a century, research efforts have focused toward development of a vaccine against infections caused by P. aeruginosa, but a licensed vaccine is not yet available. Significant advances in identifying potential vaccine antigens have been made. Immunisations via both the mucosal and systemic routes have been trialled in animal models and their effectiveness in clearing acute infections demonstrated. The challenge for translation of this research to human applications remains, since P. aeruginosa infections in the human respiratory tract can present both as an acute or chronic infection. In addition, immunisation prior to infection may not be possible for many patients with CF, nCFB or COPD. Therefore, development of a therapeutic vaccine provides an alternative approach for treatment of chronic infection. Preliminary animal and human studies suggest that mucosal immunisation may be effective as a therapeutic vaccine against P. aeruginosa respiratory infections. Nevertheless, more research is needed to improve our understanding of the basic biology of P. aeruginosa and the mechanisms needed to upregulate the induction of host immune pathways to prevent infection. Recognition of variability in the host immune responses for a range of patient health conditions at risk from P. aeruginosa infection is also required to support development of a successful vaccine delivery strategy and vaccine. Activation of mucosal immune responses may provide improved efficacy of vaccination for P. aeruginosa during both acute exacerbations and chronic infection. PMID:25483510

  19. [New Virulent Bacteriophages Active against Multiresistant Pseudomonas aeruginosa Strains].

    PubMed

    Balarjishvili, N Sh; Kvachadze, L I; Kutateladze, M I; Meskhi, T Sh; Pataridze, T K; Berishvili, T A; Tevdoradze, E Sh

    2015-01-01

    The sensitivity of 512 newly isolated Pseudomonas aeruginosa clinical strains to six classes of anti-microbial preparations has been studied. Antibiotic-resistant strains were selected and genotyped. Three new virulent bacteriophages of the families Myoviridae and Podoviridae were isolated against these strains. The parameters of the intracellular phage development cycle were established, and the influence of inactivating factors (temperature, pH, and UV exposure) on phage viability was studied. The molecular weight of the phage genome was determined. Phage DNA restriction analysis and polyacrylamide gel electrophoresis in the presence of envelope protein SDS were carried out. The plating efficacy of phages on 28 genetically distant antibiotic-resistant P. aeruginosa strains was studied. It was established that 26 of them were lysed by phages with a high efficacy. The range of antibacterial action of the studied phages and their mixtures on 427 multi-drug-resistant clinical isolates was assessed. It is shown that including these phages in one multicomponent preparation enhanced their lytic activity. PMID:26859962

  20. A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and AlgT/U activity results in the loss of alginate production

    PubMed Central

    Sautter, Robert; Ramos, Damaris; Schneper, Lisa; Ciofu, Oana; Wassermann, Tina; Heydorn, Arne; Hentzer, Morton; Høiby, Niels; Kharazmi, Arsalan; Molin, Søren; DeVries, Caroline A.; Ohman, Dennis E.; Mathee, Kalai

    2016-01-01

    Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg+) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg+ due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About forty percent of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression. PMID:22088575

  1. Pathogen Special: Vibrio Cholerae, Pseudomonas Aeruginosa and Xylella Fastidiosa

    PubMed Central

    2000-01-01

    One could almost say that it is the latest fashion to sequence a bacterial genome. However, this would belittle the efforts of those working on these important organisms, whose data will greatly help those working on the prevention of disease in the fields of medicine and agriculture. In this feature we present a guided tour of the latest additions to the ‘sequenced microbes’ club. Vibrio cholerae is the causative agent of cholera, which is still a threat in countries with poor sanitation and unsafe drinking water. Pseudomonas aeruginosa is responsible for a large proportion of opportunistic human infections, typically infecting those with compromised immune systems, particularly cystic fibrosis patients, those patients on respirators and burn victims. Xylella fastidiosa is a plant pathogen that attacks citrus fruits by blocking the xylem, resulting in juiceless fruits of no commercial value. PMID:11119308

  2. Novel Multiscale Modeling Tool Applied to Pseudomonas aeruginosa Biofilm Formation

    PubMed Central

    Biggs, Matthew B.; Papin, Jason A.

    2013-01-01

    Multiscale modeling is used to represent biological systems with increasing frequency and success. Multiscale models are often hybrids of different modeling frameworks and programming languages. We present the MATLAB-NetLogo extension (MatNet) as a novel tool for multiscale modeling. We demonstrate the utility of the tool with a multiscale model of Pseudomonas aeruginosa biofilm formation that incorporates both an agent-based model (ABM) and constraint-based metabolic modeling. The hybrid model correctly recapitulates oxygen-limited biofilm metabolic activity and predicts increased growth rate via anaerobic respiration with the addition of nitrate to the growth media. In addition, a genome-wide survey of metabolic mutants and biofilm formation exemplifies the powerful analyses that are enabled by this computational modeling tool. PMID:24147108

  3. Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated Pseudomonas aeruginosa sp. Strain JQ-41.

    PubMed

    Song, Haihai; Zhou, Zhiren; Liu, Yuanxiu; Deng, Si; Xu, Heng

    2015-09-01

    A soil bacterium designated strain JQ-41, capable of growth on fenpropathrin as the sole carbon source and energy source, was isolated from a long-term pyrethroid insecticide-treated orchard. Based on the morphology, physio-biochemical characteristics, and 16S rDNA gene analysis, as well as the G+C content of the genomic DNA, the strain JQ-41 was identified as Pseudomonas aeruginosa. Up to 92.3% of 50 mg l(-1) fenpropathrin was degraded by P. aeruginosa strain at 30°C and pH 7 within 7 days. The kinetic parameters q max, K s, and K i were established to be 1.14 day(-1), 38.41 mg l(-1), and 137.67 mg l(-1), respectively, and the critical inhibitor concentration was determined to be 72.72 mg l(-1). Cell surface hydrophobicity of P. aeruginosa strain was enhanced during growth on fenpropathrin. Three metabolites from fenpropathrin degradation were identified by gas chromatography mass spectrometry, and then a possible degradation pathway was proposed. In addition, this isolate was also able to degrade a wide range of synthetic pyrethroid insecticides including cypermethrin, deltamethrin, bifenthrin, and cyhalothrin with the degradation process following the first-order kinetic model. Taken together, our results provide insights into the kinetics and mechanism of fenpropathrin degradation by P. aeruginosa strain and also highlight its promising potential in bioremediation of pyrethroid-contaminated environment. PMID:26068594

  4. Therapeutic effect of Pseudomonas aeruginosa phage YH30 on mink hemorrhagic pneumonia.

    PubMed

    Gu, Jingmin; Li, Xinwei; Yang, Mei; Du, Chongtao; Cui, Ziyin; Gong, Pengjuan; Xia, Feifei; Song, Jun; Zhang, Lei; Li, Juecheng; Yu, Chuang; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu

    2016-07-15

    Hemorrhagic pneumonia caused by Pseudomonas aeruginosa remains one of the most costly infectious diseases among farmed mink and commonly leads to large economic losses during mink production. The objective of this study was to investigate the potential of using phages as a therapy against hemorrhagic pneumonia in mink. A broad-host-range phage from the Podoviridae family, YH30, was isolated using the mink-originating P. aeruginosa (serotype G) D7 strain as a host. The genome of YH30 was 72,192bp (54.92% G+C), contained 86 open reading frames and lacked regions encoding known virulence factors, integration-related proteins or antibiotic resistance determinants. These characteristics make YH30 eligible for use in phage therapy. The results of a curative treatment experiment demonstrated that a single intranasal administration of YH30 was sufficient to cure hemorrhagic pneumonia in mink. The mean colony count of P. aeruginosa in the blood and lung of YH30-protected mink was less than 10(3) CFU/mL (g) within 24h of bacterial challenge and ultimately became undetectable, whereas that in unprotected mink reached more than 10(8) CFU/mL (g). Additionally, YH30 dramatically improved the pathological manifestations of lung injury in mink with hemorrhagic pneumonia. Our work demonstrates the potential of phages to treat P. aeruginosa-caused hemorrhagic pneumonia in mink. PMID:27283850

  5. The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa.

    PubMed

    Llamas, María A; Sparrius, Marion; Kloet, Roy; Jiménez, Connie R; Vandenbroucke-Grauls, Christina; Bitter, Wilbert

    2006-03-01

    Pseudomonas aeruginosa secretes two siderophores, pyoverdine and pyochelin, under iron-limiting conditions. These siderophores are recognized at the cell surface by specific outer membrane receptors, also known as TonB-dependent receptors. In addition, this bacterium is also able to incorporate many heterologous siderophores of bacterial or fungal origin, which is reflected by the presence of 32 additional genes encoding putative TonB-dependent receptors. In this work, we have used a proteomic approach to identify the inducing conditions for P. aeruginosa TonB-dependent receptors. In total, 11 of these receptors could be discerned under various conditions. Two of them are only produced in the presence of the hydroxamate siderophores ferrioxamine B and ferrichrome. Regulation of their synthesis is affected by both iron and the presence of a cognate siderophore. Analysis of the P. aeruginosa genome showed that both receptor genes are located next to a regulatory locus encoding an extracytoplasmic function sigma factor and a transmembrane sensor. The involvement of this putative regulatory locus in the specific induction of the ferrioxamine B and ferrichrome receptors has been demonstrated. These results show that P. aeruginosa has evolved multiple specific regulatory systems to allow the regulation of TonB-dependent receptors. PMID:16484199

  6. ENVIRONMENTAL AND MOLECULAR CHARACTERIZATION OF SYSTEMS WHICH ALTER GENOME STABILITY IN PSEUDOMONAS

    EPA Science Inventory

    Pseudomonas aeruginosa was used a model organism to study mechanisms that lead to genome alteration in freshwater microbial populations. ur studies demonstrated horizontal transmission by both transduction and conjugation in freshwater ecosystems and provided data that suggest th...

  7. Responses of Pseudomonas aeruginosa to antimicrobials

    PubMed Central

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2014-01-01

    Infections caused by Pseudomonas aeruginosa often are hard to treat; inappropriate chemotherapy readily selects multidrug-resistant P. aeruginosa. This organism can be exposed to a wide range of concentrations of antimicrobials during treatment; learning more about the responses of P. aeruginosa to antimicrobials is therefore important. We review here responses of the bacterium P. aeruginosa upon exposure to antimicrobials at levels below the inhibitory concentration. Carbapenems (e.g., imipenem) have been shown to induce the formation of thicker and more robust biofilms, while fluoroquinolones (e.g., ciprofloxacin) and aminoglycosides (e.g., tobramycin) have been shown to induce biofilm formation. Ciprofloxacin also has been demonstrated to enhance the frequency of mutation to carbapenem resistance. Conversely, although macrolides (e.g., azithromycin) typically are not effective against P. aeruginosa because of the pseudomonal outer-membrane impermeability and efflux, macrolides do lead to a reduction in virulence factor production. Similarly, tetracycline is not very effective against this organism, but is known to induce the type-III secretion system and consequently enhance cytotoxicity of P. aeruginosa in vivo. Of special note are the effects of antibacterials and disinfectants on pseudomonal efflux systems. Sub-inhibitory concentrations of protein synthesis inhibitors (aminoglycosides, tetracycline, chloramphenicol, etc.) induce the MexXY multidrug efflux system. This response is known to be mediated by interference with the translation of the leader peptide PA5471.1, with consequent effects on expression of the PA5471 gene product. Additionally, induction of the MexCD-OprJ multidrug efflux system is observed upon exposure to sub-inhibitory concentrations of disinfectants such as chlorhexidine and benzalkonium. This response is known to be dependent upon the AlgU stress response factor. Altogether, these biological responses of P. aeruginosa provide useful

  8. Responses of Pseudomonas aeruginosa to antimicrobials.

    PubMed

    Morita, Yuji; Tomida, Junko; Kawamura, Yoshiaki

    2014-01-01

    Infections caused by Pseudomonas aeruginosa often are hard to treat; inappropriate chemotherapy readily selects multidrug-resistant P. aeruginosa. This organism can be exposed to a wide range of concentrations of antimicrobials during treatment; learning more about the responses of P. aeruginosa to antimicrobials is therefore important. We review here responses of the bacterium P. aeruginosa upon exposure to antimicrobials at levels below the inhibitory concentration. Carbapenems (e.g., imipenem) have been shown to induce the formation of thicker and more robust biofilms, while fluoroquinolones (e.g., ciprofloxacin) and aminoglycosides (e.g., tobramycin) have been shown to induce biofilm formation. Ciprofloxacin also has been demonstrated to enhance the frequency of mutation to carbapenem resistance. Conversely, although macrolides (e.g., azithromycin) typically are not effective against P. aeruginosa because of the pseudomonal outer-membrane impermeability and efflux, macrolides do lead to a reduction in virulence factor production. Similarly, tetracycline is not very effective against this organism, but is known to induce the type-III secretion system and consequently enhance cytotoxicity of P. aeruginosa in vivo. Of special note are the effects of antibacterials and disinfectants on pseudomonal efflux systems. Sub-inhibitory concentrations of protein synthesis inhibitors (aminoglycosides, tetracycline, chloramphenicol, etc.) induce the MexXY multidrug efflux system. This response is known to be mediated by interference with the translation of the leader peptide PA5471.1, with consequent effects on expression of the PA5471 gene product. Additionally, induction of the MexCD-OprJ multidrug efflux system is observed upon exposure to sub-inhibitory concentrations of disinfectants such as chlorhexidine and benzalkonium. This response is known to be dependent upon the AlgU stress response factor. Altogether, these biological responses of P. aeruginosa provide useful

  9. Pseudomonas aeruginosa Dose-Response and Bathing Water Infection

    EPA Science Inventory

    Pseudomonas aeruginosa is the most commonly identified opportunistic pathogen associated with pool acquired bather disease. To better understand why this microorganism poses this protracted problem we recently appraised P. aeruginosa pool risk management. Much is known about the ...

  10. Developing an international Pseudomonas aeruginosa reference panel

    PubMed Central

    De Soyza, Anthony; Hall, Amanda J; Mahenthiralingam, Eshwar; Drevinek, Pavel; Kaca, Wieslaw; Drulis-Kawa, Zuzanna; Stoitsova, Stoyanka R; Toth, Veronika; Coenye, Tom; Zlosnik, James E A; Burns, Jane L; Sá-Correia, Isabel; De Vos, Daniel; Pirnay, Jean-Paul; Kidd, Timothy J; Reid, David; Manos, Jim; Klockgether, Jens; Wiehlmann, Lutz; Tümmler, Burkhard; McClean, Siobhán; Winstanley, Craig

    2013-01-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents. PMID:24214409

  11. Clonal complex Pseudomonas aeruginosa in horses.

    PubMed

    Kidd, Timothy J; Gibson, Justine S; Moss, Susan; Greer, Ristan M; Cobbold, Rowland N; Wright, John D; Ramsay, Kay A; Grimwood, Keith; Bell, Scott C

    2011-05-01

    Pseudomonas aeruginosa is associated with infectious endometritis in horses. Although infectious endometritis is often considered a venereal infection, there is relatively limited genotypic-based evidence to support this mode of transmission. The study sought to determine the relatedness between genital P. aeruginosa isolates collected from a limited geographical region using molecular strain typing. Enterobacterial repetitive intergenic consensus PCR typing was performed on 93 isolates collected between 2005 and 2009 from 2058 thoroughbred horses (including 18 stallions) at 66 studs. While P. aeruginosa was not detected in the stallions, 53/93 (57%) mares harbouring P. aeruginosa had clonally related strains, which included a single dominant genotype detected in 42 (45%) mares from 13 different studs. These novel findings suggest that most equine genital P. aeruginosa infections in this region may have been acquired from mechanisms other than direct horse to horse transmission. Instead, other potential acquisition pathways, as well as strain specific adaptation to the equine genital tract, should be investigated. PMID:21183294

  12. Comparison of the complete genome sequences of Pseudomonassyringae pv. syringae B728a and pv. tomato DC3000.

    SciTech Connect

    Feil, Helene; Feil, William S.; Chain, Patrick; Larimer, Frank; DiBartolo, Genevieve; Copeland, Alex; Lykidis, Athanasios; Trong,Stephen; Nolan, Matt; Goltsman, Eugene; Thiel, James; Malfatti,Stephanie; Loper, Joyce E.; Lapidus, Alla; Detter, John C.; Land, Miriam; Richardson, Paul M.; Kyrpides, Nikos C.; Ivanova, Natalia; Lindow, StevenE.