scholarly journals Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1407-1415 ◽  
Author(s):  
James E. A. Zlosnik ◽  
Gholam Reza Tavankar ◽  
Jacob G. Bundy ◽  
Dimitris Mossialos ◽  
Ronan O'Toole ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Toxic Agent ◽  
Stationary Phase Culture ◽  
Prolonged Incubation ◽  
Mutant Strains ◽  
Electron Sink ◽  
Culture Supernatants

Pseudomonas aeruginosa is an opportunistic pathogen which demonstrates considerable respiratory versatility, possessing up to five terminal oxidases. One oxidase, the cyanide-insensitive oxidase (CIO), has been previously shown to be resistant to the potent respiratory inhibitor cyanide, a toxin that is synthesized by this bacterium. This study investigated the physiological relationship between hydrogen cyanide production and the CIO. It was found that cyanide is produced in P. aeruginosa at similar levels irrespective of its complement of CIO, indicating that the CIO is not an obligatory electron sink for cyanide synthesis. However, MICs for cyanide and growth in its presence demonstrated that the CIO provides P. aeruginosa with protection against the effects of exogenous cyanide. Nevertheless, the presence of cyanide did not affect the viability of cio mutant strains compared to the wild-type during prolonged incubation in stationary phase. The detection of the fermentation end products acetate and succinate in stationary-phase culture supernatants suggests that P. aeruginosa, irrespective of its CIO complement, may in part rely upon fermentation for energy generation in stationary phase. Furthermore, the decrease in cyanide levels during incubation in sealed flasks suggested that active breakdown of HCN by the culture was taking place. To investigate the possibility that the CIO may play a role in pathogenicity, wild-type and cio mutant strains were tested in the paralytic killing model of Caenorhabditis elegans, a model in which cyanide is the principal toxic agent leading to nematode death. The CIO mutant had delayed killing kinetics, demonstrating that the CIO is required for full pathogenicity of P. aeruginosa in this animal model.

scholarly journals Pyocyanin Alters Redox Homeostasis and Carbon Flux through Central Metabolic Pathways in Pseudomonas aeruginosa PA14

2007 ◽  
Vol 189 (17) ◽  
pp. 6372-6381 ◽  
Author(s):  
Alexa Price-Whelan ◽  
Lars E. P. Dietrich ◽  
Dianne K. Newman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Metabolic Pathways ◽  
Carbon Flux ◽  
Redox Homeostasis ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Physiological Relevance ◽  
In The Wild ◽  
Phenazine Production

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with P. aeruginosa infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by P. aeruginosa PA14 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NAD+ in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in P. aeruginosa PA14, indicating that pyocyanin may also influence the intracellular redox state by decreasing carbon flux through central metabolic pathways.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1311-1322 ◽  
Author(s):  
Amanda S. Nouwens ◽  
Scott A. Beatson ◽  
Cynthia B. Whitchurch ◽  
Bradley J. Walsh ◽  
Herbert P. Schweizer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Proteome Analysis ◽  
Hypothetical Protein ◽  
Extracellular Proteins ◽  
Wild Type ◽  
Two Dimensional Gel Electrophoresis ◽  
Stationary Phase Culture ◽  
Chitin Binding Protein ◽  
Culture Supernatants

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique ‘hypothetical’ protein (PA0572), which could not be detected in the culture supernatants of Δlas mutants, although they were unaffected in Δrhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

Antibacterial effect of hyaluronan/chitosan nanofilm in the initial adhesion of Pseudomonas aeruginosa wild type, and IV pili and LPS mutant strains

2021 ◽  
pp. 101415
Author(s):  
Jacobo Hernandez-Montelongo ◽  
Gianlucca G. Nicastro ◽  
Thays de O. Pereira ◽  
Mariana Zavarize ◽  
Marisa M. Beppu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Effect ◽  
Wild Type ◽  
Initial Adhesion ◽  
Mutant Strains

scholarly journals TheprrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

2014 ◽  
Vol 83 (3) ◽  
pp. 863-875 ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Daniel A. Powell ◽  
Angela T. Nguyen ◽  
Maura O'Neill ◽  
Louise Djapgne ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Iron Homeostasis ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Genes Encoding ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Heme Regulation ◽  
Heme Binding Protein

Pseudomonas aeruginosais an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part ofP. aeruginosa'siron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem inP. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identifiedphuS, encoding a heme binding protein involved in heme acquisition, andvreR, encoding a previously identified regulator ofP. aeruginosavirulence genes, as novel targets ofprrF-mediated heme regulation. Finally, we showed that theprrFlocus encoding the PrrF and PrrH sRNAs is required forP. aeruginosavirulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2deletion mutant protects against future challenge with wild-typeP. aeruginosa. Combined, these data demonstrate that theprrF-encoded sRNAs are critical regulators ofP. aeruginosavirulence.

scholarly journals Yersinia pestis YbtU and YbtT Are Involved in Synthesis of the Siderophore Yersiniabactin but Have Different Effects on Regulation

2000 ◽  
Vol 68 (8) ◽  
pp. 4452-4461 ◽  
Author(s):  
Valerie A. Geoffroy ◽  
Jacqueline D. Fetherston ◽  
Robert D. Perry
Keyword(s):  
Yersinia Pestis ◽  
Iron Transport ◽  
Significant Loss ◽  
Gene Products ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Iron Deficient ◽  
High Pathogenicity ◽  
Mutant Strains ◽  
Culture Supernatants

ABSTRACT One prerequisite for the virulence of Yersinia pestis, causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of theY. pestis chromosome. Several gene products within the HPI have demonstrated functions in the synthesis or transport of Ybt. Here we examine the roles of ybtU and ybtT. In-frame mutations in ybtT or ybtU yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37°C but could be cross-fed by culture supernatants from a Ybt-producing strain of Y. pestis. TheybtU mutant failed to express four indicator Ybt proteins (HMWP1, HMWP2, YbtE, and Psn), a pattern similar to those for otherybt biosynthetic mutants. In contrast, strains carrying mutations in ybtT or ybtS (a previously identified gene required for Ybt biosynthesis) produced all four proteins at wild-type levels under iron-deprived conditions. To assess the effects of ybtT, -U, and -Smutations on transcription of ybt genes, reporter plasmids with ybtP or psn promoters controllinglacZ expression were introduced into these mutants. Normal iron-regulated β-galactosidase activity was observed in theybtT and ybtS mutants, whereas a significant loss of expression occurred in the ΔybtU strain. These results show that ybtT and ybtU genes are involved in the biosynthesis of the Ybt siderophore and that aybtU mutation but not ybtT or ybtSmutations affects transcription from the ybtP andpsn promoters.

scholarly journals Adaptation of Aerobically Growing Pseudomonas aeruginosa to Copper Starvation

2008 ◽  
Vol 190 (20) ◽  
pp. 6706-6717 ◽  
Author(s):  
Emanuela Frangipani ◽  
Vera I. Slaveykova ◽  
Cornelia Reimmann ◽  
Dieter Haas
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Cellular Respiration ◽  
Wild Type ◽  
Respiratory Enzymes ◽  
Terminal Oxidases ◽  
Copper Status ◽  
In The Wild ◽  
Quadruple Mutant ◽  
Heme Copper Oxidases

ABSTRACT Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA′-′lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases.

scholarly journals PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in Salmonella spp

1999 ◽  
Vol 67 (4) ◽  
pp. 1614-1622 ◽  
Author(s):  
Jennifer C. van Velkinburgh ◽  
John S. Gunn
Keyword(s):  
Salmonella Typhi ◽  
Enteric Pathogens ◽  
Wild Type ◽  
Salmonella Spp ◽  
Prolonged Incubation ◽  
Mutant Strains ◽  
Bile Resistance ◽  
Sense And Respond ◽  
Virulence Regulator ◽  
Type Strains

ABSTRACT As enteric pathogens, Salmonella spp. are resistant to the actions of bile. Salmonella typhimurium andSalmonella typhi strains were examined to better define the bile resistance phenotype. The MICs of bile for wild-typeS. typhimurium and S. typhi were 18 and 12%, respectively, and pretreatment of log-phase S. typhimurium with 15% bile dramatically increased bile resistance. Mutant strains of S. typhimurium andS. typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60%. PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both S. typhimurium and S. typhi in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance. However, of the PhoP-activated or -repressed genes tested, two (prgC andprgH) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon. These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp. in the intestine or gallbladder.

scholarly journals Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Ian T. Hill ◽  
Thomas Tallo ◽  
Matthew J. Dorman ◽  
Simon L. Dove
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Toxic Effects ◽  
Growth Defect ◽  
Wild Type ◽  
Rna Chaperone ◽  
Small Protein ◽  
Content Type ◽  
Transcription Regulator ◽  
Mutant Cells

ABSTRACT Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

scholarly journals Scnn1b-Transgenic BALB/c Mice as a Model of Pseudomonas aeruginosa Infections of the Cystic Fibrosis Lung

2020 ◽  
Vol 88 (9) ◽  
Author(s):  
Kristen J. Brao ◽  
Brendan P. Wille ◽  
Joshua Lieberman ◽  
Robert K. Ernst ◽  
Mark E. Shirtliff ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Immune Cell ◽  
Opportunistic Pathogen ◽  
Sodium Absorption ◽  
Lung Pathology ◽  
Wild Type ◽  
Content Type ◽  
Lung Infections ◽  
Content Modeling

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1β (IL-1β), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.

scholarly journals Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

2016 ◽  
Vol 12 ◽  
pp. 1428-1433 ◽  
Author(s):  
Bernardas Morkunas ◽  
Balint Gal ◽  
Warren R J D Galloway ◽  
James T Hodgkinson ◽  
Brett M Ibbeson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Cell Signalling ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Wide Range ◽  
Potential Applications ◽  
Therapeutic Context ◽  
Pyocyanin Production

Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

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