The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.

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

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.12.137 ◽

2016 ◽

Vol 12 ◽

pp. 1428-1433 ◽

Cited By ~ 8

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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The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin

Microbiology ◽

10.1099/mic.0.031971-0 ◽

2010 ◽

Vol 156 (3) ◽

pp. 678-686 ◽

Cited By ~ 53

Author(s):

Tiffany Vinckx ◽

Qing Wei ◽

Sandra Matthijs ◽

Pierre Cornelis

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Protective Action ◽

Opportunistic Pathogen ◽

Single Copy ◽

Protective Role ◽

Phenotypic Change ◽

Wild Type ◽

Redox Active ◽

Pyocyanin Production

The LysR-type transcriptional regulator (LTTR) OxyR orchestrates the defence of the opportunistic pathogen Pseudomonas aeruginosa against reactive oxygen species. In previous work we also demonstrated that OxyR is needed for the utilization of the ferrisiderophore pyoverdine, stressing the importance of this regulator. Here, we show that an oxyR mutant is unable to swarm on agar plates, probably as a consequence of absence of production of rhamnolipid surfactant molecules. Another obvious phenotypic change was the increased production of the phenazine redox-active molecule pyocyanin in the oxyR mutant. As already described, the oxyR mutant could not grow in LB medium, unless high numbers of cells (>108 ml−1) were inoculated. However, its growth in Pseudomonas P agar (King's A), a medium inducing pyocyanin production, was like that of the wild-type, suggesting a protective action of this redox-active phenazine compound. This was confirmed by the restoration of the capacity to grow in LB medium upon addition of pure pyocyanin. Although both rhamnolipid and pyocyanin production are controlled by quorum sensing, no obvious changes were observed in the production of N-acylhomoserine lactones or the Pseudomonas quinolone signal (PQS). Complementation of rhamnolipid production and motility, and restoration of normal pyocyanin levels, was only possible when the oxyR gene was in single copy, while pyocyanin levels were increased when oxyR was present in a multicopy vector. Conversely, plating efficiency was increased only when the oxyR gene was present in multicopy, but not when in single copy in the chromosome, due to lower expression of oxyR compared with the wild-type, suggesting that some phenotypes are differently affected in function to the levels of OxyR molecules in the cell. Analysis of transcripts of oxidative stress-response enzymes showed a strong decrease of katB, ahpC and ahpB expression in the oxyR mutant grown in LB, but this was not the case when the mutant was grown on P agar, suggesting that the OxyR dependency for the transcription of these genes is not total.

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

Microbiology ◽

10.1099/mic.0.28396-0 ◽

2006 ◽

Vol 152 (5) ◽

pp. 1407-1415 ◽

Cited By ~ 23

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.

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

Infection and Immunity ◽

10.1128/iai.02707-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 863-875 ◽

Cited By ~ 41

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.

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Differential Regulation of Twitching Motility and Elastase Production by Vfr in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.184.13.3605-3613.2002 ◽

2002 ◽

Vol 184 (13) ◽

pp. 3605-3613 ◽

Cited By ~ 127

Author(s):

Scott A. Beatson ◽

Cynthia B. Whitchurch ◽

Jennifer L. Sargent ◽

Roger C. Levesque ◽

John S. Mattick

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Binding Pocket ◽

Receptor Protein ◽

Twitching Motility ◽

Wild Type ◽

Type Iv ◽

Allelic Deletion ◽

Pyocyanin Production ◽

Null Mutants

ABSTRACT Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

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Adaptation of Aerobically Growing Pseudomonas aeruginosa to Copper Starvation

Journal of Bacteriology ◽

10.1128/jb.00450-08 ◽

2008 ◽

Vol 190 (20) ◽

pp. 6706-6717 ◽

Cited By ~ 33

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.

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DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus

Journal of Fungi ◽

10.3390/jof7090738 ◽

2021 ◽

Vol 7 (9) ◽

pp. 738

Author(s):

Macario Osorio-Concepción ◽

Carlos Lax ◽

Eusebio Navarro ◽

Francisco E. Nicolás ◽

Victoriano Garre

Keyword(s):

Wild Type Strain ◽

Pathogenic Fungi ◽

Infection Process ◽

Deletion Mutants ◽

Polar Growth ◽

Wild Type ◽

C Elegans ◽

Double Deletion ◽

Multiple Processes ◽

Causative Agents

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process.

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DNA Methylation on N6-Adenine Regulates the Hyphal Development During Dimorphism in the Early-Diverging Fungus Mucor lusitanicus

10.20944/preprints202107.0525.v1 ◽

2021 ◽

Author(s):

Macario Osorio-Concepción ◽

Carlos Lax ◽

Eusebio Navarro ◽

Francisco E. Nicolás ◽

Victoriano Garre

Keyword(s):

Wild Type Strain ◽

Pathogenic Fungi ◽

Infection Process ◽

Deletion Mutants ◽

Polar Growth ◽

Wild Type ◽

C Elegans ◽

Double Deletion ◽

Multiple Processes ◽

Causative Agents

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales; causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxyadenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins homologs to C. elegans NMAD-1 and ALKBH1, respectively. The function of the three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase of 6mA levels during the dimorphic transition, suggesting that 6mA regulates dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction of polar growth, suggesting that 6mA also has a role during the spore-macrophage interaction that could be important in the infection process.

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

Journal of Bacteriology ◽

10.1128/jb.00232-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 2

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.

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

Infection and Immunity ◽

10.1128/iai.00237-20 ◽

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.

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