Mechanisms of nitric oxide-mediated biofilm dispersal in Pseudomonas aeruginosa

ABSTRACT Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, ΔnirS) did not disperse, whereas a NO reductase mutant (ΔnorCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

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The Nitrite Transporter Facilitates Biofilm Formation via Suppression of Nitrite Reductase and Is a New Antibiofilm Target in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00878-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Ji-Su Park ◽

Ha-Young Choi ◽

Won-Gon Kim

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Nitrite Reductase ◽

Biofilm Formation ◽

No Production ◽

Content Type ◽

A Genome ◽

Biofilm Dispersal ◽

Gmp Production

ABSTRACT Biofilm-forming bacteria, including the Gram-negative Pseudomonas aeruginosa, cause multiple types of chronic infections and are responsible for serious health burdens in humans, animals, and plants. Nitric oxide (NO) has been shown to induce biofilm dispersal via triggering a reduction in cyclic-di-GMP levels in a variety of bacteria. However, how NO, at homeostatic levels, also facilitates biofilm formation is unknown. Here, we found that complestatin, a structural analog of vancomycin isolated from Streptomyces, inhibits P. aeruginosa biofilm formation by upregulating NO production via nitrite reductase (NIR) induction and c-di-GMP degradation via phosphodiesterase (PDE) stimulation. The complestatin protein target was identified as a nitrite transporter from a genome-wide screen using the Keio Escherichia coli knockout library and confirmed using nitrite transporter knockout and overexpression strains. We demonstrated that the nitrite transporter stimulated biofilm formation by controlled NO production via appropriate NIR suppression and subsequent diguanylate cyclase (DGC) activation, not PDE activity, and c-di-GMP production in E. coli and P. aeruginosa. Thus, this study provides a mechanism for NO-mediated biofilm formation, which was previously not understood. IMPORTANCE Bacterial biofilms play roles in infections and avoidance of host defense mechanisms of medically important pathogens and increase the antibiotic resistance of the bacteria. Nitric oxide (NO) is reported to be involved in both biofilm formation and dispersal, which are conflicting processes. The mechanism by which NO regulates biofilm dispersal is relatively understood, but there are no reports about how NO is involved in biofilm formation. Here, by investigating the mechanism by which complestatin inhibits biofilm formation, we describe a novel mechanism for governing biofilm formation in Escherichia coli and Pseudomonas aeruginosa. Nitrite transporter is required for biofilm formation via regulation of NO levels and subsequent c-di-GMP production. Additionally, the nitrite transporter contributes more to P. aeruginosa virulence than quorum sensing. Thus, this study identifies nitrite transporters as new antibiofilm targets for future practical and therapeutic agent development.

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Faculty Opinions recommendation of Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1053001.504958 ◽

2006 ◽

Author(s):

Eric V Stabb

Keyword(s):

Nitric Oxide ◽

Pseudomonas Aeruginosa ◽

Biofilm Dispersal

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Nitric Oxide Signaling in Pseudomonas aeruginosa Biofilms Mediates Phosphodiesterase Activity, Decreased Cyclic Di-GMP Levels, and Enhanced Dispersal

Journal of Bacteriology ◽

10.1128/jb.00975-09 ◽

2009 ◽

Vol 191 (23) ◽

pp. 7333-7342 ◽

Cited By ~ 305

Author(s):

Nicolas Barraud ◽

David Schleheck ◽

Janosch Klebensberger ◽

Jeremy S. Webb ◽

Daniel J. Hassett ◽

...

Keyword(s):

Nitric Oxide ◽

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Site Directed Mutagenesis ◽

Nitric Oxide Signaling ◽

Physiological Characterization ◽

Secondary Messenger ◽

Increase Cell Motility ◽

Biofilm Dispersal ◽

Gene Expression Studies

ABSTRACT Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO.

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Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz378 ◽

2019 ◽

Vol 75 (1) ◽

pp. 117-125 ◽

Cited By ~ 8

Author(s):

Odel Soren ◽

Ardeshir Rineh ◽

Diogo G Silva ◽

Yuming Cai ◽

Robert P Howlin ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Clinical Isolates ◽

Nitric Oxide Donor ◽

Confocal Laser ◽

Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

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Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa

PLoS ONE ◽

10.1371/journal.pone.0091813 ◽

2014 ◽

Vol 9 (3) ◽

pp. e91813 ◽

Cited By ~ 21

Author(s):

Shengchang Su ◽

Warunya Panmanee ◽

Jeffrey J. Wilson ◽

Harry K. Mahtani ◽

Qian Li ◽

...

Keyword(s):

Nitric Oxide ◽

Pseudomonas Aeruginosa

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Endogenous nitric oxide-generating surfaces via polydopamine-copper coatings for preventing biofilm dispersal and promoting microbial killing

Materials Science and Engineering C ◽

10.1016/j.msec.2021.112297 ◽

2021 ◽

pp. 112297

Author(s):

Jayanti Mendhi ◽

Srinivas Sulugodu Ramachandra ◽

Indira Prasadam ◽

Saso Ivanovski ◽

Ying Yang ◽

...

Keyword(s):

Nitric Oxide ◽

Copper Coatings ◽

Endogenous Nitric Oxide ◽

Biofilm Dispersal

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Direct detection of blood nitric oxide reveals a burn-dependent decrease of nitric oxide in response to Pseudomonas aeruginosa infection

Burns ◽

10.1016/j.burns.2016.05.005 ◽

2016 ◽

Vol 42 (7) ◽

pp. 1522-1527 ◽

Cited By ~ 6

Author(s):

Julia L.M. Dunn ◽

Rebecca A. Hunter ◽

Karli Gast ◽

Robert Maile ◽

Bruce A. Cairns ◽

...

Keyword(s):

Nitric Oxide ◽

Pseudomonas Aeruginosa ◽

Direct Detection ◽

Pseudomonas Aeruginosa Infection

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New insights into the activity of Pseudomonas aeruginosa cd1 nitrite reductase

Biochemical Society Transactions ◽

10.1042/bst0361155 ◽

2008 ◽

Vol 36 (6) ◽

pp. 1155-1159 ◽

Cited By ~ 14

Author(s):

Serena Rinaldo ◽

Alessandro Arcovito ◽

Giorgio Giardina ◽

Nicoletta Castiglione ◽

Maurizio Brunori ◽

...

Keyword(s):

Nitric Oxide ◽

Pseudomonas Aeruginosa ◽

Nitrite Reductase ◽

Nitrite Reduction ◽

Nitrite Reductases ◽

Cytochrome Cd1 ◽

Haem Iron ◽

Shed Light ◽

Denitrification Process

The cytochrome cd1 nitrite reductases are enzymes that catalyse the reduction of nitrite to nitric oxide (NO) in the bacterial energy conversion denitrification process. These enzymes contain two different redox centres: one covalently bound c-haem, which is reduced by external donors, and one peculiar d1-haem, where catalysis occurs. In the present paper, we summarize the current understanding of the reaction of nitrite reduction in the light of the most recent results on the enzyme from Pseudomonas aeruginosa and discuss the differences between enzymes from different organisms. We have evidence that release of NO from the ferrous d1-haem occurs rapidly enough to be fully compatible with the turnover, in contrast with previous hypotheses, and that the substrate nitrite is able to displace NO from the d1-haem iron. These results shed light on the mechanistic details of the activity of cd1 nitrite reductases and on the biological role of the d1-haem, whose presence in this class of enzymes has to date been unexplained.

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Effect of Nitroxides on Swarming Motility and Biofilm Formation, Multicellular Behaviors in Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01381-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4877-4881 ◽

Cited By ~ 45

Author(s):

César de la Fuente-Núñez ◽

Fany Reffuveille ◽

Kathryn E. Fairfull-Smith ◽

Robert E. W. Hancock

Keyword(s):

Nitric Oxide ◽

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Wild Type ◽

Planktonic Cells ◽

Swarming Motility ◽

Content Type ◽

Exogenous Addition ◽

Biofilm Dispersion ◽

Dispersal Activity

ABSTRACTThe ability of nitric oxide (NO) to induce biofilm dispersion has been well established. Here, we investigated the effect of nitroxides (sterically hindered nitric oxide analogues) on biofilm formation and swarming motility inPseudomonas aeruginosa. A transposon mutant unable to produce nitric oxide endogenously (nirS) was deficient in swarming motility relative to the wild type and the complemented strain. Moreover, expression of thenirSgene was upregulated by 9.65-fold in wild-type swarming cells compared to planktonic cells. Wild-type swarming levels were substantially restored upon the exogenous addition of nitroxide containing compounds, a finding consistent with the hypothesis that NO is necessary for swarming motility. Here, we showed that nitroxides not only mimicked the dispersal activity of NO but also prevented biofilms from forming in flow cell chambers. In addition, anirStransposon mutant was deficient in biofilm formation relative to the wild type and the complemented strain, thus implicating NO in the formation of biofilms. Intriguingly, despite its stand-alone action in inhibiting biofilm formation and promoting dispersal, a nitroxide partially restored the ability of anirSmutant to form biofilms.

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