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

mBio ◽  
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.

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

2013 ◽  
Vol 57 (10) ◽  
pp. 4877-4881 ◽  
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.

scholarly journals Induction of native c-di-GMP phosphodiesterases leads to dispersal of Pseudomonas aeruginosa biofilms

2021 ◽  
Author(s):  
Jens Bo Andersen ◽  
Kasper Nørskov Kragh ◽  
Louise Dahl Hultqvist ◽  
Morten Rybtke ◽  
Martin Nilsson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Single Cell ◽  
Biofilm Formation ◽  
Ectopic Expression ◽  
Second Messenger ◽  
Flow Cell ◽  
Diguanylate Cyclases ◽  
Biofilm Dispersal ◽  
High Level

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. C-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We have previously presented evidence that ectopic expression in Pseudomonas aeruginosa of the Escherichia coli phosphodiesterase YhjH results in biofilm dispersal. More recently, however, evidence has been presented that induction of native c-di-GMP phosphodiesterases does not lead to dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for development of anti-biofilm drugs. However, here we demonstrate that induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed does result in dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and in a flow-cell biofilm system.

scholarly journals Subtilase Cytotoxin Enhances Escherichia coli Survival in Macrophages by Suppression of Nitric Oxide Production through the Inhibition of NF-κB Activation

2012 ◽  
Vol 80 (11) ◽  
pp. 3939-3951 ◽  
Author(s):  
Hiroyasu Tsutsuki ◽  
Kinnosuke Yahiro ◽  
Kotaro Suzuki ◽  
Akira Suto ◽  
Kohei Ogura ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Inos Expression ◽  
Inos Mrna ◽  
No Production ◽  
Content Type ◽  
Monocyte Chemoattractant Protein 1 ◽  
E Coli ◽  
Inos Promoter ◽  
Subtilase Cytotoxin

ABSTRACTSubtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenicEscherichia coli(STEC), cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress and caspase-dependent apoptosis. SubAB alters the innate immune response. SubAB pretreatment of macrophages inhibited lipopolysaccharide (LPS)-induced production of both monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor α (TNF-α). We investigated here the mechanism by which SubAB inhibits nitric oxide (NO) production by mouse macrophages. SubAB suppressed LPS-induced NO production through inhibition of inducible NO synthase (iNOS) mRNA and protein expression. Further, SubAB inhibited LPS-induced IκB-α phosphorylation and nuclear localization of the nuclear factor-κB (NF-κB) p65/p50 heterodimer. Reporter gene and chromatin immunoprecipitation (ChIP) assays revealed that SubAB reduced LPS-induced NF-κB p65/p50 heterodimer binding to an NF-κB binding site on the iNOS promoter. In contrast to the native toxin, a catalytically inactivated SubAB mutant slightly enhanced LPS-induced iNOS expression and binding of NF-κB subunits to the iNOS promoter. The SubAB effect on LPS-induced iNOS expression was significantly reduced in macrophages from NF-κB1 (p50)-deficient mice, which lacked a DNA-binding subunit of the p65/p50 heterodimer, suggesting that p50 was involved in SubAB-mediated inhibition of iNOS expression. Treatment of macrophages with an NOS inhibitor or expression of SubAB byE. coliincreasedE. colisurvival in macrophages, suggesting that NO generated by macrophages resulted in efficient killing of the bacteria and SubAB contributed toE. colisurvival in macrophages. Thus, we hypothesize that SubAB might represent a novel bacterial strategy to circumvent host defense during STEC infection.

scholarly journals BolA Is a Transcriptional Switch That Turns Off Motility and Turns On Biofilm Development

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Clémentine Dressaire ◽  
Ricardo Neves Moreira ◽  
Susana Barahona ◽  
António Pedro Alves de Matos ◽  
Cecília Maria Arraiano
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Extracellular Polysaccharides ◽  
Bacterial Cells ◽  
Content Type ◽  
Bacterial Transcription ◽  
A Genome ◽  
Exact Function ◽  
Transcriptional Switch ◽  
Biofilm Development

ABSTRACTBacteria are extremely versatile organisms that rapidly adapt to changing environments. When bacterial cells switch from planktonic growth to biofilm, flagellum formation is turned off and the production of fimbriae and extracellular polysaccharides is switched on. BolA is present in most Gram-negative bacteria, and homologues can be found from proteobacteria to eukaryotes. Here, we show that BolA is a new bacterial transcription factor that modulates the switch from a planktonic to a sessile lifestyle. It negatively modulates flagellar biosynthesis and swimming capacity inEscherichia coli. Furthermore, BolA overexpression favors biofilm formation, involving the production of fimbria-like adhesins and curli. Our results also demonstrate that BolA is a protein with high affinity to DNA and is able to regulate many genes on a genome-wide scale. Moreover, we show that the most significant targets of this protein involve a complex network of genes encoding proteins related to biofilm development. Herein, we propose that BolA is a motile/adhesive transcriptional switch, specifically involved in the transition between the planktonic and the attachment stage of biofilm formation.IMPORTANCEEscherichia colicells possess several mechanisms to cope with stresses. BolA has been described as a protein important for survival in late stages of bacterial growth and under harsh environmental conditions. BolA-like proteins are widely conserved from prokaryotes to eukaryotes. Although their exact function is not fully established at the molecular level, they seem to be involved in cell proliferation or cell cycle regulation. Here, we unraveled the role of BolA in biofilm development and bacterial motility. Our work suggests that BolA actively contributes to the decision of bacteria to arrest flagellar production and initiate the attachment to form structured communities, such as biofilms. The molecular studies of different lifestyles coupled with the comprehension of the BolA functions may be an important step for future perspectives, with health care and biotechnology applications.

scholarly journals Flagellar Stators Stimulate c-di-GMP Production by Pseudomonas aeruginosa

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Amy E. Baker ◽  
Shanice S. Webster ◽  
Andreas Diepold ◽  
Sherry L. Kuchma ◽  
Eric Bordeleau ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Feedback Loop ◽  
Bacterial Cells ◽  
Flagellar Motility ◽  
Swarming Motility ◽  
Surface Attachment ◽  
Content Type ◽  
Bidirectional Interactions ◽  
Gmp Production

ABSTRACT Flagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in Pseudomonas aeruginosa and other microbes is cyclic diguanylate (c-di-GMP). High levels of this second messenger repress motility and stimulate biofilm formation. c-di-GMP levels regulate motility in P. aeruginosa in part by influencing the localization of its two flagellar stator sets, MotAB and MotCD. Here, we show that while c-di-GMP can influence stator localization, stators can in turn impact c-di-GMP levels. We demonstrate that the swarming motility-driving stator MotC physically interacts with the transmembrane region of the diguanylate cyclase SadC. Furthermore, we demonstrate that this interaction is capable of stimulating SadC activity. We propose a model by which the MotCD stator set interacts with SadC to stimulate c-di-GMP production under conditions not permissive to motility. This regulation implies a positive-feedback loop in which c-di-GMP signaling events cause MotCD stators to disengage from the motor; then disengaged stators stimulate c-di-GMP production to reinforce a biofilm mode of growth. Our studies help to define the bidirectional interactions between c-di-GMP and the flagellar machinery. IMPORTANCE The ability of bacterial cells to control motility during early steps in biofilm formation is critical for the transition to a nonmotile, biofilm lifestyle. Recent studies have clearly demonstrated the ability of c-di-GMP to control motility via a number of mechanisms, including through controlling transcription of motility-related genes and modulating motor function. Here, we provide evidence that motor components can in turn impact c-di-GMP levels. We propose that communication between motor components and the c-di-GMP synthesis machinery allows the cell to have a robust and sensitive switching mechanism to control motility during early events in biofilm formation.

scholarly journals Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Judith H. Merritt ◽  
Dae-Gon Ha ◽  
Kimberly N. Cowles ◽  
Wenyun Lu ◽  
Diana K. Morales ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Critical Question ◽  
Cyclic Diguanylate ◽  
Critical Signal ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Samuel Fenn ◽  
Jean-Frédéric Dubern ◽  
Cristina Cigana ◽  
Maura De Simone ◽  
James Lazenby ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Disease Models ◽  
Attractive Alternative ◽  
Dependent Manner ◽  
Content Type ◽  
Isogenic Mutant ◽  
Virulence Traits ◽  
Infection Models

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

scholarly journals Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 880-890 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Toshiyuki Ishizuka ◽  
Shuhei Hotta ◽  
Michiko Aoki ◽  
Tomohiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Cell Aggregation ◽  
Master Regulator ◽  
Gene Encoding ◽  
Content Type ◽  
Promoter Probe ◽  
K 12 ◽  
Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

scholarly journals Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

2012 ◽  
Vol 78 (15) ◽  
pp. 5060-5069 ◽  
Author(s):  
Morten T. Rybtke ◽  
Bradley R. Borlee ◽  
Keiji Murakami ◽  
Yasuhiko Irie ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Subsequent Development ◽  
Cellular Level ◽  
Host Immune System ◽  
Chronic Infections ◽  
Content Type ◽  
Genes Encoding ◽  
Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

Sign in / Sign up

Export Citation Format

Share Document