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

2009 ◽  
Vol 191 (23) ◽  
pp. 7333-7342 ◽  
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.

scholarly journals Involvement of Nitric Oxide in Biofilm Dispersal of Pseudomonas aeruginosa

2006 ◽  
Vol 188 (21) ◽  
pp. 7344-7353 ◽  
Author(s):  
Nicolas Barraud ◽  
Daniel J. Hassett ◽  
Sung-Hei Hwang ◽  
Scott A. Rice ◽  
Staffan Kjelleberg ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agents ◽  
Nitrosative Stress ◽  
Sodium Dodecyl ◽  
Anaerobic Respiration ◽  
Opportunistic Pathogen ◽  
No Donor ◽  
Biofilm Dispersal ◽  
Novel Strategy

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.

scholarly journals Structure of heme d 1-free cd 1 nitrite reductase NirS

2020 ◽  
Vol 76 (6) ◽  
pp. 250-256
Author(s):  
Thomas Klünemann ◽  
Wulf Blankenfeldt
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Active Site ◽  
Opportunistic Pathogen ◽  
Active Enzyme ◽  
Free Form ◽  
Nitrate Respiration ◽  
Gram Negative ◽  
Open Conformation

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd 1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d 1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d 1-bound form. The heme d 1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d 1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.

scholarly journals Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa

2019 ◽  
Author(s):  
Yu-Ming Cai ◽  
Andrew Hutchin ◽  
Jack Craddock ◽  
Martin A. Walsh ◽  
Jeremy Stephen Webb ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Specific Protein ◽  
Cellular Functions ◽  
Environmental Stimuli ◽  
Secondary Messenger ◽  
Domain Structures ◽  
Biofilm Dispersal ◽  
Opportunistic Human Pathogen ◽  
Homology Domains

AbstractBacteria typically occur either as free-swimming planktonic cells or within a sessile, biofilm mode of growth. In Pseudomonas aeruginosa, the transition between these lifestyles is known to be modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP). We are interested in the control of distinct biofilm-relevant phenotypes in P. aeruginosa through the modulation of intracellular c-di-GMP. Here, we characterise motility and associated biofilm formation and dispersal in two pairs of related multi-domain proteins with putative c-di-GMP turnover domains, selected to contain additional PAS (Per-Arnt-Sim) homology domains known for their ability to process environmental stimuli. The enzymes PA0861 (RbdA) and PA2072 have distinct functions despite their similar domain structures. The ΔrbdA deletion mutant showed significantly increased biofilm formation while biofilm formation was impaired in ΔPA2072. Using a GFP transcriptional reporter fused to the cyclic di-GMP-responsive cdrA promoter, we show correlation between biofilm phenotype and c-di-GMP levels. Both proteins are shown to play a role in nitric oxide (NO) induced biofilm dispersal. We further studied pseudo-enzymes of similar architecture. PA5017 (DipA) is an inactive cyclase, and PA4959 (FimX) is described here as an inactive cyclase/phosphodiesterase. Loss of swimming and twitching motilities, respectively, is observed in deletion variants, which correlated with NO-induced biofilm dispersal phenotypes, as ΔdipA dispersed less well while ΔfimX dispersed better than wild type. The study highlights how Pseudomonas differentiates c-di-GMP output – in this case motility – using structurally very similar proteins and underlines a significant role for pseudo-enzymes in motility regulation and associated biofilm dispersal.ImportanceBacterial biofilms exert pervasive economic and societal impact across a range of environmental, engineered and clinical contexts. The secondary messenger cyclic guanosine di-phosphate, c-di-GMP, is known to control the ability of many bacteria to form biofilms. The opportunistic human pathogen Pseudomonas aeruginosa PAO1 has 38 putative enzymes that can regulate c-di-GMP turnover, and these proteins modulate various cellular functions and influence bacterial lifestyle. The specific protein sensory domains and mechanisms of motility that lead to biofilm dispersal remain to be fully understood. Here we studied multi-domain proteins with the PAS (Per-Arnt-Sim) homology domains, these being classic sensors to environmental stimuli. Our study demonstrates the significant roles for the pseudo-enzymes PA4959 (FimX) and PA5017 (DipA) in regulation of biofilm phenotype and motility. Further, enzymes with highly homologous structures, such as PA0861 (RbdA) and PA2072, have almost orthogonal function in biofilm and motility control.

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 Genetic Analysis of the Regulation of Type IV Pilus Function by the Chp Chemosensory System of Pseudomonas aeruginosa

2009 ◽  
Vol 192 (4) ◽  
pp. 994-1010 ◽  
Author(s):  
Jacob J. Bertrand ◽  
Joyce T. West ◽  
Joanne N. Engel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Histidine Kinase ◽  
Opportunistic Pathogen ◽  
Negative Regulator ◽  
Site Directed Mutagenesis ◽  
Type Iv Pilus ◽  
Chemosensory System ◽  
Type Iv ◽  
Chp System

ABSTRACT The virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of many virulence factors, including type IV pili, which are required for colonization of host tissues and for twitching motility. Type IV pilus function is controlled in part by the Chp chemosensory system, which includes a histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. How the Chp components interface with the type IV pilus motor proteins PilB, PilT, and PilU is unknown. We present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and the role of PilH and PilT in regulating pilus retraction. Using informative double and triple mutants, we show that (i) ChpA, PilG, and PilB function upstream of PilH, PilT, and PilU; (ii) that PilH enhances PilT function; and (iii) that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. By site-directed mutagenesis, we demonstrate that the histidine kinase domain of ChpA and the phosphoacceptor sites of both PilG and PilH are required for type IV pilus function, suggesting that they form a phosphorelay system important in the regulation of pilus extension and retraction. Finally, we present evidence suggesting that pilA transcription is regulated by intracellular PilA levels. We show that PilA is a negative regulator of pilA transcription in P. aeruginosa and that the Chp system functionally regulates pilA transcription by controlling PilA import and export.

scholarly journals Heterologous NNR-Mediated Nitric Oxide Signaling inEscherichia coli

2000 ◽  
Vol 182 (22) ◽  
pp. 6434-6439 ◽  
Author(s):  
Matthew I. Hutchings ◽  
Neil Shearer ◽  
Sarah Wastell ◽  
Rob J. M. van Spanning ◽  
Stephen Spiro
Keyword(s):  
Nitric Oxide ◽  
Paracoccus Denitrificans ◽  
Culture Media ◽  
Reductase Activity ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Reporter System ◽  
Nitric Oxide Signaling ◽  
Cell Extracts ◽  
Nitrate And Nitrite

ABSTRACT The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melRpromoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO+ donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a “positive control” phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

scholarly journals Substrate recognition by the Pseudomonas aeruginosa EF-Tu methyltransferase EftM

2019 ◽  
Author(s):  
Emily G. Kuiper ◽  
Debayan Dey ◽  
Paige A. LaMore ◽  
Joshua P. Owings ◽  
Samantha M. Prezioso ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Conformational Changes ◽  
Substrate Recognition ◽  
Elongation Factor ◽  
Homology Model ◽  
Opportunistic Pathogen ◽  
Docking Studies ◽  
Protein Docking ◽  
Site Directed Mutagenesis ◽  
Severe Burns

ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen and a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, and severe burns. During infection, P. aeruginosa adhesion to host epithelial cells is enhanced by surface exposed translation elongation factor EF-Tu carrying a Lys5 trimethylation. This modification is incorporated by the S-adenosyl-L-methionine-dependent methyltransferase EftM. Thus, EF-Tu modification by EftM may represent a novel target to restrict the establishment of P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM action by defining the molecular mechanism of EF-Tu substrate recognition by this enzyme. First, following the observation that EftM can bind to EF-Tu lacking an N-terminal peptide (encompassing the Lys5 target site), an EftM homology model was generated and used in protein-protein docking studies to predict EftM:EF-Tu interactions. The predicted protein-protein interface was then experimentally validated using site-directed mutagenesis of residues in both proteins coupled with binding and methyltransferase activity assays. We also show that EftM is unable to methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to allow placement of the first 5-6 amino acids of EF-Tu into the conserved peptide binding channel. In this channel, a group of residues that are highly conserved in EftM family proteins position the N-terminal sequence to facilitate modification of Lys5. Our findings provide detailed insights into substrate recognition by this lysine methyltransferase, paving the way for a deeper understanding of EftM’s mechanism of action on EF-Tu.

The In Vitro Antibiofilm Activity of Water Leaf Extract of Papaya (Carica papaya L.) against Pseudomonas aeruginosa

2017 ◽  
Vol 2 (3) ◽  
pp. 150-163
Author(s):  
Ekajayanti Kining ◽  
Syamsul Falah ◽  
Novik Nurhidayat
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Contact Time ◽  
Cell Attachment ◽  
Water Extract ◽  
Opportunistic Pathogen ◽  
Bacterial Biofilm ◽  
Antibiofilm Activity ◽  
Bacterial Survival ◽  
Optimum Conditions

Pseudomonas aeruginosa is one of opportunistic pathogen forming bacterial biofilm. The biofilm sustains the bacterial survival and infections. This study aimed to assess the activity of water extract of papaya leaves on inhibition of cells attachment, growth and degradation of the biofilm using crystal violet (CV) biofilm assay. Research results showed that water extract of papaya leaves contains alkaloids, tanins, flavonoids, and steroids/terpenoids and showed antibacterial activity and antibiofilm against P. aeruginosa. Addition of extract can inhibit the cell attachment and was able to degrade the biofilm of 40.92% and 48.058% respectively at optimum conditions: extract concentration of 25% (v/v), temperature 37.5 °C and contact time 45 minutes. With a concentration of 25% (v/v), temperature of 50 °C and the contact time of 3 days, extract of papaya leaves can inhibit the growth of biofilms of 39.837% v/v.

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