scholarly journals Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

2016 ◽  
Vol 2 (5) ◽  
pp. e1501632 ◽  
Author(s):  
Perrin Baker ◽  
Preston J. Hill ◽  
Brendan D. Snarr ◽  
Noor Alnabelseya ◽  
Matthew J. Pestrak ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Sublethal Concentration ◽  
Glycoside Hydrolases ◽  
Host Defenses ◽  
Half Maximal Effective Concentration ◽  
Bacterial Exopolysaccharide ◽  
Opportunistic Human Pathogen ◽  
Potential Therapeutics

Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.

scholarly journals Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and preventPseudomonas aeruginosabiofilms

2015 ◽  
Author(s):  
Perrin Baker ◽  
Preston J HIll ◽  
Brendan D Snarr ◽  
Noor Alnabelseya ◽  
Mathew J Pestrak ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Glycoside Hydrolases ◽  
Bacterial Biofilms ◽  
Human Pathogen ◽  
Host Defenses ◽  
Bacterial Exopolysaccharide ◽  
Opportunistic Human Pathogen ◽  
Potential Therapeutics

Bacterial biofilms are a significant medical challenge as they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogenPseudomonas aeruginosais the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases - PelAhand PslGh- encoded in thepelandpslbiosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to targetP. aeruginosabiofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix and that nanomolar concentrations of these enzymes can both prevent biofilm formation as well as rapidly disrupt preexisting biofilms in vitro. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58-94%. These non-cytotoxic enzymes potentiated antibiotics as the addition of either enzyme to a sub-lethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude. Additionally, PelAhwas able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases and synthetic biology to develop novel anti-biofilm therapeutics.

scholarly journals A Novel Infection Protocol in Zebrafish Embryo to Assess Pseudomonas aeruginosa Virulence and Validate Efficacy of a Quorum Sensing Inhibitor In Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 401
Author(s):  
Pauline Nogaret ◽  
Fatima El El Garah ◽  
Anne-Béatrice Blanc-Potard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Animal Model ◽  
Quorum Sensing ◽  
Drug Testing ◽  
Drug Efficacy ◽  
Embryo Viability ◽  
Immersion Method ◽  
Opportunistic Human Pathogen

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, new therapeutic strategies against P. aeruginosa are urgently needed. In this context, we aimed to develop a simple vertebrate animal model to rapidly assess in vivo drug efficacy against P. aeruginosa. Zebrafish are increasingly considered for modeling human infections caused by bacterial pathogens, which are commonly microinjected in embryos. In the present study, we established a novel protocol for zebrafish infection by P. aeruginosa based on bath immersion in 96-well plates of tail-injured embryos. The immersion method, followed by a 48-hour survey of embryo viability, was first validated to assess the virulence of P. aeruginosa wild-type PAO1 and a known attenuated mutant. We then validated its relevance for antipseudomonal drug testing by first using a clinically used antibiotic, ciprofloxacin. Secondly, we used a novel quorum sensing (QS) inhibitory molecule, N-(2-pyrimidyl)butanamide (C11), the activity of which had been validated in vitro but not previously tested in any animal model. A significant protective effect of C11 was observed on infected embryos, supporting the ability of C11 to attenuate in vivo P. aeruginosa pathogenicity. In conclusion, we present here a new and reliable method to compare the virulence of P. aeruginosa strains in vivo and to rapidly assess the efficacy of clinically relevant drugs against P. aeruginosa, including new antivirulence compounds.

scholarly journals Staphylococcus aureus Serves as an Iron Source for Pseudomonas aeruginosa during In Vivo Coculture

2005 ◽  
Vol 187 (2) ◽  
pp. 554-566 ◽  
Author(s):  
Lauren M. Mashburn ◽  
Amy M. Jett ◽  
Darrin R. Akins ◽  
Marvin Whiteley
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Iron Acquisition ◽  
Low Oxygen ◽  
Dialysis Membrane ◽  
Opportunistic Human Pathogen ◽  
The Impact

ABSTRACT Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in this environment. We propose a model where P. aeruginosa lyses S. aureus and uses released iron for growth in low-iron environments.

scholarly journals Screening of a Library of Oligosaccharides Targeting Lectin LecB of Pseudomonas Aeruginosa and Synthesis of High Affinity Oligoglycoclusters

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3073 ◽  
Author(s):  
Lucie Dupin ◽  
Mathieu Noël ◽  
Silvère Bonnet ◽  
Albert Meyer ◽  
Thomas Géhin ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Biofilm Formation ◽  
Selective Advantage ◽  
Host Cells ◽  
Host Defenses ◽  
Negative Bacterium ◽  
High Affinity ◽  
Structure Relationship ◽  
Relationship Of

The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.

scholarly journals Pseudomonas aeruginosa PqsA Is an Anthranilate-Coenzyme A Ligase

2007 ◽  
Vol 190 (4) ◽  
pp. 1247-1255 ◽  
Author(s):  
James P. Coleman ◽  
L. Lynn Hudson ◽  
Susan L. McKnight ◽  
John M. Farrow ◽  
M. Worth Calfee ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Coenzyme A ◽  
Intercellular Signaling ◽  
Signaling Systems ◽  
Coa Ligase ◽  
Coenzyme A Ligase ◽  
Opportunistic Human Pathogen ◽  
Acyl Coenzyme A

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroanthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.

Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 373-383 ◽  
Author(s):  
Thomas Bjarnsholt ◽  
Peter Østrup Jensen ◽  
Mette Burmølle ◽  
Morten Hentzer ◽  
Janus A. J. Haagensen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Polymorphonuclear Leukocytes ◽  
Present Report ◽  
Cell Communication ◽  
A Cell ◽  
Opportunistic Human Pathogen ◽  
Conventional Antibiotics

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell–cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2, and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

scholarly journals Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Patrick R. Secor ◽  
Lia A. Michaels ◽  
Kate S. Smigiel ◽  
Maryam G. Rohani ◽  
Laura K. Jennings ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inflammatory Response ◽  
Chronic Infection ◽  
Bacterial Invasion ◽  
Airway Epithelial ◽  
Content Type ◽  
Epithelial Cultures ◽  
Opportunistic Human Pathogen

ABSTRACT Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

scholarly journals Attenuation of Quorum Sensing Controlled Virulence Factors and Biofilm Formation by Edible Fruit Extract of Coccinia indica against Pseudomonas aeruginosa

2021 ◽  
pp. 756-764
Author(s):  
R. Shruthi Devi ◽  
P. Sankar Ganesh ◽  
A. S. Smiline Girija ◽  
J. Vijayashree Priyadharshini
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Crystal Violet Staining ◽  
Chronic Infections ◽  
Fruit Extract ◽  
Fluorescent Microscope ◽  
Edible Fruit ◽  
Coccinia Indica ◽  
Opportunistic Human Pathogen

Background: Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that mainly infects immunocompromised individuals and patients with urinary tract infection and chronic infections of the respiratory pathways, including cystic fibrosis. Many quorum sensing (QS) controlled components such as bio surfactants and swarming motilities play an important role in the establishment of biofilms. Targeting these factors through anti-QS strategies prevent biofilm formation and treating infections. Coccinia indica commonly called little gourd is used to treat diabetes, wound, burn infections and has antioxidant, antibacterial and antitussive properties. Methods: The methanolic fruit extract of C. indica was prepared and screened for anti-QS and anti-biofilm formation activity. Pyocyanin inhibition, rhamnolipid, crystal violet staining assay tests was performed and the extract was observed under fluorescent microscope. Results: The results obtained are as follows - the fruit extract inhibits the pyocyanin at 58.13% and 42.27% at 0.5 mg/ml and 1.0 mg/ml, biofilm at 69.86% and 49.06% at 0.5 mg/ml and 1.0 mg/ml, inhibits rhamnolipid assay and under fluorescent microscope it is seen scattered whereas control produce biofilm matrix like appearance. Conclusion: Since less study has been made on the quorum sensing and biofilm activity of C.indica our study aimed to fulfil it and it was found that it exhibits good biofilm formation and thus can be used for treating infections.

scholarly journals Cephalosporins target quorum sensing and suppress virulence of Pseudomonas aeruginosa in Caenorhabditis elegans infection model

2020 ◽  
Author(s):  
Lokender Kumar ◽  
Nathanael Brenner ◽  
John Brice ◽  
Judith Klein-Seetharaman ◽  
Susanta K. Sarkar
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Caenorhabditis Elegans ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Host Cells ◽  
Infection Model ◽  
Host Immune System ◽  
Bacterial Cells

ABSTRACTPseudomonas aeruginosa utilizes a chemical social networking system referred to as quorum sensing (QS) to strategically co-ordinate the expression of virulence factors and biofilm formation. Virulence attributes damage the host cells, impair the host immune system, and protect bacterial cells from antibiotic attack. Thus, anti-QS agents may act as novel anti-infective therapeutics to treat P. aeruginosa infections. The present study was performed to evaluate the anti-QS, anti-biofilm, and anti-virulence activity of β-lactam antibiotics (carbapenems and cephalosporins) against P. aeruginosa. The anti-QS activity was quantified using Chromobacterium violaceum CV026 as a QS reporter strain. Our results showed that cephalosporins including cefepime (CP), ceftazidime (CF), and ceftriaxone (CT) exhibited potent anti-QS and anti-virulence activities against P. aeruginosa PAO1. These antibiotics significantly impaired motility phenotypes, decreased pyocyanin production, and reduced the biofilm formation by P. aeruginosa PAO1. In the present study, we studied isogenic QS mutants of PAO1: ΔLasR, ΔRhlR, ΔPqsA, and ΔPqsR and found that the levels of virulence factors of antibiotic-treated PAO1 were comparable to QS mutant strains. Molecular docking predicted high binding affinities of cephalosporins for the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). In addition, our results showed that the anti-microbial activity of aminoglycosides increased in the presence of sub-inhibitory concentrations (sub-MICs) of CP against P. aeruginosa PAO1. Further, utilizing Caenorhabditis elegans as an animal model for the in vivo anti-virulence effects of antibiotics, cephalosporins showed a significant increase in C. elegans survival by suppressing virulence factor production in P. aeruginosa. Thus, our results indicate that cephalosporins might provide a viable anti-virulence therapy in the treatment of infections caused by multi-drug resistant P. aeruginosa.

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