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1. Pseudomonas aeruginosa Biofilm Dispersion by the Human Atrial Natriuretic Peptide

Author

Mélissande Louis, Thomas Clamens, Ali Tahrioui, Florie Desriac, Sophie Rodrigues, Thibaut Rosay, Nicholas Harmer, Suraya Diaz, Magalie Barreau, Pierre‐Jean Racine, Eric Kipnis, Teddy Grandjean, Julien Vieillard, Emeline Bouffartigues, Pierre Cornelis, Sylvie Chevalier, Marc G. J. Feuilloley, and Olivier Lesouhaitier

Subjects
ami pathway, antibiotics, bacterial adaptation, bacterial sensor, biofilm dispersion, hANP, Science
Abstract

Abstract Pseudomonas aeruginosa biofilms cause chronic, antibiotic tolerant infections in wounds and lungs. Numerous recent studies demonstrate that bacteria can detect human communication compounds through specific sensor/receptor tools that modulate bacterial physiology. Consequently, interfering with these mechanisms offers an exciting opportunity to directly affect the infection process. It is shown that the human hormone Atrial Natriuretic Peptide (hANP) both prevents the formation of P. aeruginosa biofilms and strongly disperses established P. aeruginosa biofilms. This hANP action is dose‐dependent with a strong effect at low nanomolar concentrations and takes effect in 30–120 min. Furthermore, although hANP has no antimicrobial effect, it acts as an antibiotic adjuvant. hANP enhances the antibiofilm action of antibiotics with diverse modes of action, allowing almost full biofilm eradication. The hANP effect requires the presence of the P. aeruginosa sensor AmiC and the AmiR antiterminator regulator, indicating a specific mode of action. These data establish the activation of the ami pathway as a potential mechanism for P. aeruginosa biofilm dispersion. hANP appears to be devoid of toxicity, does not enhance bacterial pathogenicity, and acts synergistically with antibiotics. These data show that hANP is a promising powerful antibiofilm weapon against established P. aeruginosa biofilms in chronic infections.

Published
2022
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2. Contribution of the Pseudomonas fluorescens MFE01 Type VI Secretion System to Biofilm Formation.

Author

Mathias Gallique, Victorien Decoin, Corinne Barbey, Thibaut Rosay, Marc G J Feuilloley, Nicole Orange, and Annabelle Merieau

Subjects
Medicine, Science
Abstract

Type VI secretion systems (T6SSs) are widespread in Gram-negative bacteria, including Pseudomonas. These macromolecular machineries inject toxins directly into prokaryotic or eukaryotic prey cells. Hcp proteins are structural components of the extracellular part of this machinery. We recently reported that MFE01, an avirulent strain of Pseudomonas fluorescens, possesses at least two hcp genes, hcp1 and hcp2, encoding proteins playing important roles in interbacterial interactions. Indeed, P. fluorescens MFE01 can immobilise and kill diverse bacteria of various origins through the action of the Hcp1 or Hcp2 proteins of the T6SS. We show here that another Hcp protein, Hcp3, is involved in killing prey cells during co-culture on solid medium. Even after the mutation of hcp1, hcp2, or hcp3, MFE01 impaired biofilm formation by MFP05, a P. fluorescens strain isolated from human skin. These mutations did not reduce P. fluorescens MFE01 biofilm formation, but the three Hcp proteins were required for the completion of biofilm maturation. Moreover, a mutant with a disruption of one of the unique core component genes, MFE01ΔtssC, was unable to produce its own biofilm or inhibit MFP05 biofilm formation. Finally, MFE01 did not produce detectable N-acyl-homoserine lactones for quorum sensing, a phenomenon reported for many other P. fluorescens strains. Our results suggest a role for the T6SS in communication between bacterial cells, in this strain, under biofilm conditions.

Published
2017
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3. Different Dose-Dependent Modes of Action of C-Type Natriuretic Peptide on Pseudomonas aeruginosa Biofilm Formation

Author

Florie Desriac, Thomas Clamens, Thibaut Rosay, Sophie Rodrigues, Ali Tahrioui, Jérémy Enault, Lucille Roquigny, Pierre-Jean Racine, Laure Taupin, Alexis Bazire, Alain Dufour, Jérôme Leprince, Emeline Bouffartigues, Sylvie Chevalier, Marc G. J. Feuilloley, and Olivier Lesouhaitier

Subjects
biofilm, hormones, peptides, bacterial adaptation, lung, cystic fibrosis, Medicine
Abstract

We have previously shown that the C-type Natriuretic Peptide (CNP), a peptide produced by lungs, is able to impact Pseudomonas aeruginosa physiology. In the present work, the effect of CNP at different concentrations on P. aeruginosa biofilm formation was studied and the mechanisms of action of this human hormone on P. aeruginosa were deciphered. CNP was shown to inhibit dynamic biofilm formation in a dose-dependent manner without affecting the bacterial growth at any tested concentrations. The most effective concentrations were 1 and 0.1 µM. At 0.1 µM, the biofilm formation inhibition was fully dependent on the CNP sensor protein AmiC, whereas it was only partially AmiC-dependent at 1 µM, revealing the existence of a second AmiC-independent mode of action of CNP on P. aeruginosa. At 1 µM, CNP reduced both P. aeruginosa adhesion on glass and di-rhamnolipid production and also increased the bacterial membrane fluidity. The various effects of CNP at 1 µM and 0.1 µM on P. aeruginosa shown here should have major consequences to design drugs for biofilm treatment or prevention.

Published
2018
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4. Pseudomonas aeruginosa Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation

Author

Thibaut Rosay, Alexis Bazire, Suraya Diaz, Thomas Clamens, Anne-Sophie Blier, Lily Mijouin, Brice Hoffmann, Jacques-Aurélien Sergent, Emeline Bouffartigues, Wilfrid Boireau, Julien Vieillard, Christian Hulen, Alain Dufour, Nicholas J. Harmer, Marc G. J. Feuilloley, and Olivier Lesouhaitier

Subjects
Microbiology, QR1-502
Abstract

ABSTRACT Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression. IMPORTANCE The bacterium Pseudomonas aeruginosa is a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites of P. aeruginosa infection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this study, we showed that the CNP hormone counteracts P. aeruginosa biofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein, which is an ortholog of the human receptor NPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factor production while reducing biofilm formation.

Published
2015
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5. The extra-cytoplasmic function sigma factor sigX modulates biofilm and virulence-related properties in Pseudomonas aeruginosa.

Author

Gwendoline Gicquel, Emeline Bouffartigues, Manjeet Bains, Virginie Oxaran, Thibaut Rosay, Olivier Lesouhaitier, Nathalie Connil, Alexis Bazire, Olivier Maillot, Magalie Bénard, Pierre Cornelis, Robert E W Hancock, Alain Dufour, Marc G J Feuilloley, Nicole Orange, Eric Déziel, and Sylvie Chevalier

Subjects
Medicine, Science
Abstract

SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the "chaperones and heat shock proteins", "antibiotic resistance and susceptibility", "energy metabolism", "protein secretion/export apparatus", and "secreted factors", and "motility and attachment" classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes.

Published
2013
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6. Neuropeptides and bacterial interactions: The example of the impact of the C-type natriuretic peptide (CNP) on Pseudomonas aeruginosa biofilm formation

Author

Olivier Lesouhaitier, Thomas Clamens, Florie Desriac, Thibaut Rosay, Louis Mélissande, Sophie Rodrigues, Alexis Bazire, Alain Dufour, Jérôme Leprince, Emeline Bouffartigues, Pierre Cornelis, sylvie CHEVALIER, Marc Feuilloley, Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Laboratoire de Biotechnologie et Chimie Marines (LBCM), Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Plate-Forme de Recherche en Imagerie Cellulaire de Haute-Normandie (PRIMACEN), Normandie Université (NU)-Normandie Université (NU)-Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Lesouhaitier, Olivier

Subjects
[SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience; The opening of a new field of research known as microbial endocrinology, has prompted us to investigate the impact of several neuropeptides produced by the host on various bacteria isolated from skin or pathogens in the respiratory tract. We observed that substance P and CGRP two skin nerves neuropeptides are able to modify Staphylococcus epidermidis, S. aureus and Bacillus cereus biofilm formation and virulence regulation through binding to specific bacterial sensors. We also demonstrated that the lung produced C-type Natriuretic Peptide (CNP) enhances Pseudomonas aeruginosa virulence. In the present study, we focus on the CNP action mechanism on P. aeruginosa biofilm formation and on the identification of the bacterial sensor for this human hormone. We observed that exposure of P. aeruginosa to graded concentrations of CNP (10-9 to 10-6 M) induced a dose-dependent inhibition of biofilm formation. This effect was totally abolished by isatin, an antagonist of eukaryotic cells natriuretic peptide receptors. Screening and comparing 3D structures of human natriuretic peptides receptors and P. aeruginosa proteins revealed that the bacterial protein AmiC shows significant homology with the human C-type eukaryotic natriuretic peptide receptor (hNPR-C). Recombinant protein AmiC was obtained and the protein interactions assessed using microscale thermophoresis. The results showed that both CNP and the hNPR-C agonist osteocrin bind AmiC with respective K D of 2 µM and 5 nM confirming the role of AmiC as natriuretic peptides sensor. The amiC gene belongs to the ami operon where amiE encodes the AmiE amidase which hydrolyses short-chain aliphatic amides to their corresponding organic acids. We further observed that over expression of AmiE resulted in altered biofilm formation and strongly reduced bacterial virulence, suggesting that AmiE should be the final effector of the CNP effects on P. aeruginosa. Taken together, these data show that neuropeptides, firstly identified as host signal molecules, could act as bacterial modulator and in certain conditions act as potential antimicrobial peptides.

Published
2018

7. Different Dose-Dependent Modes of Action of C-Type Natriuretic Peptide on Pseudomonas aeruginosa Biofilm Formation

Author

Thomas Clamens, Jérôme Leprince, Ali Tahrioui, Sophie Rodrigues, Olivier Lesouhaitier, Jérémy Enault, Lucille Roquigny, Alexis Bazire, Alain Dufour, Sylvie Chevalier, Thibaut Rosay, Florie Desriac, Laure Taupin, Marc G. J. Feuilloley, Emeline Bouffartigues, Pierre-Jean Racine, Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Laboratoire de Biotechnologie et Chimie Marines (LBCM), Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Différenciation et communication neuronale et neuroendocrine (DC2N), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Microbiologie du Froid – Signaux et Micro-Environnement (LMDF-SME), and Neuroendocrinologie cellulaire et moléculaire

Subjects
0301 basic medicine, Microbiology (medical), medicine.drug_class, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, 030106 microbiology, bacterial adaptation, lcsh:Medicine, Peptide, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Bacterial growth, medicine.disease_cause, biofilm, lung, cystic fibrosis, 03 medical and health sciences, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Membrane fluidity, medicine, Natriuretic peptide, Immunology and Allergy, Mode of action, Molecular Biology, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, General Immunology and Microbiology, hormones, Pseudomonas aeruginosa, lcsh:R, Biofilm, Adhesion, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, peptides, Infectious Diseases, chemistry, Biophysics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology
Abstract

International audience; We have previously shown that the C-type Natriuretic Peptide (CNP), a peptide produced by lungs, is able to impact Pseudomonasaeruginosa physiology. In the present work, the effect of CNP at different concentrations on P. aeruginosa biofilm formation was studied and the mechanisms of action of this human hormone on P. aeruginosa were deciphered. CNP was shown to inhibit dynamic biofilm formation in a dose-dependent manner without affecting the bacterial growth at any tested concentrations. The most effective concentrations were 1 and 0.1 µM. At 0.1 µM, the biofilm formation inhibition was fully dependent on the CNP sensor protein AmiC, whereas it was only partially AmiC-dependent at 1 µM, revealing the existence of a second AmiC-independent mode of action of CNP on P. aeruginosa. At 1 µM, CNP reduced both P. aeruginosa adhesion on glass and di-rhamnolipid production and also increased the bacterial membrane fluidity. The various effects of CNP at 1 µM and 0.1 µM on P. aeruginosa shown here should have major consequences to design drugs for biofilm treatment or prevention.

Published
2018

8. Host Peptidic Hormones Affecting Bacterial Biofilm Formation and Virulence

Author

Sophie Rodrigues, Mélissande Louis, Thibaut Rosay, Pierre Cornelis, Emeline Bouffartigues, Marc G. J. Feuilloley, Florie Desriac, A. V. Gannesen, Alexis Bazire, Sylvie Chevalier, Ali Tahrioui, Vladimir K. Plakunov, Alain Dufour, Thomas Clamens, Olivier Lesouhaitier, Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Russian Academy of Sciences [Moscow] (RAS), Lomonosov Moscow State University (MSU), Laboratoire de Biotechnologie et Chimie Marines (LBCM), Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), and Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, medicine.drug_class, Calcitonin Gene-Related Peptide, Peptide Hormones, Antibiotics, Virulence, Biology, medicine.disease_cause, Gram-Positive Bacteria, Dynorphins, Microbiology, 03 medical and health sciences, 0302 clinical medicine, Gram-Negative Bacteria, medicine, Immunology and Allergy, Humans, Natriuretic Peptides, ComputingMilieux_MISCELLANEOUS, Pseudomonas aeruginosa, Host (biology), Neuropeptides, Biofilm, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, 030104 developmental biology, 030228 respiratory system, Biofilms, Somatostatin, Bacteria, Hormone
Abstract

Bacterial biofilms constitute a critical problem in hospitals, especially in resuscitation units or for immunocompromised patients, since bacteria embedded in their own matrix are not only protected against antibiotics but also develop resistant variant strains. In the last decade, an original approach to prevent biofilm formation has consisted of studying the antibacterial potential of host communication molecules. Thus, some of these compounds have been identified for their ability to modify the biofilm formation of both Gram-negative and Gram-positive bacteria. In addition to their effect on biofilm production, a detailed study of the mechanism of action of these human hormones on bacterial physiology has allowed the identification of new bacterial pathways involved in biofilm formation. In this review, we focus on the impact of neuropeptidic hormones on bacteria, address some future therapeutic issues, and provide a new view of inter-kingdom communication.

Published
2018

9. Contribution of the Pseudomonas fluorescens MFE01 Type VI Secretion System to Biofilm Formation

Author

Nicole Orange, Marc G. J. Feuilloley, Annabelle Merieau, Corinne Barbey, Mathias Gallique, Thibaut Rosay, Victorien Decoin, Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), Université de Rouen Normandie (UNIROUEN), and Normandie Université (NU)-Normandie Université (NU)

Subjects
0301 basic medicine, Agrobacteria, Molecular biology, lcsh:Medicine, Plant Science, medicine.disease_cause, Pathology and Laboratory Medicine, Plant Microbiology, Medicine and Health Sciences, lcsh:Science, ComputingMilieux_MISCELLANEOUS, Skin, Multidisciplinary, biology, Chemistry, Antimicrobials, Pseudomonas, Pseudomonas Aeruginosa, Drugs, Detectors, Bacterial Pathogens, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Medical Microbiology, Engineering and Technology, Pathogens, Research Article, Pseudomonas Fluorescens, 030106 microbiology, Equipment, Pseudomonas fluorescens, DNA construction, Microbiology, Agrobacterium Tumefaciens, 03 medical and health sciences, Microbial Control, medicine, Humans, Secretion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Microbial Pathogens, Type VI secretion system, Pharmacology, Bacteria, Pseudomonas aeruginosa, lcsh:R, Biofilm, Organisms, Biology and Life Sciences, Bacteriology, biology.organism_classification, Research and analysis methods, Quorum sensing, 030104 developmental biology, Biosensors, Molecular biology techniques, Biofilms, Plasmid Construction, Mutation, Antibacterials, lcsh:Q, Bacterial Biofilms
Abstract

Type VI secretion systems (T6SSs) are widespread in Gram-negative bacteria, including Pseudomonas. These macromolecular machineries inject toxins directly into prokaryotic or eukaryotic prey cells. Hcp proteins are structural components of the extracellular part of this machinery. We recently reported that MFE01, an avirulent strain of Pseudomonas fluorescens, possesses at least two hcp genes, hcp1 and hcp2, encoding proteins playing important roles in interbacterial interactions. Indeed, P. fluorescens MFE01 can immobilise and kill diverse bacteria of various origins through the action of the Hcp1 or Hcp2 proteins of the T6SS. We show here that another Hcp protein, Hcp3, is involved in killing prey cells during co-culture on solid medium. Even after the mutation of hcp1, hcp2, or hcp3, MFE01 impaired biofilm formation by MFP05, a P. fluorescens strain isolated from human skin. These mutations did not reduce P. fluorescens MFE01 biofilm formation, but the three Hcp proteins were required for the completion of biofilm maturation. Moreover, a mutant with a disruption of one of the unique core component genes, MFE01ΔtssC, was unable to produce its own biofilm or inhibit MFP05 biofilm formation. Finally, MFE01 did not produce detectable N-acyl-homoserine lactones for quorum sensing, a phenomenon reported for many other P. fluorescens strains. Our results suggest a role for the T6SS in communication between bacterial cells, in this strain, under biofilm conditions.

Published
2017

10. The aliphatic amidase AmiE is involved in regulation of Pseudomonas aeruginosa virulence

Author

Pascal Cosette, Alain Dufour, Alexandre Crépin, Sylvie Chevalier, Pierre Cornelis, Julie Hardouin, Thibaut Rosay, Julien Vieillard, Perrine Bortolotti, Thomas Clamens, Joerg Overhage, Benoit Guery, Fergal O'Gara, Takfarinas Kentache, Anke Neidig, Teddy Grandjean, Marc G. J. Feuilloley, Marlies J. Mooij, Emeline Bouffartigues, Mélanie Hillion, Olivier Lesouhaitier, Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Laboratoire de Biotechnologie et Chimie Marines (LBCM), Université de Brest (UBO)-Université de Bretagne Sud (UBS)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Recherche translationelle relations hôte-pathogènes, Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Polymères Biopolymères Surfaces (PBS), Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS), Université Lille 2 - Faculté de Médecine, Karlsruhe Institute of Technology (KIT), University College Cork (UCC), Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Plate-forme de Protéomique PISSARO, Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biologie et physiologie des états septiques, IFR114-Université de Lille, Droit et Santé, This work was supported by grants from the Communauté d’Agglomération d’Evreux, the Conseil Général de l′Eure, European Union (FEDER), the French Association 'Vaincre la Mucoviscidose' and the Partenariats Hubert Curien (PHC Ulysses). Financial support by the BioInterfaces (BIF) Program of the Karlsruhe Institute of Technology (KIT) in the Helmholtz Association is gratefully acknowledged., We wish to thank Magalie Barreau and Olivier Maillot for technical assistance. T. Clamens and T. Rosay are recipients of a doctoral fellowship from the French Ministry of Research (MRE)., Vriendenkring VUB, Microbiology, Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Faculté de Médecine Henri Warembourg - Université de Lille, Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-High-tech Research Infrastructures for Life Sciences (HeRacLeS), Normandie Université (NU)-Normandie Université (NU)-Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Bazire, Alexis

Subjects
Male, 0301 basic medicine, Virulence regulation, MESH: Pseudomonas Infections/microbiology, Proteome, health care facilities, manpower, and services, MESH: Dictyostelium/microbiology, MESH: Virulence, medicine.disease_cause, MESH: Quorum Sensing, Pilus, chemistry.chemical_compound, MESH: Caenorhabditis elegans/microbiology, MESH: Amidohydrolases/metabolism, AmiE overproduction, Dictyostelium, MESH: Animals, Lung, health care economics and organizations, MESH: Pseudomonas aeruginosa/enzymology, Multidisciplinary, Virulence, Quorum Sensing, MESH: Proteome, Pseudomonas aeruginosa, Female, Life sciences, biology, MESH: Virulence Factors, Virulence Factors, 030106 microbiology, Homoserine, MESH: Biofilms, Biology, Microbiology, Article, Amidohydrolases, Amidase, 03 medical and health sciences, Pyocyanin, MESH: Mice, Inbred C57BL, ddc:570, Journal Article, medicine, Animals, Pseudomonas Infections, cardiovascular diseases, Caenorhabditis elegans, MESH: Lung/microbiology, MESH: Pseudomonas aeruginosa/pathogenicity, Biofilm, Bacteriology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Male, MESH: Pseudomonas Infections/enzymology, Mice, Inbred C57BL, Quorum sensing, chemistry, Biofilms, FOS: Biological sciences, Pseudomonas aeruginosa virulence, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Female
Abstract

We have previously shown that the eukaryotic C-type natriuretic peptide hormone (CNP) regulates Pseudomonas aeruginosa virulence and biofilm formation after binding on the AmiC sensor, triggering the amiE transcription. Herein, the involvement of the aliphatic amidase AmiE in P. aeruginosa virulence regulation has been investigated. The proteome analysis of an AmiE over-producing strain (AmiE+) revealed an expression change for 138 proteins, including some that are involved in motility, synthesis of quorum sensing compounds and virulence regulation. We observed that the AmiE+ strain produced less biofilm compared to the wild type, and over-produced rhamnolipids. In the same line, AmiE is involved in P. aeruginosa motilities (swarming and twitching) and production of the quorum sensing molecules N-acyl homoserine lactones and Pseudomonas Quinolone Signal (PQS). We observed that AmiE overproduction reduced levels of HCN and pyocyanin causing a decreased virulence in different hosts (i.e. Dictyostelium discoideum and Caenorhabditis elegans). This phenotype was further confirmed in a mouse model of acute lung infection, in which AmiE overproduction resulted in an almost fully virulence decrease. Taken together, our data suggest that, in addition to its role in bacterial secondary metabolism, AmiE is involved in P. aeruginosa virulence regulation by modulating pilus synthesis and cell-to-cell communication.

Published
2017

11. <named-content content-type='genus-species'>Pseudomonas aeruginosa</named-content> Expresses a Functional Human Natriuretic Peptide Receptor Ortholog: Involvement in Biofilm Formation

Author

Lily Mijouin, Emeline Bouffartigues, Thomas Clamens, Olivier Lesouhaitier, Thibaut Rosay, Alexis Bazire, Alain Dufour, Christian Hulen, Nicholas J. Harmer, Marc G. J. Feuilloley, Jacques-Aurélien Sergent, Anne-Sophie Blier, Wilfrid Boireau, Julien Vieillard, Suraya Diaz, Brice Hoffmann, Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), EA3884, Université de Bretagne Sud (UBS), School of Biosciences, University of Kent [Canterbury], Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Centre National de la Recherche Scientifique (CNRS), Université de Cergy Pontoise (UCP), Université Paris-Seine, Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut de Chimie Organique Fine (IRCOF), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), and Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Receptors, Peptide, Virulence Factors, medicine.drug_class, Molecular Conformation, Virulence, Biology, Crystallography, X-Ray, medicine.disease_cause, Microbiology, Virulence factor, [SPI.MAT]Engineering Sciences [physics]/Materials, 03 medical and health sciences, Virology, Natriuretic Peptide, Brain, Natriuretic peptide, medicine, Humans, Computer Simulation, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Receptor, 030304 developmental biology, [SPI.ACOU]Engineering Sciences [physics]/Acoustics [physics.class-ph], 0303 health sciences, 030306 microbiology, Pseudomonas aeruginosa, Biofilm, Natriuretic Peptide, C-Type, NPR1, NPR2, Peptide Fragments, QR1-502, 3. Good health, Biofilms, Periplasmic Binding Proteins, Atrial Natriuretic Factor, Research Article
Abstract

Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic-resistant pathogen Pseudomonas aeruginosa can detect the human hormones brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. The possibility of identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa, while sANP, the NPR-A receptor agonist, appears to be weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar three-dimensional (3D) structures and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa. Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrated that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses structural and pharmacological profiles similar to those of the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression., IMPORTANCE The bacterium Pseudomonas aeruginosa is a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites of P. aeruginosa infection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this study, we showed that the CNP hormone counteracts P. aeruginosa biofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein, which is an ortholog of the human receptor NPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factor production while reducing biofilm formation.

Published
2015

12. The absence of the Pseudomonas aeruginosa OprF protein leads to increased biofilm formation through variation in c-di-GMP level

Author

Alain Dufour, Olivier Lesouhaitier, Joerg Overhage, Gwendoline Gicquel, Gerald Brenner-Weiss, Alexis Bazire, Joana A. Moscoso, Olivier Maillot, Rachel Duchesne, Alain Filloux, Sylvie Chevalier, Nicole Orange, Thibaut Rosay, Magalie Bénard, Patrice Lerouge, Marc G. J. Feuilloley, Emeline Bouffartigues, Laurène Fito-Boncompte, Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Imperial College London, Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plate-Forme de Recherche en Imagerie Cellulaire de Haute-Normandie (PRIMACEN), Normandie Université (NU)-Normandie Université (NU)-Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biotechnologie et Chimie Marines (LBCM), Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Karlsruhe Institute of Technology (KIT), Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV), Laboratoire de Biotechnologie et Chimie Marine (LBCM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-High-tech Research Infrastructures for Life Sciences (HeRacLeS), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Bazire, Alexis, Laboratoire de Microbiologie du Froid – Signaux et Micro-Environnement ( LMDF-SME ), Université de Rouen Normandie ( UNIROUEN ), Normandie Université ( NU ) -Normandie Université ( NU ), Reproduction et développement des plantes ( RDP ), Centre National de la Recherche Scientifique ( CNRS ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique ( INRA ) -École normale supérieure - Lyon ( ENS Lyon ), Laboratoire de Biotechnologie et Chimie Marines ( LBCM ), Université de Bretagne Sud ( UBS ) -Université de Brest ( UBO ), Institute for Research and Innovation in Biomedicine ( IRIB ), Normandie Université ( NU ) -Normandie Université ( NU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Karlsruhe Institute of Technology ( KIT ), Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale ( Glyco-MEV ), UR DER Développement et économie rurale, Institut Supérieur d'Agriculture Rhône-Alpes, Laboratoire Interdisciplinaire Carnot de Bourgogne ( LICB ), Université de Bourgogne ( UB ) -Centre National de la Recherche Scientifique ( CNRS ), and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
SIGMA-FACTOR SIGX, EXPRESSION, Microbiology (medical), OUTER-MEMBRANE PROTEIN, Mutant, lcsh:QR1-502, Biology, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, medicine.disease_cause, Microbiology, lcsh:Microbiology, biofilm, Sigma factor, medicine, ECF, NATURAL sciences & mathematics, ComputingMilieux_MISCELLANEOUS, Original Research, Science & Technology, CYSTIC-FIBROSIS, ROLES, RECEPTOR, Pseudomonas aeruginosa, exopolysaccharides, Biofilm, OprF, SigX, c-di-GMP, biochemical phenomena, metabolism, and nutrition, EXOPOLYSACCHARIDE PRODUCTION, GENE, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell aggregation, POLYSACCHARIDE, Regulon, Porin, ddc:500, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Bacterial outer membrane, Life Sciences & Biomedicine, MATRIX
Abstract

OprF is the major outer membrane porin in bacteria belonging to the Pseudomonas genus. In previous studies, we have shown that OprF is required for full virulence expression of the opportunistic pathogen Pseudomonas aeruginosa. Here, we describe molecular insights on the nature of this relationship and report that the absence of OprF leads to increased biofilm formation and production of the Pel exopolysaccharide. Accordingly, the level of c-di-GMP, a key second messenger in biofilm control, is elevated in an oprF mutant. By decreasing c-di-GMP levels in this mutant, both biofilm formation and pel gene expression phenotypes were restored to wild-type levels. We further investigated the impact on two small RNAs, which are associated with the biofilm lifestyle, and found that expression of rsmZ but not of rsmY was increased in the oprF mutant and this occurs in a c-di-GMP-dependent manner. Finally, the extracytoplasmic function (ECF) sigma factors AlgU and SigX displayed higher activity levels in the oprF mutant. Two genes of the SigX regulon involved in c-di-GMP metabolism, PA1181 and adcA (PA4843), were up-regulated in the oprF mutant, partly explaining the increased c-di-GMP level. We hypothesized that the absence of OprF leads to a cell envelope stress that activates SigX and results in a c-di-GMP elevated level due to higher expression of adcA and PA1181. The c-di-GMP level can in turn stimulate Pel synthesis via increased rsmZ sRNA levels and pel mRNA, thus affecting Pel-dependent phenotypes such as cell aggregation and biofilm formation. This work highlights the connection between OprF and c-di-GMP regulatory networks, likely via SigX (ECF), on the regulation of biofilm phenotypes.

Published
2015

13. The extra-cytoplasmic function sigma factor sigX modulates biofilm and virulence-related properties in Pseudomonas aeruginosa

Author

Pierre Cornelis, Alexis Bazire, Alain Dufour, Thibaut Rosay, Eric Déziel, Manjeet Bains, Gwendoline Gicquel, Emeline Bouffartigues, Magalie Bénard, Nathalie Connil, Marc G. J. Feuilloley, Sylvie Chevalier, Olivier Maillot, Virginie Oxaran, Robert E. W. Hancock, Olivier Lesouhaitier, Nicole Orange, Microbiology, Laboratoire de Microbiologie du Froid – Signaux et Micro-Environnement (LMDF-SME), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Centre for Microbal Diseases and Immunity Research, University of British Columbia (UBC), Laboratoire de Biotechnologie et Chimie Marines, Université européenne de Bretagne - European University of Brittany (UEB)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS), Plate-Forme de Recherche en Imagerie Cellulaire de Haute-Normandie (PRIMACEN), Normandie Université (NU)-Normandie Université (NU)-Institute for Research and Innovation in Biomedicine (IRIB), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-High-tech Research Infrastructures for Life Sciences (HeRacLeS), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Department of Bioengineering Sciences, Research group Microbiology, VIB Department of Structural Biology, Vrije Universiteit Brussel (VUB), Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), This work was supported by the French government and Région Haute Normandie, France (part of the doctoral fellowship to G. Gicquel), the Grand Evreux Agglomération (financial support of O. Maillot), the European FEDER funds number 32789 for the LMSM (post doctoral fellowships to E. Bouffartigues and V. Oxaran) and by Canadian Institutes for Health Research (CIHR) grants to ED and REWH. REWH was further supported by a Cystic Fibrosis Canada grant. ED holds a tier 2 Canada Research Chair and REWH holds a tier 1 Canada Research Chair., Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rouen Normandie (UNIROUEN), and Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
SigX, PAOSX, MESH: Transcriptome/genetics, Cytoplasm, Transcription, Genetic, Mutant, lcsh:Medicine, MESH: Cytoplasm/genetics, Swarming motility, MESH: Sigma Factor/genetics, medicine.disease_cause, Sigma factor, lcsh:Science, Caenorhabditis elegans, Genetics, 0303 health sciences, Multidisciplinary, Virulence, biology, MESH: Transcription, Genetic/genetics, MESH: Pseudomonas aeruginosa/genetics, Pseudomonas aeruginosa, MESH: Caco-2 Cells, MESH: Energy Metabolism/genetics, Bacterial outer membrane, Research Article, MESH: Cell Line, Tumor, Virulence Factors, Sigma Factor, MESH: Biofilms, MESH: Bacterial Proteins/genetics, 03 medical and health sciences, Bacterial Proteins, Cell Line, Tumor, MESH: Mutation/genetics, medicine, Humans, MESH: Virulence/genetics, 030304 developmental biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Humans, 030306 microbiology, lcsh:R, Wild type, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Biofilms, Mutation, lcsh:Q, Caco-2 Cells, Energy Metabolism, Transcriptome, MESH: Pseudomonas aeruginosa/physiology
Abstract

International audience; SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the "chaperones and heat shock proteins", "antibiotic resistance and susceptibility", "energy metabolism", "protein secretion/export apparatus", and "secreted factors", and "motility and attachment" classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes.

Published
2013

14. TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa

Author

Anke, Neidig, Amy T Y, Yeung, Thibaut, Rosay, Beatrix, Tettmann, Nikola, Strempel, Martina, Rueger, Olivier, Lesouhaitier, and Joerg, Overhage

Subjects
Virulence, Virulence Factors, Pathogen, Macrophage, Gene Expression Profiling, Macrophages, TypA, Biofilm, Resistance, Dictyostelium discoideum, Real-Time Polymerase Chain Reaction, Bacterial Adhesion, Endocytosis, GTP Phosphohydrolases, Type III secretion system, Biofilms, Drug Resistance, Bacterial, Mutation, Pseudomonas aeruginosa, Humans, Amoeba, Cells, Cultured, Research Article
Abstract

Background Pseudomonas aeruginosa is an important opportunistic human pathogen and is extremely difficult to treat due to its high intrinsic and adaptive antibiotic resistance, ability to form biofilms in chronic infections and broad arsenal of virulence factors, which are finely regulated. TypA is a GTPase that has recently been identified to modulate virulence in enteric Gram-negative pathogens. Results Here, we demonstrate that mutation of typA in P. aeruginosa resulted in reduced virulence in phagocytic amoebae and human macrophage models of infection. In addition, the typA mutant was attenuated in rapid cell attachment to surfaces and biofilm formation, and exhibited reduced antibiotic resistance to ß-lactam, tetracycline and antimicrobial peptide antibiotics. Quantitative RT-PCR revealed the down-regulation, in a typA mutant, of important virulence-related genes such as those involved in regulation and assembly of the Type III secretion system, consistent with the observed phenotypes and role in virulence of P. aeruginosa. Conclusions These data suggest that TypA is a newly identified modulator of pathogenesis in P. aeruginosa and is involved in multiple virulence-related characteristics.

Published
2012

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