Natriuretic peptides modify Pseudomonas fluorescens cytotoxicity by regulating cyclic nucleotides and modifying LPS structure

<p>Abstract</p> <p>Background</p> <p>Nervous tissues express various communication molecules including natriuretic peptides, <it>i.e</it>. Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP). These molecules share structural similarities with cyclic antibacterial peptides. CNP and to a lesser extent BNP can modify the cytotoxicity of the opportunistic pathogen <it>Pseudomonas aeruginosa</it>. The psychrotrophic environmental species <it>Pseudomonas fluorescens </it>also binds to and kills neurons and glial cells, cell types that both produce natriuretic peptides. In the present study, we investigated the sensitivity of <it>Pseudomonas fluorescens </it>to natriuretic peptides and evaluated the distribution and variability of putative natriuretic peptide-dependent sensor systems in the <it>Pseudomonas </it>genus.</p> <p>Results</p> <p>Neither BNP nor CNP modified <it>P. fluorescens </it>MF37 growth or cultivability. However, pre-treatment of <it>P. fluorescens </it>MF37 with BNP or CNP provoked a decrease of the apoptotic effect of the bacterium on glial cells and an increase of its necrotic activity. By homology with eukaryotes, where natriuretic peptides act through receptors coupled to cyclases, we observed that cell-permeable stable analogues of cyclic AMP (dbcAMP) and cyclic GMP (8BcGMP) mimicked the effect of BNP and CNP on bacteria. Intra-bacterial concentrations of cAMP and cGMP were measured to study the involvement of bacterial cyclases in the regulation of <it>P. fluorescens </it>cytotoxicity by BNP or CNP. BNP provoked an increase (+49%) of the cAMP concentration in <it>P. fluorescens</it>, and CNP increased the intra-bacterial concentrations of cGMP (+136%). The effect of BNP and CNP on the virulence of <it>P. fluorescens </it>was independent of the potential of the bacteria to bind to glial cells. Conversely, LPS extracted from MF37 pre-treated with dbcAMP showed a higher necrotic activity than the LPS from untreated or 8BcGMP-pre-treated bacteria. Capillary electrophoresis analysis suggests that these different effects of the LPS may be due, at least in part, to variations in the structure of the macromolecule.</p> <p>Conclusion</p> <p>These observations support the hypothesis that <it>P. fluorescens </it>responds to natriuretic peptides through a putative sensor system coupled to a cyclase that could interfere with LPS synthesis and thereby modify the overall virulence of the micro-organism.</p

Pseudomonas fluorescens alters epithelial permeability and translocates across Caco-2/TC7 intestinal cells

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas fluorescens </it>has long been considered as a psychrotrophic microorganism. Recently, we have shown that clinical strains of <it>P. fluorescens </it>(biovar 1) are able to adapt at a growth temperature of 37°C or above and induce a specific inflammatory response. Interestingly, a highly specific antigen of <it>P. fluorescens</it>, I2, is detected in the serum of patients with Crohn's disease but the possible role of this bacterium in the disease has not yet been explored. In the present study, we examined the ability of a psychrotrophic and a clinical strain of <it>P. fluorescens </it>to modulate the permeability of a Caco-2/TC7 intestinal epithelial model, reorganize the actin cytoskeleton, invade the target cells and translocate across the epithelium. The behaviour of these two strains was compared to that of the well known opportunistic pathogen <it>P. aeruginosa </it>PAO1.</p> <p>Results</p> <p>Both strains of <it>P. fluorescens </it>were found to decrease the transepithelial resistance (TER) of Caco-2/TC7 differentiated monolayers. This was associated with an increase in paracellular permeability and F-actin microfilaments rearrangements. Moreover, the invasion and translocation tests demonstrated that the two strains used in this study can invade and translocate across the differentiated Caco-2/TC7 cell monolayers.</p> <p>Conclusions</p> <p>The present work shows for the first time, that <it>P. fluorescens </it>is able to alter the intestinal epithelial barrier function by disorganizing the F-actin microfilament network. Moreover, we reveal that independently of their origins, the two <it>P. fluorescens </it>strains can translocate across differentiated Caco-2/TC7 cell monolayers by using the transcellular pathway. These findings could, at least in part, explain the presence of the <it>P. fluorescens </it>specific I2 antigen in the serum of patients with Crohn's disease.</p

The Hidden Face of Nitrogen Oxides Species: From Toxic Effects to Potential Cure?

Nitrogen oxide (NOx) species represent ones of the most threatening air pollutants due to their prevalence and harmful impact on the environment and human health. The term NOx gathers mainly nitric oxide (NO) and nitrogen dioxide (NO2), mostly produced by anthropogenic activities such as transport and industries. Several cellular constituents were already described as NOx targets. These include membranes, proteins, respiratory chain enzymes, lipids, and DNA. Such damages lead to pathologies of lungs, cardiovascular system, and skin because these organs represent the first barrier toward the environment. On the other hand, NOx is also naturally synthetized by several organisms, playing a mediator role in essential cellular functions. However, few data are yet available on NOx activity toward microorganisms. Here, we review data concerning the double face of NOx, including their use in the medical field against pathogens’ infections that highlight the versatility of these compounds

Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas fluorescens </it>is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of <it>P. fluorescens </it>biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions.</p> <p>Results</p> <p>We found that MFN1032 secreted extracellular factors with a lytic potential at least as high as that of MF37, a psychrotrophic strain of <it>P. fluorescens </it>or the mesophilic opportunistic pathogen, <it>Pseudomonas aeruginosa </it>PAO1. We demonstrated the direct, and indirect – through increases in biosurfactant release – involvement of a phospholipase C in the hemolytic activity of this bacterium. Sequence analysis assigned this phospholipase C to a new group of phospholipases C different from those produced by <it>P. aeruginosa</it>. We show that changes in PlcC production have pleiotropic effects and that <it>plcC </it>overexpression and <it>plcC </it>extinction increase MFN1032 toxicity and colonization, respectively.</p> <p>Conclusion</p> <p>This study provides the first demonstration that a PLC is involved in the secreted hemolytic activity of a clinical strain of <it>Pseudomonas fluorescens</it>. Moreover, this phospholipase C seems to belong to a complex biological network associated with the biosurfactant production.</p

Development of preservative-free nanoparticles-based emulsions: Effects of NP surface properties and sterilization process

International audienceModel emulsions were developed with or without commercial titanium dioxide nanoparticles (NP) carrying various surface treatments in order to get close physicochemical properties whatever the NP surface polarity (hydrophilic and hydrophobic). Rheology and texturometry highlighted that the macroscopic properties of the three formulated emulsions were similar. However, characterizations by optical microscopy, static light scattering and zetametry showed that their microstructures reflected the diversity of the incorporated NP surface properties. In order to use these model emulsions as tools for biological evaluations of the NP in use, they had to show the lowest initial microbiological charge and, specifically for the NP-free emulsion, the lowest bactericidal effect. Hence, formulae were developed preservative-free and a thermal sterilization step was conducted. Efficiency of the sterilization and its impact on the emulsion integrity were monitored. Results highlighted the effect of the NP surface properties: only the control emulsion and the emulsion containing hydrophilic NP fulfilled both requirements. To ensure the usability of these model emulsions as tools to evaluate the 'NP effect' on representative bacteria of the skin microflora (S. aureus and P. fluorescens), impact on the bacterial growth was measured on voluntary inoculated formulae

Cell-associated hemolysis activity in the clinical strain of Pseudomonas fluorescens MFN1032

<p>Abstract</p> <p>Background</p> <p>MFN1032 is a clinical <it>Pseudomonas fluorescens </it>strain able to grow at 37°C. MFN1032 cells induce necrosis and apoptosis in rat glial cells at this temperature. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides. Under laboratory conditions, this activity is not expressed at 37°C. This activity is tightly regulated and is subject to phase variation.</p> <p>Results</p> <p>We found that MFN1032 displays a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis was expressed at 37°C and was only detected <it>in vitro </it>in mid log growth phase in the presence of erythrocytes. We studied the regulation of this activity in the wild-type strain and in a mutant defective in the Gac two-component pathway. GacS/GacA is a negative regulator of this activity. In contrast to the <it>Pseudomonas fluorescens </it>strains PfO-1 and Pf5, whose genomes have been sequenced, the MFN1032 strain has the type III secretion-like genes <it>hrc</it>RST belonging to the <it>hrpU </it>operon. We showed that disruption of this operon abolished cell-associated hemolytic activity. This activity was not detected in <it>P.fluorescens </it>strains carrying similar <it>hrc </it>genes, as for the <it>P. fluorescens </it>psychrotrophic strain MF37.</p> <p>Conclusions</p> <p>To our knowledge this the first demonstration of cell-associated hemolytic activity of a clinical strain of <it>Pseudomonas fluorescens</it>. Moreover, this activity seems to be related to a functional <it>hrpU </it>operon and is independent of biosurfactant production. Precise link between a functional <it>hrpU </it>operon and cell-associated hemolytic activity remains to be elucidated.</p

Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence Regulating Activity

The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10−5 M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies

IMPACT OF A TIO2 [NANO] COSMETICS GRADE ON THE CUTANEOUS MICROBIOTA: NANOPARTICLES BEHAVIOR IN EMULSION AND BACTERIOTOXICITY

International audienceTiO2-nanoparticles (NP) commonly used in cosmetics as physical UV-filters, are surface-modified by applying a first coating in order to quench photo-produced radicals. In addition, a second coating is sometimes added to raise their dispersibility in emulsion. Whereas European regulation mentions the obligation to demonstrate the safety of NP, very few studies took interest on their behavior in use. During formulation and storage, NP are exposed to a variety of environmental conditions which can accelerate aging and induce particles aggregation, adsorption of formula’s compounds or coatings deterioration. Consequently, a crucial question remains their safety while layered on skin and particularly their effect on the skin microflora. We will present the impact of TiO2-nanoparticles on the skin microflora in relation to their physicochemical properties in cosmetic emulsions during aging.A series of cosmetic emulsions were formulated with using conventional cosmetic ingredients, with or without Nanoparticles (NPs): one without NPs considered as blank, and two with hydrophilic and hydrophobic commercial TiO2-NPs, respectively. An emulsification process was developed and optimized to obtain a blank emulsion, physically stable, with improved NPs dispersions in emulsion. Then, emulsions were submitted to accelerate aging.Particles were extracted from the fresh/aged emulsions by original protocols allowing recovering particles with possible adsorbed compounds or cleaned surfaces.Skin bacteria growths were evaluated in emulsions or dispersions to check the toxicity of nanoparticles in use. Then, because of NP opacity, protocols were adapted to measure their effect on bacteria virulence toward skin cells.Microscopic and macroscopic characterizations revealed comparable structures and functional properties between the three fresh emulsions. Then, during aging, different behavior were registered and could be related to nanoparticle coating nature.Extracted NP from the fresh/aged formulas showed quick surface modifications in terms of chemical structures and physical properties that might be caused by adsorption or deterioration at their surfaces.For fresh emulsions, both nanoparticles did not have any impact on skin bacteria strains, S. aureus and P. fluorescens. In the case of aged emulsions, part of NPs seemed to allow the S. aureus growth, whereas others still remained with no effect on microflora. Finally, bacteria generation times and virulence toward skin cells differed depending on NPs surface treatments and exposition time.This work dealt with the impact of nanoparticles on skin microflora along cosmetic emulsion shelf-life. This new approach allows taking into account their safety when used in emulsion.First, through innovative extraction and characterization protocols, it was highlighted that chemical nature and physical properties of nanoparticle surfaces might be altered once in emulsion. These physicochemical observations were related to microbiological tests on skin representative bacteria. Effect of some of the NPs on bacteria growth and virulence evolved after aging. Finally, modification of NPs surface treatment seemed to have an effect on skin microflora

The absence of SigX may result in a nutritional stress response in Pseudomonas aeruginosa

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Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH

International audienceThe translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae and Bacteria, in which it plays several important functions including membrane biogenesis, signaling and stress response. A tspo homologue gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. (B) Co-transcription of Pfl01_2810 and tspo by RT-PCR assay. RT-PCR was assayed on tspo (1), Pfl01_2810 (2) and on the putative operonic structure Pfl01_2810-tspo (3), using primers located into tspo (1) or Pfl01_2810 (2) ORFs, or into both tspo and Pfl01_2810 ORFs (3). RT-PCR achieved on total RNA did not lead to a PCR fragment (line 4, negative control). L: Ladder 1kb+ (Biorad ®), (C) Growth curves in microtitre wells in LB medium at 28 C and relative luminescence (RL) activity of pTSPO and pHK-TSPO in P. fluorescens Pf0-1. The tspo gene (Pfl01_2811) of P. fluorescens Pf0-1 forms an operonic structure with Pfl01_2810 and is expressed transiently during the bacterial growth. (A) Schematic representation of the genomic environment of tspo and of transcriptional fusions pTSPO and pHK-TSPO. Grey bars represent the beginning of the luxCDABE reporter cassette of the promoterless pAB133 vector. The position of the studied promoter region is indicated relatively to the translational initiation start of each ORF. Pfl01_ 2809 lux CDABE pHK-TSPO-253 bp lux CDABE pTSPO-227 bp P. fluorescens Pf0-1 chromosome lux transcriptional fusions Pfl01_2810 tsp