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1.  A Pseudomonas fluorescens type 6 secretion system is related to mucoidy, motility and bacterial competition 
BMC Microbiology  2015;15:72.
Pseudomonas fluorescens strain MFE01 secretes in abundance two Hcp proteins (haemolysin co-regulated proteins) Hcp1 and Hcp2, characteristic of a functional type 6 secretion system. Phenotypic studies have shown that MFE01 has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinically relevant bacteria. Mutagenesis of the hcp2 gene abolishes or reduces, depending on the target strain, MFE01 antibacterial activity. Hcp1, encoded by hcp1, may also be involved in bacterial competition. We therefore assessed the contribution of Hcp1 to competition of P. fluorescens MFE01 with other bacteria, by studying MFE01 mutants in various competitive conditions.
Mutation of hcp1 had pleiotropic effects on the MFE01 phenotype. It affected mucoidy of the strain and its motility and was associated with the loss of flagella, which were restored by introduction of plasmid expressing hcp1. The hcp1 mutation had no effect on bacterial competition during incubation in solid medium. MFE01 was able to sequester another P. fluorescens strain, MFN1032, under swimming conditions. The hcp2 mutant but not the hcp1 mutant conserved this ability. In competition assays on swarming medium, MFE01 impaired MFN1032 swarming and displayed killing activity. The hcp2 mutant, but not the hcp1 mutant, was able to reduce MFN1032 swarming. The hcp1 and hcp2 mutations each abolished killing activity in these conditions.
Our findings implicate type 6 secretion of Hcp1 in mucoidy and motility of MFE01. Our study is the first to establish a link between a type 6 secretion system and flagellin and mucoidy. Hcp1 also appears to contribute to limiting the motility of prey cells to facilitate killing mediated by Hcp2. Inhibition of motility associated with an Hcp protein has never been described. With this work, we illustrate the importance and versatility of type 6 secretion systems in bacterial adaptation and fitness.
PMCID: PMC4379610  PMID: 25886496
Pseudomonas fluorescens; Type 6 secretion system; Hcp protein; Competitive inhibition; Motility; Mucoidy; Exopolysaccharides
2.  Virulence of the Pseudomonas fluorescens clinical strain MFN1032 towards Dictyostelium discoideum and macrophages in relation with type III secretion system 
BMC Microbiology  2012;12:223.
Pseudomonas fluorescens biovar I MFN1032 is a clinical isolate able to grow at 37°C. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides, and a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis is independent of biosurfactant production and remains in a gacA mutant. Disruption of the hrpU-like operon (the basal part of type III secretion system from rhizospheric strains) suppresses this activity. We hypothesized that this phenotype could reflect evolution of an ancestral mechanism involved in the survival of this species in its natural niche. In this study, we evaluated the hrpU-like operon’s contribution to other virulence mechanisms using a panel of Pseudomonas strains from various sources.
We found that MFN1032 inhibited the growth of the amoebae Dictyostelium discoideum and that this inhibition involved the hrpU-like operon and was absent in a gacA mutant. MFN1032 was capable of causing macrophage lysis, if the hrpU-like operon was intact, and this cytotoxicity remained in a gacA mutant. Cell-associated hemolytic activity and macrophage necrosis were found in other P. fluorescens clinical isolates, but not in biocontrol P. fluorescens strains harbouring hrpU-like operon. The growth of Dictyostelium discoideum was inhibited to a different extent by P. fluorescens strains without correlation between this inhibition and hrpU-like operon sequences.
In P. fluorescens MFN1032, the basal part of type III secretion system plays a role in D. discoideum growth inhibition and macrophage necrosis. The inhibition of D. discoideum growth is dependent on the GacS/GacA system, while cell-associated hemolytic activity and macrophage lysis are not. Virulence against eukaryotic cells based on the hrpU-like operon may be more than just a stochastic evolution of a conserved system dedicated to survival in competition with natural predators such as amoebae. It may also mean that there are some important modifications of other type III secretion system components, which remain unknown. Cell-associated hemolysis might be a good indicator of the virulence of Pseudomonas fluorescens strain.
PMCID: PMC3489880  PMID: 23020706
Pseudomonas fluorescens clinical strains; Type III secretion system; Dictyostelium discoideum; Macrophage necrosis; Cell-associated hemolytic activity
3.  The clinical Pseudomonas fluorescens MFN1032 strain exerts a cytotoxic effect on epithelial intestinal cells and induces Interleukin-8 via the AP-1 signaling pathway 
BMC Microbiology  2010;10:215.
Pseudomonas fluorescens is present in low number in the intestinal lumen and has been proposed to play a role in Crohn's disease (CD). Indeed, a highly specific antigen, I2, has been detected in CD patients and correlated to the severity of the disease. We aimed to determine whether P. fluorescens was able to adhere to human intestinal epithelial cells (IECs), induce cytotoxicity and activate a proinflammatory response.
Behaviour of the clinical strain P. fluorescens MFN1032 was compared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunistic pathogen P. aeruginosa PAO1. Both strains of P. fluorescens were found to adhere on Caco-2/TC7 and HT-29 cells. Their cytotoxicity towards these two cell lines determined by LDH release assays was dose-dependent and higher for the clinical strain MFN1032 than for MF37 but lower than P. aeruginosa PAO1. The two strains of P. fluorescens also induced IL-8 secretion by Caco-2/TC7 and HT-29 cells via the AP-1 signaling pathway whereas P. aeruginosa PAO1 potentially used the NF-κB pathway.
The present work shows, for the first time, that P. fluorescens MFN1032 is able to adhere to IECs, exert cytotoxic effects and induce a proinflammatory reaction. Our results are consistent with a possible contribution of P. fluorescens in CD and could explain the presence of specific antibodies against this bacterium in the blood of patients.
PMCID: PMC2933668  PMID: 20698984
4.  Cell-associated hemolysis activity in the clinical strain of Pseudomonas fluorescens MFN1032 
BMC Microbiology  2010;10:124.
MFN1032 is a clinical Pseudomonas fluorescens 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.
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 in vitro 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 Pseudomonas fluorescens strains PfO-1 and Pf5, whose genomes have been sequenced, the MFN1032 strain has the type III secretion-like genes hrcRST belonging to the hrpU operon. We showed that disruption of this operon abolished cell-associated hemolytic activity. This activity was not detected in P.fluorescens strains carrying similar hrc genes, as for the P. fluorescens psychrotrophic strain MF37.
To our knowledge this the first demonstration of cell-associated hemolytic activity of a clinical strain of Pseudomonas fluorescens. Moreover, this activity seems to be related to a functional hrpU operon and is independent of biosurfactant production. Precise link between a functional hrpU operon and cell-associated hemolytic activity remains to be elucidated.
PMCID: PMC2871272  PMID: 20416103
5.  Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens 
BMC Microbiology  2008;8:189.
Pseudomonas fluorescens 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 P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions.
We found that MFN1032 secreted extracellular factors with a lytic potential at least as high as that of MF37, a psychrotrophic strain of P. fluorescens or the mesophilic opportunistic pathogen, Pseudomonas aeruginosa 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 P. aeruginosa. We show that changes in PlcC production have pleiotropic effects and that plcC overexpression and plcC extinction increase MFN1032 toxicity and colonization, respectively.
This study provides the first demonstration that a PLC is involved in the secreted hemolytic activity of a clinical strain of Pseudomonas fluorescens. Moreover, this phospholipase C seems to belong to a complex biological network associated with the biosurfactant production.
PMCID: PMC2613904  PMID: 18973676
6.  Natriuretic peptides modify Pseudomonas fluorescens cytotoxicity by regulating cyclic nucleotides and modifying LPS structure 
BMC Microbiology  2008;8:114.
Nervous tissues express various communication molecules including natriuretic peptides, i.e. 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 Pseudomonas aeruginosa. The psychrotrophic environmental species Pseudomonas fluorescens 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 Pseudomonas fluorescens to natriuretic peptides and evaluated the distribution and variability of putative natriuretic peptide-dependent sensor systems in the Pseudomonas genus.
Neither BNP nor CNP modified P. fluorescens MF37 growth or cultivability. However, pre-treatment of P. fluorescens 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 P. fluorescens cytotoxicity by BNP or CNP. BNP provoked an increase (+49%) of the cAMP concentration in P. fluorescens, and CNP increased the intra-bacterial concentrations of cGMP (+136%). The effect of BNP and CNP on the virulence of P. fluorescens 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.
These observations support the hypothesis that P. fluorescens 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.
PMCID: PMC2488351  PMID: 18613967

Results 1-6 (6)