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

BACKGROUND: MFN1032 is a clinical Pseudomonas fluorescens strain able to grow at 37 degrees 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 degrees C. This activity is tightly regulated and is subject to phase variation. RESULTS: We found that MFN1032 displays a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis was expressed at 37 degrees 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. CONCLUSIONS: 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.

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

BACKGROUND: 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 degrees 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 degrees C in laboratory conditions. RESULTS: 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. CONCLUSION: 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.

Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences.

The major surface protein of the genus Pseudomonas, OprF, is a non-specific porin that plays an important role in maintenance of cell shape, in growth in a low osmolarity environment, and in adhesion to various supports. The objectives of our study were (i) to carry out a comparative analysis of phylogenies obtained from the OprF protein and from the 16S rRNA gene in 41 isolates from various sources (water, soil, milk and the hospital) and (ii) to investigate the physiological characteristics correlated with the phylogeny of OprF. We report here an important incongruence between the phylogenies of the 16S rRNA gene and the OprF protein. Phylogenetic analysis of 16S rRNA genes grouped Pseudomonas fluorescens isolates into one cluster (termed fluorescens r-cluster) whilst the phylogeny of the OprF protein divided Pseudomonas fluorescens isolates into two quite distinct clusters (termed fluorescens 1 o-cluster and fluorescens 2 o-cluster) that may be related to the original habitat of the strain. The fluorescens 1 o-cluster contained the majority of non-rhizospheric soil isolates, while the fluorescens 2 o-cluster contained all our clinical isolates and most of the rhizospheric isolates (which are fixed to the roots). In order to check this correlation, we studied two physiological characteristics: the range of growth temperature and the capacity for non-specific adhesion to polystyrene. The temperature range study for strains did not explain the existence of the two o-clusters but it did confirm the capacity of certain P. fluorescens strains to grow at 37 degrees C. The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche.

Regulation of the cytotoxic effects of Pseudomonas fluorescens by growth temperature.

We had previously shown that the psychrotrophic bacterium Pseudomonas fluorescens can act as a pathogen, inducing apoptosis and necrosis in neurons and glial cells. In the present study, we investigated the influence of the growth temperature of P. fluorescens on its infectious potential. Adherence of P. fluorescens to glial cells was found to be maximal with bacteria grown at a low temperature (8 degrees C). At that temperature the swimming behaviour was markedly reduced. An increase in the growth temperature to 19, 28 or 32 degrees C strongly diminished the binding of bacteria to host cells. Thus, the adhesion phenotype of P. fluorescens appears to be independent of the motility of the bacteria. The apoptotic effect of P. fluorescens, determined by morphological (nuclear condensation) and biochemical (induction of nitric oxide synthase activity) indicators, correlated well with its binding activity on glial cells. In contrast, there was a clear dissociation between maximum binding and maximal necrotic action (measured by the release of lactate dehydrogenase) observed with bacteria grown at 19 degrees C. As suggested by capillary electrophoresis analysis, the differences in apoptotic effects may be related to variations in the molecular structure of LPS originating from bacteria grown at low and high temperatures, whereas the necrotic effect, which was maximal at the optimum temperature for the secretion of exoenzymes, could reflect variations in the metabolic activity of bacteria.