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

Abstract

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.

Figure 1

Growth and extracellular activities of MFN1032. A- Growth of MFN1032 on LB agar plates after 24 hours at 37°C. B- Secreted hemolytic activity of MFN1032 on sheep red blood agar plate after 48 hours of incubation at 28°C (1), and comparison with PAO1 (2) and MF37 (3). C- MFN1032 lecithinase activity on egg-yolk agar plate after 48 hours of incubation at 28°C.

Figure 2

Effect of growth temperature on the cytotoxicity of MFN1032 (white square), MF37 (grey square), and PAO1 (black square). After 15 generations in LB medium, aliquots of cell-free supernatants were assayed for glial cell lysis (A) or hemolysis (B), as described in the materials and methods. For assays of glial cell cytotoxicity, supernatants were concentrated with an Amicon ultra-15 filter, and then resuspended in glial cell medium. Each experiment was performed at least three times in triplicate. Nd: not determined.

Figure 3

Zymogram on egg-yolk agar of a silver-stained SDS-PAGE gel of MFN1032 supernatant. PLC activity was detected as an opaque band on the plate (arrow), the molecular mass of which was deduced. Concentrated MFN1032 supernatant was obtained following growth at 17°C in LB medium. 1: Front of a silver-stained gel placed on an egg-yolk agar plate (zymogram); 2: corresponding silver-stained gel; 3: other side of the zymogram; M: molecular size markers.

Figure 4

SDS-PAGE of total extracellular proteins of MFN032 and the PlcC mutants MFN1037 and MFN1038. Supernatants of cultures at 17°C in LB medium were concentrated on an Amicon ultra-15 filter and subjected to SDS-PAGE in a 10% acrylamide gel. The gel was silver-stained (lanes 1 to 3) and placed against an egg-yolk agar plate (lanes 4 to 6) to reveal lecithinase activity (the opaque band on the plate). 1 and 6: MFN1032; 2 and 5: MFN1037 (plcC-deficient MFN1032); 3 and 4: MFN1038 (plcC complemented MFN1037). M- molecular size markers. a: PlcC; b: flagellin.

Figure 5

Motility assays for MFN1032, MFN1036 and MFN1037. The swimming motility of MFN1032 (A), MFN1036 (B) and MFN1037 (C) on 0.3% LB agar plates and the swarming motility of MFN1032 (D) and MFN1037 (E) on 0.6% LB agar plates after 16 h of incubation at 28°C. MFN1032 (wild type) and MFN1037 (plcC-deficient MFN1032) presented concentric halos on 0.3% agar, corresponding to swimming motility, wherease MFN1036 (plcC-overexpressing MFN1032) displayed a dendritic pattern indicative of swarming motility. The same pattern of MFN1037 mobility was obtained with strain MFN1038 (data not shown).

Figure 6

Transmission electron microscopy (TEM) of MFN1032 (A) and MFN1037 (B) cells. (C) Biofilm quantification of MFN1032 and MFN1037 after 24 h of growth at 37°C in a polystyrene microtiter plate. Biofilm formation was quantified as the percentage relative to that observed with MFN1032 grown at 37°C.

Figure 7

Genomic organization and flanking regions of the plcC gene from MFN1032 and the putative phospholipase C genes in P. fluorescens Pf0-1 and Pf5. HP: hypothetical protein