Role of flagella in pathogenesis of Pseudomonas aeruginosa pulmonary infection

Abstract

Pseudomonas aeruginosa strains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens, P. aeruginosa cells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence of fliC mutants in a neonatal mouse model of pneumonia. In the absence of fliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to a pilA mutant PAK/NP (P < 0.0001). The fliC mutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that the fliC mutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung. 125I-labeled flagellin bound to the glycolipids GM1 and GD1a and to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type or fliC mutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla+ organisms but not fliC mutants were efficiently taken up by murine macrophages. P. aeruginosa flagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.

FIG. 1

Construction of fliC mutants demonstrated by Southern hybridization. A fliC probe consisting of a 659-bp EcoRI-SalI DNA fragment from pMS590 was used to detect homologous DNA cleaved with EcoRI and NruI in pMS590 (fliC interrupted by a Gm cassette) (lane A), ³²P-λ HindIII-labeled molecular weight markers (lane B), PAK genomic digest (lane C), PAKfliC (lane D), and PAK/NPfliC (lane E).

FIG. 2

(a) Purified flagellin. Lanes: A, molecular weight markers; B, gel-purified flagellin isolated from PAO/NP electrophoresed on a 12% polyacrylamide gel and stained with Coomassie blue. (b) Binding of ¹²⁵I-labeled flagellin to glycolipids. Lanes: A, autoradiograph of ¹²⁵I-labeled flagellin from PAO/NP overlaid on the glycolipids separated by thin-layer chromatography; B, visualization of the glycolipids with orcinol-ferric chloride following autoradiography. AGM₁, asialoGM1.

FIG. 3

Binding of flagellin and pilin to GM1 and asialoGM1 quantified by ELISA. (A) Pilin binding to microtiter plates coated with either asialoGM1 (AGM1) or GM1, detected by using antipilin antisera and alkaline phosphatase-conjugated goat anti-rabbit antibody and plotted by the relative OD₄₀₅ determined in an ELISA reader. (B) Flagellin binding to asialoGM1 and GM1, similarly compared by using antiflagellin antisera and alkaline phosphatase-conjugated goat anti-rabbit antibody as measured by ELISA.

FIG. 4

Bacterial adherence to CHO cells in the presence or absence of exogenous GM1. (A) The percentage of CHO Lec-2 cells, preincubated with or without exogenous GM1 (n = 10,000), which had adherent PAO1 (labeled with GFP) was quantified by flow cytometry under control conditions. The amount of GM1 incorporation into the CHO cells was estimated by measuring the percentage of cells which could be labeled with FITC-conjugated CTB, a GM1-specific ligand. (B) Binding of ³⁵S-labeled PAK, PAK/NP, PAK/fliC, and PAK/NP/fliC mutants to the CHO cells under control conditions or with added GM1 (as in panel A) was quantified. For these strains, the percent bound reflects the percentage of the bacterial inoculum added which bound to the CHO Lec-2 cells. The error bars are contained within the graph for many of the data points.

FIG. 5

P. aeruginosa adherence to 1HAEo- cells. The adherence of ³⁵S-labeled PAK and mutant strains (from an inoculum of 5 × 10⁸ CFU/ml) was quantified. (Some of the error bars fall within the data bar.)

FIG. 6

Virulence of fliC mutants in a neonatal BALBc/ByJ mice. The percentage of mice inoculated which developed pneumonia (recovery of >10³ CFU/lung), bacteremia, or mortality is indicated. Numbers of mice inoculated with the various strains: PAK, 30; PAK/NP, 29; PAK/fliC, 28; and PAK/NP/fliC, 25.

FIG. 7

Pathology associated with infection due to fliC mutants. (A) Inoculation of the mice with PAK/fliC resulted in a focal infiltrate involving a very limited portion of the lung. (B) Infection with wild-type PAK shown at higher magnification (×100) demonstrates the typical response to P. aeruginosa with edema, infiltration of PMNs, and some hemorrhage. A small area of relatively normal appearing lung is visible at the upper right. (C) Pathology induced by the introduction of purified flagellin into the mouse lung.

FIG. 7

Pathology associated with infection due to fliC mutants. (A) Inoculation of the mice with PAK/fliC resulted in a focal infiltrate involving a very limited portion of the lung. (B) Infection with wild-type PAK shown at higher magnification (×100) demonstrates the typical response to P. aeruginosa with edema, infiltration of PMNs, and some hemorrhage. A small area of relatively normal appearing lung is visible at the upper right. (C) Pathology induced by the introduction of purified flagellin into the mouse lung.

FIG. 8

Adherence and uptake of PAK strains by RAW 264.7 cells. PAK strains transformed with a plasmid encoding GFP were incubated with RAW cells for 15 min, and the mean fluorescence of RAW cells with associated bacteria was quantified by flow cytometry. Mean fluorescence was normalized by dividing the fluorescence of the RAW cells plus bacteria by the mean fluorescence of each bacterial strain.

FIG. 9

Model of respiratory tract infection. Stage 1, acquisition of organisms from the environment. Motile, piliated organisms are inhaled and usually killed by local defensins (arrows) and removed by mucociliary clearance before penetrating the glycocalyx. Stage 2, immunostimulatory phase. In CF, due to diminished defensin activity, organisms produce exoproducts which destroy the protective glycocalyx, allowing access to the epithelial surface. Flagella may function to tether the bacteria to the mucosal surface. The close apposition of the organism to glycolipid receptors found predominantly on cells with mutant CF transmembrane conductance regulator function allows pilin-mediated attachment to occur, followed by stimulation of epithelial IL-8 expression and migration of PMNs to the airway. Stage 3, adaptation and chronic infection. In response to the immune pressure of the host, mutants which do not express flagella as well as the alginate-producing strains are selected and predominate due to their ability to avoid phagocytic clearance.