Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.

FIG. 1.

Relative expression levels of exsA (A), exoS (B), exoT (C), and pcrV (D). The results are representative of three independent experiments carried out in triplicate. The error bars indicate standard deviations.

FIG. 2.

Analysis of ExoS (A) and PcrV (B) production (white bars) and secretion (black bars) from PAO1 and isogenic LPS mutant strains. The results are representative of three independent experiments. The error bars indicate standard deviations.

FIG. 3.

Cytotoxicity assay using BEAS-2B lung epithelial cells and standardized inocula of PAO1 and isogenic LPS O-antigen mutant strains. Black bars, 4 h postinfection; white bars, 6 h postinfection. The results are representative of at least two independent experiments carried out in triplicate. The error bars indicate standard deviations.

FIG. 4.

In vivo evaluation of lung injury in BALB/c mice infected with PAO1 and an isogenic LPS mutant. (A) Measure of lung edema. (B) Epithelial permeability measurement. (C) Body temperature. The results represent the average measurement for four mice in each group. The error bars indicate standard deviations.

FIG. 5.

Immunoblot analysis of ExoU (A) and PcrV (B) production and secretion by clinical isolates cultured as either planktonic or biofilm cultures. The results are representative of at least three independent experiments. The error bars indicate standard deviations.