Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by human airway epithelia

Abstract

Mammalian airways protect themselves from bacterial infection by using multiple defense mechanisms including antimicrobial peptides, mucociliary clearance, and phagocytic cells. We asked whether airways might also target a key bacterial cell-cell communication system, quorum-sensing. The opportunistic pathogen Pseudomonas aeruginosa uses two quorum-sensing molecules, N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) and N-butanoyl-l-homoserine lactone (C4-HSL), to control production of extracellular virulence factors and biofilm formation. We found that differentiated human airway epithelia inactivated 3OC12-HSL. Inactivation was selective for acyl-HSLs with certain acyl side chains, and C4-HSL was not inactivated. In addition, the capacity for inactivation varied widely in different cell types. 3OC12-HSL was inactivated by a cell-associated activity rather than a secreted factor. These data suggest that the ability of human airway epithelia to inactivate quorum-sensing signal molecules could play a role in the innate defense against bacterial infection.

Fig. 1.

Inactivation of 3OC12-HSL, but not C4-HSL, by human airway epithelia. (A) The concentration of C4-HSL in the apical (filled circles) and basolateral (open circles) solutions of primary cultures of differentiated human airway epithelia over time after addition of 1 μM C4-HSL to the apical surface. (B) The total amount of C4-HSL in the apical and basolateral solutions of the airway epithelium (open squares) and the total amount of C4-HSL in medium without cells (filled squares). Values are percent of the initial C4-HSL. (C) The concentration of 3OC12-HSL in the apical and basolateral solutions of airway epithelia after addition of 10 μM C4-HSL to the apical surface. (D) The total amount of 3OC12-HSL in apical and basolateral solutions and the total amount of 3OC12-HSL in medium without cells as a percent of initial 3OC12-HSL. Data are means ± SEM of three independent experiments.

Fig. 2.

The specificity of acyl-HSL degradation by human airway epithelia. Shown is the amount of C4-, C6-, 3OC6-, C12-, and 3OC12-HSL remaining in differentiated human airway epithelia after 1 h. Values represent the percent of acyl-HSL remaining compared to the initial level. Data are means ± SEM from three to six independent experiments.

Fig. 3.

Degradation of 3OC12-HSL by cultured mammalian cells. Shown are the amounts of C4-HSL (gray bars) and 3OC12-HSL (black bars) remaining after a 1-h exposure to various mammalian cells in culture. Cell lines included are A549 cells, established from human bronchoalveolar carcinoma, CaCo-2 from human colon carcinoma, MDCK from canine kidney, CHO from Chinese hamster ovary, 293T from human embryonic kidney, HeLa from human cervix, COS-7 from monkey kidney, and primary cultures of human lung fibroblasts (HLF). Data are means ± SEM (n = 8 replicates).

Fig. 4.

Inactivation of 3OC12-HSL by A549 cell lysates. Shown is the amount of 3OC12-HSL remaining after a 1-h exposure to A549 cells; conditioned medium collected after a 1-h incubation on A549 cells (Cond. Medium); cell lysate clarified by low-speed centrifugation; and the supernatant or the particulate (Pellet) fraction of the cell lysate after ultracentrifugation. Values are the percentage of 3OC12-HSL remaining after 1 h compared to the initial concentration. Data are means ± SEM from three independent experiments.

Fig. 5.

The dependence of 3OC12-HSL degradation by A549 cell lysates on time and lysate dilution. (A) Time course of 3OC12-HSL degradation. 3OC12-HSL (10 μM) was added to A549 cell extracts for the indicated times. Data are percent of 3OC12-HSL remaining. The black circle shows the percent of 3OC12-HSL remaining in boiled A549 cell extract after 1 h. (B) A lysate dilution series (the incubation time was 1 h). Data are means ± SEM of three independent experiments; in some cases, error bars are hidden by symbols.