The type III pseudomonal exotoxin U activates the c-Jun NH2-terminal kinase pathway and increases human epithelial interleukin-8 production

Abstract

Microbial interactions with host cell signaling pathways are key determinants of the host cell response to infection. Many toxins secreted by bacterial type III secretion systems either stimulate or inhibit the host inflammatory response. We investigated the role of type III secreted toxins of the lung pathogen Pseudomonas aeruginosa in the inflammatory response of human respiratory epithelial cells to infection. Using bacteria with specific gene deletions, we found that interleukin-8 production by these cells was almost entirely dependent on bacterial type III secretion of exotoxin U (ExoU), a phospholipase, although other bacterial factors are involved. ExoU activated the c-Jun NH(2)-terminal kinase pathway, stimulating the phosphorylation and activation of mitogen-activated kinase kinase 4, c-Jun NH(2)-terminal kinase, and c-Jun. This in turn increased levels of transcriptionally competent activator protein-1. Although this pathway was dependent on the lipase activity of ExoU, it was independent of cell death. Activation of mitogen-activated kinase signaling by ExoU in this fashion is a novel mechanism by which a bacterial product can initiate a host inflammatory response, and it may result in increased epithelial permeability and bacterial spread.

FIG. 1.

Cytotoxicity following infection of 16HBE14o− cells. A. Differentiated cells were infected at a low MOI (1 CFU per 5 × 10³ cells) as detailed in Materials and Methods, and cytotoxicity was measured at 20 h after infection for the strains indicated using LDH release. Noninternalized bacteria were removed by washing at 6 h following infection, and gentamicin or colistin was added for the remainder of the experiment to kill extracellular bacteria. Results are the means of triplicate determinations; error bars show standard errors of the means. B. Cells were infected at a high MOI (1 CFU per 1 cell) and cytotoxicity measured at various times and with various strains as shown by LDH release. Results are the means of triplicate observations. C. Undifferentiated cells plated at 80% confluence in 24-well tissue culture dishes were infected at an MOI of 1 CFU per 1 cell and assayed for cell death 6 h after infection by trypan blue staining. Results are the means of triplicate determinations; error bars show standard errors of the means. D. Levels of secreted ExoU produced by the indicated bacterial strains. EGTA was added to induce type III secretion as indicated. Levels of ExoU produced by the PA3 ΔpcrV strain were lower than those produced by the wild type, so the panel showing secreted proteins from the PA3 ΔpcrV strain is from a longer exposure of the immunoblot.

FIG. 2.

IL-8 release following infection of 16HBE14o− cells with strains as indicated. IL-8 production was measured from either the apical compartment or the basal compartment at 20 h following infection. Results are the means of triplicate determinations; error bars are standard errors of the means. Background values of IL-8 released from uninfected cells have been subtracted. The experiment was repeated five times with identical results. Significant differences for the corresponding values obtained with the wild-type PA3 strain are marked with an asterisk (P < 0.05, t test).

FIG. 3.

IL-8 production following infection of 16HBE14o− cells. Total IL-8 produced over time following infection with the different bacterial inocula of wild-type PA3 is shown. Results are means of triplicate determinations; error bars are standard errors of the means. The differences between IL-8 produced by the different bacterial inocula compared to no infection were significant as compared using two-way ANOVA (P < 0.05).

FIG. 4.

Complementation of ΔexoU strain with active exoU restores epithelial IL-8 secretion. A. Total IL-8 production (basal plus apical) was measured 20 h after infection at low MOI with wild-type PA3, the isogenic ΔexoU mutant, the ΔexoU mutant complemented with wild-type exoU (ΔexoU:exoU), and the ΔexoU mutant complemented with the lipase-inactive S142A mutant of ΔexoU (ΔexoU:S142AexoU). In each case, the plasmid used to complement the ΔexoU strains also carried the chaperone, spcU. Results are the means of two or three measurements expressed as percentages of the total IL-8 secretion produced by wild-type PA3; error bars are standard errors of the means. The asterisks indicate results significantly different from those for the wild-type PA3 strain (P < 0.05, t test). B. Total IL-8 production (basal plus apical) was measured 20 h after infection at low MOI with wild-type PA103ΔUΔT, PA103ΔUΔT complemented with wild-type exoU (PA103ΔUΔT:exoU), and PA103ΔUΔT complemented with the lipase-inactive S142A mutant of exoU (PA103ΔUΔT:S142AexoU). Control values for PA3 are also shown. Results are the means of triplicate determinations expressed as percentages of the total IL-8 secretion produced by wild-type PA3; error bars are standard errors of the means. Asterisks indicate values significantly different from those for the PA103 ΔUΔT strain (P < 0.05, t test). In all cases, background IL-8 values from uninfected cells have been subtracted. C. ExoU secretion from the indicated bacterial strains grown in the presence of EGTA and assayed by immunoblotting.

FIG. 5.

Changes in phosphorylation status of signaling molecules following infection with wild-type PA3 and the isogenic ΔexoU mutant at high MOI (1 bacterium per 1 cell). No antibiotics were added. Results show levels of the specific proteins as indicated at various times (hours) after infection with the strains as shown. Results are representative from two separate experiments with slightly different sampling times (A and B); the experiment was repeated on two further occasions with the same results. The blots were stripped and blotted sequentially to obtain the results as shown.

FIG. 6.

Effects of JNK inhibition with different doses of the inhibitor SP600125 on total IL-8 release following infection. 16HBE14o− cells treated with the indicated doses of SP600125 were infected at low MOI with wild-type PA3, and total IL-8 was determined 20 h after infection and expressed as a percentage of maximal IL-8 secretion in the absence of inhibitor. Results are means of triplicate determinations; error bars are standard errors of the means. The drug exerted a significant inhibitory effect on IL-8 secretion as determined by ANOVA (P < 0.05). Background values from uninfected cells were subtracted in all cases.

FIG. 7.

AP-1 induction following infection of 16HBE14o− cells with wild-type (WT) or ΔexoU strains of PA3. Results show the changes in induction of AP-1 containing phospho-c-Jun bound specifically to its DNA recognition site after infection at high MOI (1 bacterium per 1 cell with no added antibiotics). Results are expressed relative to the value observed at time zero and are the means of triplicate determinations; error bars are standard errors of the means. Specificity of the assay for AP-1 binding was gauged by using a fivefold molar excess of free AP-1 binding site oligonucleotide; this reduced the signal to less than 5% of the total and was subtracted in the calculations shown here. Irrelevant sequence oligonucleotide produced no significant change in signal. Significant differences from wild-type values are shown with an asterisk (t test, P < 0.05).