Type III secretion of ExoU is critical during early Pseudomonas aeruginosa pneumonia

Abstract

The Pseudomonas aeruginosa type III secretion system has been associated with poor outcomes in both animal models and human patients. Despite a large number of studies exploring the regulation of type III secretion in vitro, little is known about the timing of secretion during mammalian infection. Here we demonstrate that the exoU gene, which encodes the highly cytotoxic type III effector ExoU, is induced early during acute P. aeruginosa pneumonia. Immunofluorescence microscopy indicated that the amount of ExoU protein in the lung also increased over time. The importance of early expression was examined using a strain of P. aeruginosa with inducible production of ExoU. Delays in expression as short as 3 h led to reduced bacterial burdens in the lungs of mice and improved survival. Our results demonstrate that early expression of exoU is critical to bacterial survival during pneumonia and suggest that therapeutic interventions that delay ExoU secretion for even short periods of time may be efficacious.

Importance: Pseudomonas aeruginosa is a major contributor to the large numbers of health care-associated infections occurring annually, particularly for immunocompromised patients. Although this organism possesses many virulence factors, the type III secretion system plays an especially important role in both animal models and humans. This system forms a needle-like apparatus that injects toxins directly into eukaryotic cells. The most toxic protein secreted by this molecular machine is ExoU, which causes rapid cell death. In this study, we demonstrated that exoU was expressed and ExoU was produced early during acute pneumonia in a mouse model. Delaying expression of exoU by as little as 3 h enhanced clearance of bacteria and survival of infected mice. Our findings highlight the importance of understanding the regulation of virulence factor expression during infection when designing therapeutic strategies to inhibit the toxic effects of these proteins.

FIG 1

The exoU gene is expressed early in the mouse lung during acute P. aeruginosa pneumonia. Total RNA was isolated from the lungs of mice infected with P. aeruginosa PA99 for the times indicated. RNA was reverse transcribed to cDNA and used in quantitative PCR to quantify exoU transcripts. exoU transcript levels were normalized between samples using a constitutively expressed bacterial gene, rpoD, and are reported relative to expression of exoU in broth culture under growth conditions that do not induce type III secretion. Each diamond represents the value for an individual mouse, and the black bars represent medians for the group of mice. Four mice were used for each time point.

FIG 2

Amounts of ExoU protein increase over time in mouse lungs during acute P. aeruginosa pneumonia. Mice were infected with either P. aeruginosa PA99null or PA99. The lungs were removed from the mice and fixed at the indicated times as described in Materials and Methods. Samples representing the entire axial cross section of both lungs were sequentially labeled with antisera specific to ExoU and PA99. (A) An image of a tissue section encompassing both lungs; analyzed regions are outlined in blue. (B to H) Representative images showing ExoU protein (green) and P. aeruginosa bacteria (red) within the lungs of infected mice. The nuclei of lung cells were stained with DAPI (blue). Images were taken at a magnification of ×200. Bars = 25 µm. (I) Quantification of total area labeled with ExoU or P. aeruginosa antibodies for an axial cross section encompassing both lungs, as shown in panel A. The analysis was completed on three independently labeled tissue sections. Error bars represent the standard errors of the means.

FIG 3

ATC-inducible ExoU-mediated cytotoxicity is dose dependent and can be delayed in vitro. (A) P. aeruginosa strains were grown in LB medium or LB medium plus ATC overnight, then diluted, and grown to exponential phase prior to infection of HeLa cells. Appropriate amounts of ATC were added to the culture wells upon infection. Each hour postinfection, aliquots of culture supernatant were removed for determination of cell lysis. Measurements were normalized to 100% lysis by Triton X-100. Error bars indicate standard errors of the means for triplicate wells. (B) Bacteria were grown overnight in LB medium, diluted, and then regrown to exponential phase prior to infection of HeLa cells. To evaluate the effect of delaying exoU expression, 100 ng/ml of ATC was added to the appropriate wells at 0, 0.5, or 1 h postinfection. Each hour postinfection, aliquots of culture supernatants were removed, and the levels of cell lysis were measured. Data are normalized to 100% lysis by Triton X-100. Error bars indicate the standard errors of the means for triplicate wells. Values that are significantly different (P value of <0.05) from the value for ATC added at 0 min are indicated by an asterisk.

FIG 4

Delaying exoU expression leads to decreased bacterial burden in the lungs of infected mice. Mice were intranasally infected with the strains indicated in the figure and given ATC intraperitoneally at the times indicated in the figure. Control mice were injected with PBS. Mice were redosed with subsequent injections 12 h following the initial dose. Mice were sacrificed at 18 h postinfection, and the number of CFU in their lungs was determined by plating. Each symbol represents the value for an individual mouse. Black bars indicate the median value for each condition. Four or five mice were used for each condition, and each experiment was repeated a total of three times. Representative results of a single experiment are shown.

FIG 5

Delayed expression of exoU is associated with improved survival in a mouse model of acute pneumonia. Mice were intranasally infected with the P. aeruginosa strains indicated in the figure and injected with ATC intraperitoneally at the times indicated in the figure. Control mice were injected with PBS. All mice receiving ATC were redosed every 12 h for the duration of the experiment. Survival was monitored for a period of 72 h postinfection. Data are the cumulative results of two separate experiments. Values that are significantly different from the value for PA99null+ptetU receiving ATC at 0 h are indicated by asterisks as follows: *, P value of ≤0.01; **, P value of <0.00001.