The Lon protease is essential for full virulence in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.

Figure 1. HBE cells infected with P. aeruginosa wild type, lon mutant (lon-) and complemented strain (lon::plon+).

HBE cells were co-cultured with bacteria cells for 3 hours to allow adherence. After 3 hours, non-adhered bacteria were removed by washing 3 times with PBS. Remaining bacteria and HBE cells were heat fixed and stained with Diff-Quick to allow microscopic analysis. The arrows indicate the bacterial filaments formed by the lon mutant and the single cells of the wild type and complemented strain.

Figure 2. Role of the Lon protease in a rat chronic lung infection model.

Equal ratios of the WT PAO1 and lon mutant (left) or WT and complemented strain (right) were embedded in agarose beads and delivered to the rat lungs via intubation. After 7 days post-infection, rats were sacrificed and lungs were recovered for CFU determinations. The competitive index was analyzed as described in Methods and is the ratio of the recovered CFU for test strain compared to the WT, corrected for the input numbers of bacteria. Each point represents the competitive index (CI) for a single animal. The lon mutant had a major attenuation in growth in this chronic model, which was statistically significant as measured with the Mann Whitney test (p<0.05).

Figure 3. Role of the Lon protease in a mouse acute lung infection model.

CD-1 mice were inoculated via intranasal route with 10⁶ CFU PA14 WT or lon mutant. Mice were sacrificed 4 hours post-infection and bronchoalveolar lavage fluids were collected for bacterial CFU determinations. Each point represents results from a single mouse and the graph is representative from 3 independent mouse experiments. The lon mutant showed a 2 log defect in virulence which was statistically significant (*p<0.05) as assessed using the Mann Whitney test for the experiment shown here and also 3 pooled experiments.

Figure 4. Role of Lon in surfing motility on mucin containing plates.

P. aeruginosa wild type and lon mutant spotted on medium containing mucin allowed for surfing motility (right), the control without mucin is seen on the left. Mucin was added to the medium in an attempt to more closely mimic the lung environment of cystic fibrosis patients.

Figure 5. Mature biofilm formation with increasing concentrations of mucin.

Cells from the wild-type strain H103, the lon mutant (lon-) and the complemented strain (lon::plon+) were incubated in 96 well microtiter plates containing LB and varying concentrations of mucin (0–0.5%) for 20 hours at 37°C. Biofilm formation was measured by crystal violet staining of the adherent cells. The bars represent the average and standard deviation of 3 independent experiments.