Burkholderia cenocepacia requires the RpoN sigma factor for biofilm formation and intracellular trafficking within macrophages

Abstract

Chronic respiratory infections by Burkholderia cenocepacia in cystic fibrosis patients are associated with increased morbidity and mortality, but virulence factors determining the persistence of the infection in the airways are not well characterized. Using a chronic pulmonary infection model, we previously identified an attenuated mutant with an insertion in a gene encoding an RpoN activator protein, suggesting that RpoN and/or components of the RpoN regulon play a role in B. cenocepacia virulence. In this study, we demonstrate that a functional rpoN gene is required for bacterial motility and biofilm formation in B. cenocepacia K56-2. Unlike other bacteria, RpoN does not control flagellar biosynthesis, as evidenced by the presence of flagella in the rpoN mutant. We also demonstrate that, in macrophages, the rpoN mutant is rapidly trafficked to lysosomes while intracellular wild-type B. cenocepacia localizes in bacterium-containing vacuoles that exhibit a pronounced delay in phagolysosomal fusion. Rapid trafficking to the lysosomes is also associated with the release of red fluorescent protein into the vacuolar lumen, indicating loss of bacterial cell envelope integrity. Although a role for RpoN in motility and biofilm formation has been previously established, this study is the first demonstration that the RpoN regulon in B. cenocepacia is involved in delaying phagolysosomal fusion, thereby prolonging bacterial intracellular survival within macrophages.

FIG. 1.

Genetic organization of the rpoN gene and flanking regions of chromosome 1 of B. cenocepacia strains J2315 and K56-2. The rpoN gene is represented by a white arrow, and the insertion site of mutagenesis plasmid pRW3 is indicated by a triangle. Flanking genes BCAL0812 and BCAL0814 are indicated in black, and they encode an RpoN modulation protein and an ABC transporter ATP-binding protein, respectively.

FIG. 2.

RpoN controls motility but not flagellar synthesis in B. cenocepacia K56-2. (A) Effect of rpoN insertional inactivation on the motility of B. cenocepacia K56-2. Each panel shows an image of the bacteria grown at 37°C for 24 h on medium solidified with 0.3% agar. (B) Electron micrograph of wild-type and rpoN mutant cells. Bars correspond to 0.5 μm.

FIG. 3.

RpoN controls biofilm production by B. cenocepacia K56-2. (A) Image of crystal violet-stained biofilms formed by K56-2, MSS13, and MSS13(pSM72). Cells were grown in LB medium for 24 h at 37°C under static conditions before crystal violet staining. (B) Quantitative comparison of biofilm formation by B. cenocepacia K56-2, MSS13, and MSS13(pSM72). Each experiment was performed at least three times in triplicate. Error bars represent the standard error of the mean.

FIG. 4.

Flagellum-mediated motility and B. cenocepacia biofilm formation. (A) Each panel shows an image of a bacterial colony grown at 37°C for 24 h on medium solidified with 0.3% agar. (B) Electron micrograph of the fliC, ΔrpoN fliC, and ΔmotA mutants. Representative bacterial cells are shown. Bars correspond to 0.5 μm. (C) Quantitative comparison of biofilms formed by wild-type strain K56-2 and the fliC, ΔrpoN fliC, and ΔmotA mutants under static conditions. Each experiment was performed at least three times in triplicate. Error bars represent the standard error of the mean.

FIG. 5.

RpoN is required for intracellular survival of B. cenocepacia K56-2. Images of RAW 264.7 macrophage cells infected for 4 h with B. cenocepacia K56-2 or MSS13 at an MOI of 50 by fluorescence and phase-contrast microscopy. (A) Macrophages were incubated with 0.5 μM LysoTracker Red prior to visualization. K56-2 bacteria are within membrane-bound vacuoles that do not colocalize with LysoTracker Red (arrows). (B) Macrophages were incubated with 250 μg/ml of TMR-dextran prior to infection. K56-2 bacteria are within membrane-bound vacuoles that do not colocalize with TMR-dextran (arrows). (C) Percentage of bacterium-containing vacuoles colocalizing with LysoTracker Red. The values correspond to the average and standard error of three experiments in which 21 fields were examined. (D) Percentage of bacterium-containing vacuoles colocalizing with TMR-dextran. The values correspond to the average and standard error of three experiments in which 21 fields were examined. DIC, differential interference contrast.

FIG. 6.

RpoN is required for cell envelope integrity of B. cenocepacia within murine macrophages. (A) Images of RAW 264.7 macrophage cells infected for 4 h with B. cenocepacia K56-2(pRedΩCm) or MSS13(pRedΩCm) at an MOI of 50 by fluorescence and phase-contrast microscopy. The rpoN mutant bacteria had compromised cell envelope permeability, as shown by the release of mRFP1 into the vacuolar lumen, while the parental bacteria retained mRFP1 within the cytoplasm. The arrows indicate bacterium-containing vacuoles. (B) Average percentages of B. cenocepacia-containing vacuoles (BcCV) containing released mRFP1 from three independent experiments. Error bars represent the standard error of the mean. DIC, differential interference contrast.