Host resistance to lung infection mediated by major vault protein in epithelial cells

Abstract

The airway epithelium plays an essential role in innate immunity to lung pathogens. Ribonucleoprotein particles primarily composed of major vault protein (MVP) are highly expressed in cells that encounter xenobiotics. However, a clear biologic function for MVP is not established. We report here that MVP is rapidly recruited to lipid rafts when human lung epithelial cells are infected with Pseudomonas aeruginosa, and maximal recruitment is dependent on bacterial binding to the cystic fibrosis transmembrane conductance regulator. MVP was also essential for optimal epithelial cell internalization and clearance of P. aeruginosa. These results suggest that MVP makes a substantial contribution to epithelial cell-mediated resistance to infection.

Fig. 1. Recruitment of MVP to lipid rafts of airway epithelial cells by P. aeruginosa

(A) Lysates of WT and homozygous ΔF508 CFTR human airway epithelial cells (CFT1-LCFSN and CFT1-LC3, respectively) left uninfected (Con) or infected with P. aeruginosa strain PA01-V (PA01) were separated on discontinuous sucrose gradients. Proteins in raft fractions were precipitated and subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis with antibodies directed against human MVP. (B) IB3-1 CF cells (ΔF508/W1282X) transfected with WT-CFTR with an N-terminal GFP tag were infected with CFP-expressing P. aeruginosa, then fixed and stained for MVP. Confocal microscopy identified (arrows) bacteria (red), CFTR (green), and MVP (blue) at the site of bacterial contact with the cell membrane. Scale bar, 10 µm. Overlay shows the merged image of the three individual channels. (C) MVP in rafts of WT-CFTR cells left uninfected (Con) or infected with P. aeruginosa (PA01) in the presence or absence of 5 mM cyclodextrin (CD). (D) MVP in rafts of WT-CFTR cells left uninfected or infected with strain PA01-V or clinical isolates of P. aeruginosa from two CF patients (N6, N8) or from a corneal infection (6294). (E) MVP in rafts of WT-CFTR cells left uninfected or infected with strain PA01-V or LPS mutants of PA01-V (algC⁻ or galU⁻). The LPS mutants lack the CFTR-binding domain on the bacterial cell surface and do not promote MVP entry into lipid rafts after P. aeruginosa infection.

Fig. 2. MVP function in the interaction of airway epithelial cells with P. aeruginosa

(A) Whole-cell lysates from WT-CFTR cells (CFT1-LCFSN) that had been treated with either a scrambled siRNA sequence or MVP-specific siRNA were subjected to electrophoresis and immunoblot analysis with MVP-specific antibodies. Specific siRNA treatment reduced the level of MVP by >90%. (B) WT-CFTR cells were treated with scrambled or MVP-specific siRNA then left uninfected (Con) or infected for 15 min with P. aeruginosa PA01-V (PA01). Cell lysates were separated on discontinuous sucrose gradients. Raft fractions were precipitated and subjected to immunoblot analysis with MVP-specific antibodies. (C) Scrambled or MVP-specific siRNA-treated WT-CFTR cells were incubated with the indicated P. aeruginosa strains, and the amount of bacterial internalization was determined. The internalization of P. aeruginosa by cells treated with MVP-specific siRNA is plotted as a percentage of that obtained for scrambled siRNA-treated cells. Error bars represent SEMs. *P < 0.05, **P < 0.01 by a two-tailed t test comparison with internalization by scrambled siRNA-treated cells.

Fig. 3. In vivo role of MVP in bacterial internalization, clearance and resistance to lethality

(A) Six hours after intranasal inoculation with ~1.5 × 10⁷ P. aeruginosa strain PA01-V, WT and MVP^−/− mice were killed (n = 9 WT, 12 MVP^−/−), and the lungs were removed and dissociated. The internalized percentage of total bacteria found in the lung is plotted. Bars represent mean values. *P < 0.0001 by unpaired two-tailed t test. (B) The lung suspensions from the same mice used in (A) were analyzed for total bacterial load. The number of bacteria per gram of lung is plotted. Error bars represent standard error of the mean. *P < 0.01 by unpaired two-tailed t test. (C) Survival curves over 96 hours after intranasal inoculation with ~4 × 10⁶ colony-forming units of P. aeruginosa strain PA01-V (n = 17 WT, 15 MVP^−/−). Hazard ratio = 2.96, 95% CI 1.27 to 13.1; P = 0.018.