Pseudomonas aeruginosa alginate promotes Burkholderia cenocepacia persistence in cystic fibrosis transmembrane conductance regulator knockout mice

Abstract

Pseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis (CF) patients, facilitates infection by other opportunistic pathogens. Burkholderia cenocepacia, which normally infects adolescent patients, encounters alginate elaborated by mucoid P. aeruginosa. To determine whether P. aeruginosa alginate facilitates B. cenocepacia infection in mice, cystic fibrosis transmembrane conductance regulator knockout mice were infected with B. cenocepacia strain BC7 suspended in either phosphate-buffered saline (BC7/PBS) or P. aeruginosa alginate (BC7/alginate), and the pulmonary bacterial load and inflammation were monitored. Mice infected with BC7/PBS cleared all of the bacteria within 3 days, and inflammation was resolved by day 5. In contrast, mice infected with BC7/alginate showed persistence of bacteria and increased cytokine levels for up to 7 days. Histological examination of the lungs indicated that there was moderate to severe inflammation and pneumonic consolidation in isolated areas at 5 and 7 days postinfection in the BC7/alginate group. Further, alginate decreased phagocytosis of B. cenocepacia by professional phagocytes both in vivo and in vitro. P. aeruginosa alginate also reduced the proinflammatory responses of CF airway epithelial cells and alveolar macrophages to B. cenocepacia infection. The observed effects are specific to P. aeruginosa alginate, because enzymatically degraded alginate or other polyuronic acids did not facilitate bacterial persistence. These observations suggest that P. aeruginosa alginate may facilitate B. cenocepacia infection by interfering with host innate defense mechanisms.

FIG. 1.

P. aeruginosa alginate increases BC7 persistence in mice. Mice infected with BC7/PBS (A and B) or BC7/alginate (C to F) were sacrificed at predetermined time points, and 10-fold serial dilutions of lung (A to D) and spleen (E and F) homogenates were plated on BCSA to determine the bacterial load. Also, CF mice were infected with BC7 suspended in alginate, ED-alginate, seaweed alginate, polygalacturonic acid, or agarose, and the bacterial density in the lungs was measured at 5 days postinfection (G). The open circles indicate individual mice, and the bars indicate the geometric means calculated for three independent experiments with a total of seven mice per group (*, different from the BC7/PBS group at the same time point; †, significantly different from BC7/alginate-infected C57BL/6 mice at the same time point [P ≤ 0.05, as determined by ANOVA on Ranks with Dunn's post hoc analysis]).

FIG. 2.

Mice infected with BC7/alginate show severe inflammation. Paraffin lung sections were prepared for mice infected with BC7/PBS (A and B) or BC7/alginate (C to F) at day 1 (A and C), day 5 (B and D), and day 7 (E and F) postinfection and stained with H&E. The insets in panels A, C, and D are magnifications of the areas in boxes to show neutrophil accumulation. Arrows in panels E and F point to areas of inflammation. The images are representative of the results for three individual animals at each time point.

FIG. 3.

Levels of inflammatory markers are increased in mice infected with BC7/P. aeruginosa alginate. CF mice infected with BC7/PBS, BC7/alginate, or BC7/ED-alginate were sacrificed at 1, 3, 5, or 7 days postinfection. MPO activity (A) was determined using whole-lung homogenates and was expressed as the fold increase compared with corresponding sham-treated animals. IL-1β (B), TNF-α (C), MIP-2 (D), and KC (E) levels in lung homogenates were measured by ELISA, and the results were corrected for the cytokine levels observed in corresponding sham-treated controls. The data are the means and standard deviations calculated from three independent experiments with a total of seven mice per group (*, different from BC7/PBS and BC7/ED-alginate groups at the same time point [P ≤ 0.05, as determined by ANOVA with Tukey-Kramer post hoc analysis]).

FIG. 4.

P. aeruginosa alginate attenuates phagocytosis in vitro and in vivo. (A to C) Alveolar macrophages were incubated with FITC-labeled BC7 suspended in medium (A), alginate (B), or ED-alginate (C) for 90 min. Unbound bacteria were removed, the fluorescence of extracellular bacteria was quenched, and cells were examined using a confocal microscope. (D) Alveolar macrophages were pretreated with cytochalasin D for 30 min, incubated with FITC-labeled BC7 suspended in medium, and examined for the presence of intracellular bacteria. Green indicates intracellular bacteria, and the images are representative of three independent experiments. (E) In some experiments, cells were detached from the wells and subjected to flow cytometry, and the results were expressed as the mean fluorescence intensity. (F) CF mice were infected with FITC-labeled BC7 suspended in either PBS, alginate, or ED-alginate, BAL was performed, fluorescence of extracellular bacteria was quenched, and the percentages of macrophages and neutrophils with intracellular bacteria were determined by fluorescence microscopy. The data are the means and standard deviations of four independent experiments performed in duplicate or triplicate (*, different from the corresponding untreated control [P = <0.05]; †, different from the BC7/PBS and BC7/ED-alginate groups [P ≤ 0.05, as determined by ANOVA with Tukey-Kramer post hoc analysis]).

FIG. 5.

P. aeruginosa alginate decreases proinflammatory responses of alveolar macrophages and human airway epithelial cells in vitro. Murine alveolar macrophages (A and B), IB3 cells (human CF bronchial epithelial cells) (C), or C38 cells (IB3 cells corrected for CFTR) (D) were incubated with BC7 in the presence or absence of alginate for 30 min (for macrophages) or 6 h (for epithelial cells), and cytokine levels in the media were measured. The data are the means and standard deviations of four independent experiments performed in triplicate (*, different from the corresponding untreated control [P = <0.05]; †, different from the BC7/PBS group [P ≤ 0.05, as determined by ANOVA with Tukey-Kramer post hoc analysis]).

FIG. 6.

Prior administration of alginate enhances BC7 persistence in mice. CF mice were treated with BC7 mixed with alginate (BC7/alginate), with PBS and then with BC7 suspended in PBS (PBS/BC7), or with alginate and then with BC7 suspended in PBS (Alginate/BC7). The pulmonary bacterial load was determined at 5 days postinfection. The open circles indicate results for individual animals, and the bars indicate geometric means calculated from two independent experiments.

FIG. 7.

P. aeruginosa alginate-facilitated bacterial persistence depends on the bacterial species. CF mice were infected with B. cepacia, B. multivorans, B. cenocepacia BC45, nontypeable H. influenzae, or S. maltophilia suspended in P. aeruginosa alginate. The bacterial load in the lungs was measured at 3 days postinfection. The symbols indicate results for individual animals, and the bars indicate geometric means calculated from two independent experiments.