Patterns of epithelial cell invasion by different species of the Burkholderia cepacia complex in well-differentiated human airway epithelia

Abstract

Burkholderia cepacia has emerged as a serious respiratory pathogen in cystic fibrosis (CF) patients. The clinical course of B. cepacia infections is variable, but approximately 20% of patients eventually succumb to the cepacia syndrome, which is characterized as a fatal necrotizing pneumonia with bacteremia. The mechanisms that permit B. cepacia to cause bacteremia are not yet known but probably involve sequential penetration of airway barriers. This study evaluated the abilities of different species of the B. cepacia complex, including a strain from the ET12 lineage (BC-7, genomovar III, cblA(+)), which is associated with most cepacia syndrome fatalities among CF populations, a genomovar IV strain (HI2258), and a genomovar II strain (J-1) to penetrate polarized, well-differentiated human airway epithelial cell cultures. As revealed by light and electron microscopy, all three B. cepacia strains tested circumvented the mechanical barriers of mucus and ciliary transport to penetrate the airway epithelium but they used different routes. The BC-7 strain (genomovar III) formed biofilms in close proximity to the apical cell surface, followed by invasion and destruction of epithelial cells. This process involved disruption of the glycocalyx and rearrangements of the actin cytoskeleton. The HI2258 strain (genomovar IV) did not form biofilms, and the majority of bacteria that penetrated the epithelium were located between epithelial cells, suggesting paracytosis. Strain J-1 penetrated the epithelium both by cell destruction and paracytosis. These studies suggest that the distinct invasion pathways employed by B. cepacia may account for differences in virulence between B. cepacia genomovars.

FIG. 1.

Noninfected and B. cepacia BC-7-infected airway epithelial cell cultures. (A) Well-differentiated human airway epithelial cell culture grown on a T-Col membrane support for 3 weeks under air-liquid interface conditions and fixed by the perfluorocarbon-osmium tetroxide technique (20). Semithin sections were stained with Richardson's stain. Note the preservation of the mucus layer. sl, superficial cell layer; bl, basal cell layer. (B to D) Small biofilms closely associated with the apical cell surface formed by B. cepacia BC-7 (∼5 × 10⁷ CFU/airway epithelial cell culture) in airway epithelial cell cultures after 24 h of incubation. Cultures were examined by LM (B) (semithin section stained with Richardson's stain), SEM (C), and TEM (ultrathin section from ruthenium red-stained culture) (D), which show bacteria being buried in the residue of their glycocalyx. Arrows (B and C), small biofilm; arrowheads (B), single (planktonic) bacteria.

FIG. 2.

Different stages of biofilm development of B. cepacia BC-7 (∼5 × 10⁷ CFU/airway epithelial cell culture) in well-differentiated airway epithelial cell cultures after 24 h of incubation. Large biofilms at late (A) and early (B) stages of penetration were examined by LM (semithin sections stained with Richardson's stain; see also Table 1). (C and D) Large biofilm at an early stage of penetration examined by TEM revealing intracellular (C) and intercellular (D) bacteria (arrowhead) in epithelial cells associated with this biofilm. Note destruction of adjacent epithelial cells.

FIG. 3.

Light micrograph of semithin section of a well-differentiated airway epithelial cell culture infected with B. stabilis HI2258 (∼5 × 10⁷ CFU/culture) for 24 h. Note bacteria within and between epithelial cells (see also Table 1).

FIG. 4.

TEM of ruthenium red-stained glycocalyx on the apical cell surface of noninfected and BC-7 (∼5 × 10⁷ CFU/airway epithelial cell culture for 24 h)-infected airway epithelial cultures (see also Table 1). (A) Glycocalyx (70 to 100 nm thick) on the apical membrane of a noninfected airway epithelium including cilia. (B) Bacteria (arrowheads) closely associated with glycocalyx of microvilli. (C) Single bacterium binding to the extended microvillus with the glycocalyx apparently destroyed. (D) Mature biofilm of BC-7. Note the absence of a glycocalyx on the apical cell surface of the biofilm-associated airway epithelium.

FIG. 5.

Visualization of B. multivorans J-1 and B. cepacia BC-7 biofilm and structure of filamentous actin in human airway epithelia. Confocal x-y scans of the apical epithelial domain from noninfected airway epithelia (A) and B. multivorans J-1- (B to D) and B. cepacia BC-7 (E to G)-infected (∼5 × 10⁷ CFU/airway epithelial cell culture for 24 h) airway epithelia. (B and E) B. cepacia biofilm (green); (C and F), actin filaments (red; note extensive cytoskeletal rearrangements); (D and G) colocalization of green biofilm and red actin filaments seen as yellow-stained area. For immunostaining noninfected and infected airway epithelial cell cultures were incubated for 1 h at 37°C with B. cepacia antiserum (1:250 dilution), followed by incubation with a goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody. For labeling the actin cytoskeleton, the immunostained airway epithelial cell cultures were incubated with BODIPY-phalloidin at a 1:25 dilution for 30 min at 25°C. Airway cultures were then visualized by confocal microscopy. Bars, 10 (A to D) and 20 μm (E to G).