In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells

Abstract

P. aeruginosa forms biofilms in the lungs of individuals with cystic fibrosis (CF); however, there have been no effective model systems for studying biofilm formation in the CF lung. We have developed a tissue culture system for growth of P. aeruginosa biofilms on CF-derived human airway cells that promotes the formation of highly antibiotic-resistant microcolonies, which produce an extracellular polysaccharide matrix and require the known abiotic biofilm formation genes flgK and pilB. Treatment of P. aeruginosa biofilms with tobramycin reduced the virulence of the biofilms both by reducing bacterial numbers and by altering virulence gene expression. We performed microarray analysis of these biofilms on epithelial cells after treatment with tobramycin, and we compared these results with gene expression of (i) tobramycin-treated planktonic P. aeruginosa and (ii) tobramycin-treated P. aeruginosa biofilms on an abiotic surface. Despite the conservation in functions required to form a biofilm, our results show that the responses to tobramycin treatment of biofilms grown on biotic versus abiotic surfaces are different, as exemplified by downregulation of genes involved in Pseudomonas quinolone signal biosynthesis specifically in epithelial cell-grown biofilms versus plastic-grown biofilms. We also identified the gene PA0913, which is upregulated by tobramycin specifically in biofilms grown on CF airway cells and codes for a probable magnesium transporter, MgtE. Mutation of the PA0913 gene increased the bacterial virulence of biofilms on the epithelial cells, consistent with a role for the gene in the suppression of bacterial virulence. Taken together, our data show that analysis of biofilms on airway cells provides new insights into the interaction of these microbial communities with the host.

FIG. 1.

Arginine and tobramycin prevent bacterial killing of CFBE epithelial cells. (A) An uninfected monolayer. (B and C) CFBE cell monolayers 6 h after inoculation with PA14 in the absence (B) or presence (C) of 0.4% arginine. (D) Intact CFBE cell monolayer after 8 h of biofilm development with arginine followed by 24 h of incubation with 1,000 μg/ml tobramycin. The tobramycin treatment did not clear the bacteria, which typically persisted at approximately 10³ to 10⁴ CFU/well. The images were taken by phase-contrast microscopy and are representative fields of view from several wells studied. Scale bars = 120 μm. (E) Treatment of 5-h coculture biofilms with 1 μg/ml tobramycin (Tb) for 2 h resulted in a small but significant reduction in bacterial virulence toward the CFBE cells compared to untreated samples. *, P < 0.05. (F) CFU determination of the biofilm cultures in panel E revealed equal bacterial titers in tobramycin-treated and untreated biofilms. The difference between the 0-μg/ml tobramycin and 1-μg/ml tobramycin samples was not statistically significant. The error bars indicate 1 standard deviation.

FIG. 2.

PA14 forms biofilm microcolonies on a CFBE epithelial cell monolayer. (A and B) Incubation of CFBE cells with PA14/pSMC21 (GFP) resulted in clusters of GFP-PA14 scattered across the monolayer (A) that appeared as microcolonies bound to the surfaces of the cells (B). Panels A and B are phase-contrast images overlaid with the GFP channel. (C) Staining with calcofluor resulted in blue fluorescence of the microcolonies, as seen in a phase-contrast image overlaid with the blue channel. Scale bars = 120 μm (A) or 35 μm (B and C). (D) CFBE cells were inoculated with wild-type PA14 or strains carrying mutations in the flgK or pilB gene. Bacterial CFU determined at 7 h postinfection revealed >10-fold reduction in bacterial populations on cells infected with the flgK or pilB mutant. *, P < 0.05 compared to PA14. The error bars indicate 1 standard deviation.

FIG. 3.

Abiotic biofilms respond differently to tobramycin than biofilms on CFBE cells. Quantitative RT-PCR revealed that PA14 biofilms on PVC plastic plates demonstrated an increase in transcriptional expression of dnaK (A) but no change in pqsA expression (B) in response to tobramycin (Tb) treatment. On the other hand, tobramycin treatment of CFBE/PA14 biofilms resulted in a decrease in both dnaK (C) and pqsA (D) expression, as determined by qRT-PCR. *, P < 0.05. The error bars indicate 1 standard deviation.

FIG. 4.

The PA0913 (mgtE) gene affects virulence on CFBE epithelial cells. (A) Microscopic analysis revealed that monolayers infected with an isogenic deletion mutant in the PA0913 gene (right) were destroyed more quickly than monolayers infected with wild-type PA14 (left) at 8 h postinoculation. Scale bar = 120 μm. (B) The cytotoxicity of the ΔPA0913 mutant on the CFBE cells was analyzed at 5 h, as described in Materials and Methods. The ΔPA0913 mutant was significantly more cytotoxic than PA14 (*, P < 0.05), and this effect could be rescued by complementation with the full-length gene in trans (pPA0913). pMQ72 is the empty vector control. The error bars indicate 1 standard deviation. (C) Determination of the number of CFU in the biofilms on the CFBE cells demonstrated that a PA0913 isogenic deletion mutant developed bacterial CFU at 5 h postinoculation similar to that for wild-type PA14. (D) Wild-type PA14 and the ΔPA0913 mutant displayed similar biofilm formation kinetics on PVC plastic. Black diamonds, PA14; gray triangles, ΔPA0913.

FIG. 5.

Roles of T3SS and secreted virulence factors in the PA0913 (mgtE) mutant phenotype. (A) Mutation of PA0913 in the type III secretion-defective ΔpscC mutant background eliminates the cytotoxicity observed for the single ΔPA0913 strain. This cytotoxicity of the ΔPA0913 ΔpscC double mutant is similar to that of the single ΔpscC mutant. *, P < 0.05 compared to wild-type PA14; #, P < 0.05 compared to PA14 and to ΔPA0913. The error bars indicate 1 standard deviation. (B and C) We measured pyoverdine (B) and pyocyanin (C) secreted by PA14 and the ΔPA0913 mutant in overnight cultures using colorimetric assays, as described in Materials and Methods. Slight but significant decreases were seen in pyocyanin and pyoverdine production in the ΔPA0913 (mgtE) mutant. *, P < 0.05.

FIG. 6.

Tobramycin stimulates expression of the PA0913 (mgtE) gene in biofilms on CFBE cells. (A) Semiquantitative RT-PCR demonstrated that tobramycin exposure led to an increase in PA0913 transcript levels in CFBE/PA14 biofilms (left) but not in planktonic cultures (right). fbp is a constitutively expressed control gene. Tb, tobramycin; − and + indicate that cDNA was prepared from untreated samples or tobramycin-treated samples, respectively. (B) qRT-PCR revealed a 38.3-fold enhancement of PA0913 gene transcription in PA14 biofilms on CFBE cells treated with 500 μg/ml tobramycin (+Tb). *, P < 0.05. The error bars indicate 1 standard deviation.