(398) The role of carbonic anhydrase in pathogen susceptibility in cystic fibrosis

Background: : Carbonic anhydrase (CA) expression is variably decreased in the setting of CFTR dysfunction or inhibition, and genetic alterations in carbonic anhydrase 12 can lead to a clinical cystic fibrosis phenotype, including airway infections and bronchiectasis. Based on this, we hypothesized decreased CA resulting from CFTR dysfunction perturbs membrane protein composition via cytoskeletal alterations that in turn increase pathogen adherence. By identifying intermediaries relevant to infection susceptibility, we hope to identify additional targets for the treatment and prevention of infections in CF.

Methods: CRISPR/Cas9 technology was used to knock-out CA12 in 9HTEo^- tracheal epithelial cells. To perform bacterial adherence assays, control and CA12 KO cells were infected with bacteria at a multiplicity of infection of 1:10 through 1:160, gently centrifuged, and incubated (5% CO2, 37°C) for 30 minutes. Colony-Forming Units were enumerated on LB agar plates using dilutions of 1) supernatants and wash and 2) lysed, homogenized epithelial cells carrying adherent bacteria. RNA expression was analyzed in human nasal epithelial cells via RNAseq. Protein expression in 9HTEo- cells was compared by relative density on western blot. Cell-free supernatant was collected from control and CA12 KO cells to assess for antimicrobial activity of secreted factors. Gentamicin exclusion assays were used to assess for bacterial internalization.

Results: Using a multi-MOI method, clinical CF PA isolate demonstrated increased adhesion to CA12 KO tracheal epithelial cells (9HTEo-) compared to isogenic control cells at MOI 1:10 through 1:160 (n=3, p< 0.05). PDLIM1 expression was significantly increased in membranous fractions of 9HTEo- CA12 KO (n=4, p< 0.05), raising the question of whether PDLIM1 at the membrane serves as a pathogen binding target, which may also be relevant to differential bacterial internalization based on preliminary data. Staining with PDLIM1 demonstrated increased immunofluorescence within CA12KO cells compared to control. To assess for differences in secreted factors, fractions of cell-free supernatant from control vs. CA12 KO cells were incubated with PA but resulted in nearly identical growth curves over 24 hours, suggesting there was no difference in antimicrobial peptide production. PDLIM2 and PDLIM7 RNA expression were increased in primary CF human nasal epithelial cells compared to control, highlighting the potential relevance of this family of proteins, which appears to be modified by CA, in CF.

Conclusions: CA12 KO results in altered membrane protein localization and appears to increase Pseudomonas aeruginosa adherence to tracheal epithelial cells.

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