Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A) Graphical representation of respiratory epithelial co-culture biofilm assay utilized for all imaging studies. (B) CFBE41o - cells on glass coverslips were infected with GFP-producing P. aeruginosa (green) while stimulated with NS19504 (25 µM), a BK Ca channel potentiator, or 0.05% DMSO in MEM without phenol red and imaged by fluorescent microscopy at 1-, 3-, and 6-hours. CFBE41o - cell nuclei are stained with Hoescht33342 (blue). Scale bar represents 10 μm. (C) Biomass (μm 3 /μm 2 ) measurements at 1-, 3-, and 6-hours post-inoculation from three independent experiments. Line in bar represents mean value and error bars represent standard error of the mean. Statistical significance was tested by two-way ANOVA with multiple comparisons (* p<0.05). (D) P. aeruginosa biofilms grown in 96-well plates in LB and (E) SCFM with or without NS19504 measured using crystal violet absorbance at 550 nm. (F) Planktonic growth kinetics of P. aeruginosa grown in LB Lennox broth (LB), minimal essential media (MEM), and synthetic cystic fibrosis sputum media (SCFM) with or without NS19504.

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Co-Culture Assay, Biofilm Production Assay, Imaging, Infection, Microscopy, Staining

Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A, B, C) Bacterial attachment at 1 hour and average aggregate area and number at 6 hours grown on CFBE41o - cells in co-culture experiments were measured using Nikon Elements. (A) Number of bacteria attached per 20x field for epithelial cells treated with 0.05% DMSO or NS19504 (25 µM) during respiratory epithelial co-culture biofilm experiments at 1 hour. Line represents mean and error bars represent standard error of the mean. (B) Average aggregate area per 20x field measured at 6-hour time point for epithelial cells treated with 0.05% DMSO or NS19504 (25 µM) during live-cell co-culture experiments. Line represents mean and error bars represent standard error of the mean. (C) Average aggregate number per 20x field measured at 6-hour time point for epithelial cells treated with 0.05% DMSO or NS19504 (25 µM) during live-cell co-culture experiments. Line represents mean and error bars represent standard error of the mean. Line connecting data points indicates data points from same biologic replicate. Statistical significance was tested by unpaired t-test (* p<0.05) for all panels.

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Co-Culture Assay, Bacteria

Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A and B) CFBE41o - cells on glass coverslips are infected with a 1:1:1 mixture of TFP-, YFP-, and tdTomato-producing P. aeruginosa while treated with NS19504 (25 µM) or 0.05% DMSO in respiratory epithelial co-culture biofilm assay and imaged by fluorescent microscopy at 1-, 3-, and 6-hours post-infection. (A) Representative images of bacterial coalescence at 1-, 3-, and 6-hours growth. Scale bar represents 10 μm. White box indicates area of magnification in panel B. (B) Magnification of one quarter of panel A images demonstrating multicolor aggregates (polyclonal) compared to single color aggregates (monoclonal). (C) Proportion of colocalized bacteria at 6 hours measured with Nikon Elements Software. Bar represents mean with dots representing each of the four biologic replicates and error bars represent standard error of the mean. Statistical significance was determined by unpaired t-test (* p<0.05).

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Infection, Co-Culture Assay, Biofilm Production Assay, Microscopy, Bacteria, Software

Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A) CFBE41o - cells on glass coverslips were infected with wild type (WT) PAO1 or ΔkdpFABCDE GFP-producing P. aeruginosa , grown with continuous flow of MEM with either DMSO or NS19504 and imaged by fluorescent microscopy at 1-, 3-, and 6-hours. CFBE41o - cell nuclei are stained with Hoescht33342 (blue). Scale bar represents 10 μm. (B) Biomass (μm 3 /μm 2 ) (measured with Nikon Elements) at 1-, 3-, and 6-hours post-inoculation from four independent experiments. Statistical significance was tested by 2-way ANOVA with multiple comparisons (** p<0.01, *** p<0.001). (C-E) Bacterial attachment at 1 hour and average aggregate area and number at 6 hours grown on CFBE41o - cells in live-cell co-culture experiments were measured using Nikon Elements. (C) Number of bacteria attached per 20x field with WT or ΔkdpFABCDE GFP-producing P. aeruginosa during live-cell co-culture experiments at 1 hour. Line represents mean and error bars represent standard error of the mean. (D) Average aggregate area per 20x field measured at 6-hour time point when infected WT or ΔkdpFABCDE GFP-producing P. aeruginosa . Line represents mean and error bars represent standard error of the mean. (E) Average aggregate number per 20x field measured at 6-hour time point with WT or ΔkdpFABCDE GFP-producing P. aeruginosa . Line represents mean of four biologic replicates and error bars represent standard error of the mean. Statistical significance was tested by unpaired t-test (* p<0.05).

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Infection, Microscopy, Staining, Co-Culture Assay, Bacteria