Fig. 4: Fluorofolin mutants overexpress efflux pumps and have decreased virulence.
a, RNA sequencing results from an nfxB (T39P) mutant relative to wildtype PA14. P values calculated using two-tailed quasi-likelihood F-test. b, nfxB (T39P) mutants show cross-resistance to both ciprofloxacin (2× MIC) and fluorofolin (2× MIC). Curves represent average OD600 of 2 biological replicates. c, Resistance frequency of 4× MIC of a panel of antibiotics tested against PA14. Points represent independent plates. *P < 0.05; calculated using two-sided unpaired t-test using Prism 9. d, RNA sequencing results from a mexS (L46F) mutant relative to wildtype PA14. P values calculated using two-tailed quasi-likelihood F-test. e, Pyocyanin production of nfxB (T39P) and mexS (L46F) mutants. Pyocyanin levels were measured by integration of absorbances from 306 to 326 nm. A ΔpqsA PA14 mutant was included as this strain does not make pyocyanin and ΔpqsA absorbance values were used to subtract out background signal. PQS in DMSO (1 μM) was added to samples at inoculation to rescue pyocyanin production. Data are presented as mean ± s.d. of biological triplicates for each group. ****P < 0.0001, **P < 0.01; calculated using two-sided unpaired t-test using Prism 9. f, C. elegans N2 toxicity after infection with wildtype PA14 (n = 158), nfxB T39P (n = 131) or mexS L46F (n = 128) was measured over 60 h. Worms were declared dead if they lacked movement after being gently poked with forceps. P values were calculated using a Mantel–Cox test compared to wildtype PA14 using Prism 9. g, Clinical isolate resistance to fluorofolin and ciprofloxacin was tested by treating the panel with 50 μg ml−1 of either antibiotic. Growth inhibition was determined by comparing OD600 after 16 h to DMSO-treated controls. Strains with growth inhibition >80% were considered sensitive to fluorofolin treatment.
