Activation of fro promoter transcription by flow and during infection of human tissue.

(A) RNA-seq heatmap of P. aeruginosa genes that are upregulated by at least 3-fold by flow and during infection in human wounds. Datasets are from (Cornforth et al., 2018; Sanfilippo et al., 2019). Log2(FC) indicates the log2 of the fold-change. (B) Schematic of the P. aeruginosa fluorescent reporter strain AL143 that contains a transcriptional fusion of yfp to the fro promoter and expresses mCherry under a constitutive promoter. (C) Representative phase-contrast and fluorescence images and (D) corresponding fro expression as determined by the ratio of YFP to mCherry fluorescence, of P. aeruginosa strain AL143 co-incubated with neutrophils, macrophages (MΦs), or host cell-free medium for 30 minutes. Dashed lines in images indicate boundaries of host cells and white arrows indicate P. aeruginosa with intense YFP fluorescence relative to that of mCherry. Data points indicate individual P. aeruginosa cells from a single experiment and bars indicate the average. Images in (C) are representative of three independent experiments and scale bars represent 5 μm. P-values were obtained using Welch’s t-test of data from single experiments, with values of p>0.05 denoted as ns.

Conditioned medium from stimulated neutrophils induces fro expression.

(A) Schematic depicting that PMA-stimulated neutrophils produce respiratory burst products that could activate fro expression. (B) Representative phase-contrast and fluorescence images and (C) fro expression as determined by ratios of YFP to mCherry fluorescence, of P. aeruginosa strain AL143 cultured for 3 hours in medium only, medium with PMA, conditioned medium from PMA-stimulated neutrophils, or conditioned medium from unstimulated neutrophils. Unstimulated neutrophils have a value greater than 1. Scale bars represent 5 μm. Data points indicate an average from at least one hundred individual P. aeruginosa and gray columns indicate the mean of at least three independent experiments. (D) fro expression as determined by ratios of YFP to mCherry fluorescence of P. aeruginosa strain AL143 that was cultured for 3 hours in conditioned medium in which neutrophils were stimulated with 100 ng/mL PMA, pre-treated with 2mM 4-ABAH, or both. Data points indicate individual P. aeruginosa cells, with each color representing an independent experiment, bars represent the average of 3 independent experiments, each using separate neutrophil isolations, and error bars indicate standard error of the mean (SEM). P-values were obtained using Welch’s t-test, with values of p>0.05 denoted as ns.

NaOCl but neither H2O2 nor HNO3 induces fro expression.

(A) Growth profiles as measured by optical density (OD600) of P. aeruginosa treated with the indicated concentrations of NaOCl, H2O2, HNO3, or no treatment. Data points indicate a mean of at least two independent experiments and error bars indicate SEM. (B) fro expression as determined by ratios of YFP to mCherry fluorescence in P. aeruginosa after 3 hour treatment with the indicated concentrations of NaOCl or no treatment (UTR). The value for UTR is less than 1. (C) Abundance of froA transcripts in P. aeruginosa after 30 minutes of treatment with 1 µM NaOCl, 4 µM H2O2, or no treatment (UTR), as measured by RT-qPCR. Measurements were normalized to 5S ribosomal RNA and the logarithm (base 2) of the fold-change in transcription was computed relative to the untreated condition. Horizontal bars indicate the average of four independent experiments and error bars indicate SEM. (D and E) fro expression as determined by ratios of YFP to mCherry fluorescence in P. aeruginosa after 3 hour treatment with (D) 1 µM NaOCl, 1 µM NaCl, 1 µM NaOH, 1 µM or 6 mM NaOH, or no treatment, and (E) indicated concentrations of H2O2, HNO3, or no treatment (data replotted from panel B). In panels B-E, data points indicate the average from at least one hundred individual P. aeruginosa cells, horizontal bars indicate the mean of independent experiments and error bars indicate SEM. Dashed lines indicate the average of the untreated condition, which was normalized to one. (F) fro expression as determined by ratios of YFP to mCherry fluorescence of P. aeruginosa after 3 hour treatment with 0.5 µg/mL tobramycin, vehicle control (VC; sterile water), 40 µg/mL, 1 µM NaOCl, or LL-37 and 10 µg/mL histones (LL37+His). Data points indicate individual P. aeruginosa cells, with different colors representing independent experiments. Horizontal bars indicate the mean of independent experiments and error bars indicate SEM. P-values were obtained using Welch’s t-test, with values of p>0.05 denoted as ns. All data were acquired using the P. aeruginosa strain AL143.

The expression of fro is activated during corneal infection

Abundance of froA transcripts in P. aeruginosa strain PAO1F as measured by RT-qPCR, at 0, 2, or 20 hours post-inoculation onto abraded mouse corneas. Measurements were normalized to 5S ribosomal RNA and the logarithm (base 2) of the fold-change in transcription was computed relative to the initial inoculum (time=0 hrs). Log2(FC) indicates the log2 of the fold-change. Data points represent independent corneal infections. Horizontal bars indicate the average of at least six independent experiments and error bars represent SEM. P-values were obtained using Welch’s t-test, with values of p>0.05 denoted as ns.

Expression of fro is inhibited by methionine and antioxidants.

(A) Heatmap showing P. aeruginosa gene transcripts (sorted by genomic locus) that were upregulated by at least 4-fold in the wild type PA14 background (strain AL143) compared to the ΔfroR mutant (AL325) after 30 minutes of treatment with 1 µM NaOCl and that had p-values less than 0.05 (n≥3). Log2(FC) indicates the log2 of the fold-change. Raw data can be found online (Foik, Ilona P. et al., 2025) in NCBI’s Gene Expression Omnibus (see Key Resources Table). (B and C) fro expression as determined by ratios of YFP to mCherry fluorescence in strain AL143 after 3 hour (B) treatment with; vehicle control (UTR), 1 µM NaOCl, or 1 µM NaOCl with combined with methionine (Met), cysteine (Cys), or β-mercaptoethanol (βME) at the indicated concentrations, and (C) incubation in medium only (replotted from Figure 2C), or conditioned medium from stimulated neutrophils with and without 100 µM methionine. Data points indicate an average from at least one hundred individual P. aeruginosa. Gray columns indicate the mean of at least three independent experiments and error bars represent SEM. P-values were obtained using Welch’s t-test, with values of p>0.05 denoted as ns.

FroR improves P. aeruginosa growth against HOCl stress and regulates transcription of genes involved in antioxidant defense.

(A) Growth profiles of wild-type (WT) P. aeruginosa strain AL143 and the ΔfroR mutant strain AL325 treated with 4 µM NaOCl or vehicle control (VC). Data points represent an average of at least three independent experiments and error bars indicate SEM. (B-C) Statistical significance as a function of fold-change in transcript abundance in (B) WT and (C) ΔfroR after treatment with 1 µM NaOCl (n≥3), as determined by RNA sequencing. Genes in red (upregulated) and blue (downregulated) were altered by at least four-fold. The unlabeled gene loci shown have not been previously annotated. P-values were determined using the Wald test (Love et al., 2014). Greater values along the vertical axis indicate greater statistical significance. P-values were determined using the Wald test (Love et al., 2014). Greater values along the vertical axis indicate greater statistical significance and the dashed horizontal line indicates a p-value of 0.05. Raw data can be found online (Foik, Ilona P. et al., 2025) in NCBI’s Gene Expression Omnibus (see Key Resources Table). (D) Schematic that depicts a model in which RCS oxidizes methionine residues (Met) in a target sensor(s), such as the ECF anti-σ factor FroI, which in turn activates the FroR σ factor and increases the production of methionine sulfoxide reductases (MSR).