Effect of supernatants from environmental Pseudomonas isolates on the growth of 12 human opportunistic pathogens.

(A) Screen to assess the extent to which pyoverdine-containing supernatants from 320 natural Pseudomonas isolates inhibit the growth of 12 human opportunistic pathogens. The heatmap depicts relative pathogen growth in the supernatant treatment (70% CAA + 30% spent supernatant) relative to the control (70% CAA + 30% sodium chloride solution), with values ranging from stimulation (yellow) to inhibition (blue) based on four independent replicates. Grey bars above the heatmap show the number of pathogens that were at least 20% inhibited in their growth by a given supernatant. The screen returned 25 supernatant candidates that inhibited the growth of all pathogens. (B) Control screen with the 25 top supernatant candidates to check whether pyoverdine causes the observed growth inhibition. The heatmap depicts the level of growth recovery in the 12 pathogens when iron was added to the supernatant, with values ranging from no recovery (red) to full recovery (blue). Growth recovery in iron-rich medium indicates that pyoverdines are involved in growth inhibition in iron-limited medium. Grey bars above the heatmap show the number of pathogens that experienced a relative growth recovery of at least 0.2. The screen returned 7 supernatant top candidates for which growth recovery occurred for all pathogens under iron-rich conditions.

Species and strains of human opportunistic pathogens used for the pyoverdine growth inhibition assay

Chemical structure of growth-inhibitory pyoverdines and their properties compared to non-inhibitory pyoverdines.

(A) The chemical structures of the seven top candidate pyoverdines were elucidated using UHPLC-HR-MS/MS and revealed 5 unique pyoverdine structures (1-5) differing in their peptide backbone (see27 for an in-depth chemical analysis). Pyoverdine 1 is a novel structure (from isolate 3A06). Pyoverdine 2 can occur in either a linear 2a or a cyclic 2b form (from isolate 3G07). Pyoverdine 3 was found in three different isolates, originating from the same soil sample (from isolates s3b09, s3b10 and s3b12). Pyoverdine 4 and 5 are from isolate s3c13 and s3e20, respectively. (B) The CCS values of inhibitory pyoverdines are higher than for non-inhibitory pyoverdines. (C) Molecule polarity, measured as the chromatographic retention time, is higher for ferri-pyoverdines (iron loaded) than for apo-pyoverdines (iron free). (D) Iron complex stability, assessed by the collision induced unfolding of ferri-pyoverdines, was significantly higher for inhibitory than non-inhibitory pyoverdines. (E) The normalised collision energy (CE50) necessary to fragment 50% of ferri-pyoverdines was not different between inhibitory and non-inhibitory pyoverdines. Box plots show the median and the first and third quartiles across the 7 inhibitory and 6 non-inhibitory pyoverdines. Whiskers represent the 1.5x interquartile range.

Pyoverdine dose-response curves for A. baumannii, K. pneumoniae, P. aeruginosa and S. aureus.

We exposed the four human opportunistic pathogens to three pyoverdines (3A06, 3G07, s3b09) that were among the most potent ones. We used crude-purified extracts of all three pyoverdines and a HPLC-purified variant for pyoverdine s3b09. The absolute concentrations of the crude-purified extracts are unknown and therefore expressed relative to the weighed amount of 6 mg. The absolute concentration of the HPLC-purified variant is given in µg/mL. Growth values are scaled relative to the untreated control in CAA medium. Dots and error bars show mean values and standard errors, respectively, across a minimum of three replicates per concentration. Dose-response curves were fitted using 4- or 5-parameter logistic regressions.

Toxicity assays for pyoverdines from environmental Pseudomonas spp. against human cell lines, sheep erythrocytes and the host larvae of G. mellonella.

(A) We exposed mouse neuroblastoma-spinal cord (NSC-34) and human embryonic kidney 293 (HEK-293) cells to three crude-purified pyoverdines (3A06, 3G07, s3b09) that were among the most potent ones to inhibit bacterial growth. An MTT assay was used to assess the metabolic activity of cells as an indicator of cell viability and proliferation. Cell viability data are scaled relative to the pyoverdine-free treatment, whereby dots and error bars show means and standard error across three replicates, respectively. The absolute concentrations of the crude-purified pyoverdines are unknown and concentrations are therefore expressed relative to the highest one used. Coloured dots indicate pyoverdine dosages used for the in vivo experiments (pyoverdines/10 µL). Dose-response curves were fitted using 5-parameter logistic regressions. (B) We evaluated the haemolytic activity of the pyoverdines by adding them to sheep erythrocytes along a concentration gradient (range 0.002 – 1). Triton X-100 and PBS served as positive and negative control, respectively. Haemolytic activity is scaled relative to the positive control, dots and error bars show means and standard error across 6 replicates from two independent experiments, respectively. (C) To assess the toxic effects of pyoverdine on the host, we injected pyoverdines (three relative concentrations, 0.01, 0.05, 0.1) into larvae 4 hours after a mock infection with PBS. The percentage of larval survival was tracked over 48 hours post-treatment. Data stem from three independent experiments with each 10 larvae per infection and treatment.

Pyoverdine treatments significantly increase survival of the host G. mellonella when infected with A. baumannii and K. pneumoniae.

Larvae of the greater wax moth were first infected with either A. baumannii, K. pneumoniae, or P. aeruginosa and then treated with one of three pyoverdines (3A06, 3G07, or s3b09) at four relative pyoverdine concentrations (0, 0.01, 0.05, 0.1). All panels show the larvae survival over 48 hours post-treatment. Data stem from three independent experiments with each 10 larvae per infection and treatment. Asterisks indicate significant differences in larval survival between treated and untreated infections based on Cox proportional hazard regressions (p < 0.05).

Phenotypic and genotypic analysis of experimentally evolved pathogens reveal weak levels of resistance evolution against pyoverdine treatment.

(A) We exposed evolved and ancestral pathogen populations to the treatment in which they evolved in and quantified their growth (area under the curve). Growth values were scaled relative to the ancestor in untreated medium, and the panels show the scaled growth differences between evolved and ancestral populations. The shaded areas show the scaled growth difference between the ancestor and the populations evolved in untreated medium and is representative of medium adaptation. The blue and green dots represent the pathogen populations with the lowest (population 1) and highest (population 2) scaled growth difference, respectively, which were subsequently used to pick clones. The dots show mean values across two independent replicates and asterisks show significant growth increases relative to the medium-adapted control. Box plots show the median and the first and third quartiles across the 6 independently evolved populations. Whiskers represent the 1.5x interquartile range. (B) We repeated the above growth assays with 208 individual clones evolved under the pyoverdine treatments (picked from population 1 and population 2) and 24 populations evolved in growth medium alone (control). Each square represents a clone or a control population, and the number indicates the number of mutations identified based on whole-genome sequencing. The heatmap shows the fold-change in growth relative to the evolved control populations. Asterisks depict significant fold-increases in growth compared to control populations. (C) The relationship between the number of mutations and fold-change in growth across all sequenced clones (n = 52) The red line and shaded area are the regression line and 95% confidence interval, respectively. (D) Heatmap showing the number of mutations per pathogen and per functional gene categories across clones together with the respective fold-change in growth (n = 32). For this analysis, we excluded intergenic mutations and deletions larger than 600 bp. Triangle, square and cross depict growth fold-change of A. baumannii, P. aeruginosa and S. aureus, respectively.