Filamentous bacteriophage delays healing of Pseudomonas-infected wounds

Abstract

Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.

Keywords: Pf; Pseudomonas; bacteriophage; filamentous phage; immunology; microbiology; wounds.

Graphical abstract

Figure 1

Pf phages lead to decreased wound healing in murine models (A) Schematic of the full thickness delayed inoculation chronic Pa wound-infection murine model. (B) Images of representative mice from PAOΔPf4 and PAO1groups from days 1 to 13 post-wounding. (C) Weight loss of mice across all days post-wounding (for PAO1 versus PAO1ΔPf4, ∗p < 0.05; ∗∗p < 0.01; two-way ANOVA). (D) Wound healing rates for all wounds across all days from days 1 to 13 post-wounding. n = 28 wounds in PAOΔPf4 and PAO1groups; n = 14 wounds in PBS group. Results are mean % size of original wound ±SE (∗∗p < 0.005 and ∗∗∗∗p < 0.0001 by one-way ANOVA). (E) Wound healing at day 13 post-wounding measured as percentage of original wound area. (F) Area-under-curve (AUC) analysis for wound healing rates compiled for all wounds for all days 1–13 post-wounding (n = 28 wounds in PAOΔPf4 and PAO1 groups; n = 14 wounds in PBS group; comparison by one-way ANOVA). (G) Representative H&E stain of day 13 uninfected PBS wound. (H) Representative H&E stain of day 13 wound infected with PAO1ΔPf4. (I) Representative H&E stain of day 13 wound infected with PAO1. (J) Percent re-epithelialization calculated as ([day 1 epithelial gap−day 13 epithelial gap]/day 1 epithelial gap). Statistics are by one-way ANOVA.

Figure 2

Pf is associated with increased trans-epidermal water loss (TEWL) in pig burn wounds (A) Schematic for porcine burn-wound model with inoculation of PAO1 or PAOΔPf4. (B) Representative images of wounds infected with PAO1 or PAOΔPf4 at indicated times. (C) Wound area as measured by digital planimetry over time. Each independent wound represents one n, with 32 wounds per time point per each treatment group. (D) Wound area barrier integrity as measured by TEWL over time. Each independent wound represents one n for the experiment, with 6–28 wounds per time point per treatment group. Baseline measurements were done on healthy skin. ∗p < 0.05 and ∗∗p < 0.01 by unpaired Student’s t test.

Figure 3

Pf phage impedes keratinocyte migration (A) Schematic of HaCaT keratinocyte-migration assays. (B) Representative images of keratinocyte migration at 0 and 48 h following treatment with mock, Pf, LPS, or Pf + LPS. (C) Effect of Pf (1 × 10¹⁰ copy no./mL) and LPS (1 μg/mL) on HaCaT keratinocyte migration, presented as area at 48 h/area at 0 h with decreased area correlating with increased cell migration. Three biological replicates per experiment are shown. Results are representative of three independent experiments. Statistics are by one-way ANOVA. (D) Heatmaps from Luminex immunoassay performed on supernatant from HaCaT keratinocytes stimulated with Pf and LPS at 48 h. Results are presented as log 2-fold change of concentration compared with PBS control. (E and F) Interpolated values (pg/mL) of select cytokines and growth factors from Luminex assay of human primary monocytes stimulated with Pf and LPS for 24 h (E) or 48 h (F) are shown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by one-way ANOVA. ns, not significant. (G) Schematic of keratinocyte migration assays with pretreatment with CXCL1, Pf, and/or LPS. (H) Effect of CXCL1 supplementation (1 μg/mL) on HaCaT keratinocyte migration, presented as area at 48 h/area at 0 h with decreased area correlating with increased cell migration. Three biological replicates per experiment are shown. Results are representative of three independent experiments. ∗p < 0.05 and ∗∗p < 0.01 by unpaired Student’s t test.

Figure 4

Pf phage is associated with more chronic wound infections and increased wound size in humans (A) Survival analysis of Pf(+) and Pf(−) Pa wound infections, with wound healing as endpoint. Wounds that were not healed by the end of the study were censored. p = 0.013 by Gehan-Breslow-Wilcoxon test. (B) Pf(+) status is associated with increases in wound size. Pf(−): n = 11; Pf(+): n = 25; p = 0.033 by chi-square test. (C) Plot showing wound age and change in size. Wounds were plotted based on Pf(−) or Pf(+) phage status and wound age (calculated as ln(wound age in years)). Wound size change is represented as absolute ln fold change, with color indicating whether wound grew (red), healed (green), or shrank (blue).