Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection

Abstract

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.

Fig. 1.. Pf phage promote Pa wound infection.

(A and B) Representative images of human Pa-infected wounds (A) negative and (B) positive for Pf. Scale bars: 5 mm. (C) Prevalence of Pf prophage in infected human wounds. (D) Prevalence of Pf phage in human wounds younger (n = 9) and older (n = 28) than 6 months of age; two-tailed Fisher’s exact test. (E) The full-thickness wound infection model. (F) Representative images of murine wounds before (left) and after infection (middle) showing luminescent bacterial signal (right). Scale bars: 5 mm. (G) Luminescent signal reflecting wound bacterial burden after inoculation with 7.5 ± 2.5 × 10² CFU/ml PAO1 (n = 14 wounds), the same dose used in (H) to (J). (H) Nonlinear regression analysis of wound infection rate 3 days after inoculation used to calculate the IC₅₀ for PAO1 and PAO1ΔPf4. (I) Wound infection rate for PAO1 and PAO1ΔPf4 over time (n = 3 experiments, n >10 wounds each); two-way ANOVA. (J) Wound infection rate for PAO1ΔPf4, PAO1ΔPf4 supplemented with Pf4, or PAO1 at 3 days after inoculation. Summary of n = 2 experiments, n = 22 to 24 wounds/group; two-tailed Fisher’s exact test. (K) Survival (n = 18 mice/group, n = 2 experiments; log-rank Mantel-Cox test) and (L) weight loss (n = 12 mice/ group, representative of n = 2 experiments; two-tailed Student’s t test) after inoculation with 10⁷ CFU/ml of PAO1ΔPf4 or PAO1.

Fig. 2.. Pf phage inhibit phagocytosis and TNF production.

(A) Phagocytosis of live PAO1ΔPf4, PAO1, and PAO1 supplemented with exogenous Pf4 (PAO1+Pf4) by mouse BMDCs, as measured by a gentamicin protection assay. (B) Phagocytosis of live PAO1ΔPf4 and PAO1 by human U937 macrophages. (C) Phagocytosis by BMDCs of fixed E. coli particles labeled with a pH-sensitive dye (pHrodo) in the absence or presence of purified Pf4, as measured by flow cytometry. (D) Median fluorescence intensity (MFI) of E. coli pHrodo particle-positive cells from (C). (E and F) TNF production by murine BMDCs stimulated with Pf4 and (E) LPS or (F) alginate for 24 hours. (G) TNF production by human primary monocytes stimulated with Pf4 and LPS for 24 hours. (H) Phagocytosis of E. coli-pHrodo particles by BMDCs stimulated with exogenous TNF and Pf4. All graphs are representative of n ≥ 3 experiments and depict mean with SEM of n ≥ 3 replicates. Analysis: two-tailed Student’s t test.

Fig. 3.. Pf phage inhibits TNF in a type I IFN-dependent manner.

(A) TNF production by murine BMDCs stimulated with alginate and Pf4 for 48 hours. (B) TNF production over time by murine BMDCs stimulated with alginate and Pf4. (C) TNF production in BMDCs stimulated with alginate and either Pf4 or Pf1 phage for 72 hours. (D) TNF production in BMDCs stimulated with LPS and Fd1 phage for 24 hours. (E) TNF mRNA up-regulation in BMDCs stimulated with LPS and Pf4 for various time points. (F) Intracellular cytokine staining of TNF in BMDCs stimulated with Pf4 and then LPS. (G) Type I IFN production by BMDCs stimulated with alginate and Pf4 for 24 hours. (H) TNF production by WT or Ifnar^−/− BMDCs stimulated with LPS and Pf4 phage for 24 hours. (A) to (H) are each representative of n ≥ 3 experiments and depict mean with SEM of n ≥ 3 replicates. Statistics: (E, G to H) two-tailed Student’s t test; (A, C to D) one-way ANOVA with Dunnett’s multiple comparison; (B) two-way ANOVA.

Fig. 4.. Pf phage-mediated immune inhibition is TRIF- and TLR3-dependent and associated with production of phage RNA.

(A) TNF production by WT and Trif^−/− BMDCs stimulated with alginate and Pf4 for 48 hours. (B) E. coli -pHrodo-positive cells in WT and Trif^−/− Pf-stimulated BMDCs. (C) TNF production by WT and Myd88^−/− BMDCs stimulated with alginate and Pf4 for 48 hours. (D) TNF production by WT and T/r3^−/− BMDCs stimulated with alginate and Pf4 for 24 hours. (E) E. coli-pHrodo-positive cells in WT or Tlr3^−/− BMDMs stimulated with Pf4. (F) Type I IFN production by BMDCs stimulated with LPS and Pf4 for 24 hours. (A) to (F) are each representative of n ≥ 3 experiments and depict mean with SEM of n ≥ 3 replicates. Statistics: two-tailed Student’s t test. (G) Wound infection rate 3 days after infection in Tlr3^−/− and WT mice inoculated with 7.5 ± 2.5 × 10² CFU/ml PAO1 or PAOlΔPf4. Summary of n = 2 experiments, n = 30 to 34 wounds/group. Statistics: two-tailed Fisher’s exact test. (H and I) Tlr3-reporter signal in response to (H) whole Pfl and Pf4 phage; (I) RNA from Pf4 genes. (J) RNA detected in human monocytes stimulated with whole Pfl or Pf4 phage for 24 hours. Summary of n = 2 (I and J) or n ≥ 3 (H) experiments with n > 3 replicates; depicted are means and SEM.

Fig. 5.. Pf phage is actively taken up by immune cells through endocytic pathways.

(A) Flow cytometric analysis of fluorescently labeled Pf4 uptake by BMDCs. (B) Flow cytometric analysis of BMDCs stimulated with fluorescently labeled Pf4 at 4°C (adsorption control) or with extracellular fluorescence quenched with trypan blue. (C) Immunofluorescence staining of TLR3 in BMDMs stimulated with fluorescently labeled Pf4. Scale bar: 10 μm. (D and E)Transmission electron microscopydepictinggold-abeled Pf4 present in intracellular lysosomes and the cytosol in BMDCs after 3 hours (D) and 24 hours (E) of Pf4 stimulation. Scale bars: 200 nm. (F) Pf4 uptake by BMDCs treated with various endocytosis inhibitors before Pf4 stimulation. Statistics: two-tailed Student’s t test. DMSO, dimethyl sulfoxide. (G) Composition by cell type of mouse leukocytes isolated from spleen or lymph node positive for fluorescently labeled Pf4. (H) Percentage of individual immune cell populations within mouse spleen and lymph nodes that took up Pf4. (I) Composition by cell type of human PBMCs that have taken up fluorescently labeled Pf4. (J) Percentage of individual immune cellpopulationswithin humanPBMCs that took up Pf. (A), (B), and (F) to (J) are each representative of n ≥ 3 experiments. Graphs depict mean with SEM of n ≥ 3 replicates.

Fig. 6.. A model of Pf4-mediated inhibition of TNF and phagocytosis.

Bacterial ligands stimulate TNF production and phagocytosis in BMDCs. Pf4 gets taken up through endocytic pathways, where Pf RNA stimulates TLR3-mediated TRIF signaling, which induces IFN production. IFN inhibits TNF production and phagocytosis.

Fig. 7.. Antibodies against Pf phage protect against Pa colonization.

(A) Consensus sequence analysis of CoaB across 669 Pa isolates. (B) Infection rate in full-thickness wounds 3 days after inoculation with 7.5 ± 2.5 × 10² CFU/ml PAO1 in mice vaccinated against CoaB coat protein, compared to mock-vaccinated mice (n = 16 to 20 wounds/group); two-tailed Fisher's exact test. (C) Infection rate in full-thickness wounds 3 days after inoculation with 7.5 ± 2.5×10² CFU/ml CFU/ml PAO1 in mice topically treated with isotype control or mAbs directed against CoaB protein. Summary of two experiments with n = 30 to 34 wounds per experimental group; two-tailed Fisher’s exact test. (D) Phagocytosis of PAO1ΔPf4 and PAO1 by BMDCs treated with isotype control or mAbs directed against CoaB protein; two-tailed Studen’s t test. (E) Phagocytosis of PAO1 by BMDCs treated with mAbs directed against CoaB with or without Fc block; one-way ANOVA with Tukey multiple comparison. (D) and (E) are representative of n ≥ 3 experiments; depicted is mean with SEM of n ≥ 3 replicates.