Listeria exploits IFITM3 to suppress antibacterial activity in phagocytes

Abstract

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3^-/- mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target.

Fig. 1. Type I IFN suppresses antibacterial activity in phagosomes.

Time course of bacteria killing over 24 h of (a) Δhly Lm or (b) E. coli infected WT and IFNAR1 KO BMDMs with or without IFNβ pretreatment. **P = 0.0012, ***P = 0.001. Representative images (c, d) and quantification (e, f) of LAMP1-positive bacteria in BMDMs treated with or without IFNβ, infected with Δhly Lm (c, e) or E. coli (d, f) over 24 h. **P < 0.0087. Arrow denotes LAMP1-positive bacteria, arrowhead denotes LAMP1-negative bacteria. Bulk proteolysis over time (g) and slope of the curve’s linear portion (h) of DQ-BSA-labeled particles in untreated (UT) phagosomes, or treated with IFNβ or IFNγ. ***P < 0.0009. RFU relative fluorescence unit. i Measurement of ROS production in phagosomes of zymosan-fed BMDMs with and without IFNβ or IFNγ. **P = 0.0042, ****P < 0.0001. Data shown are means ± standard deviation (s.d.) for at least n = 3 independent experiments. P value was calculated using (a, b) two-way analysis of variance (ANOVA) and (e, f, h, i) one-way ANOVA. Scale bars, 3 µm.

Fig. 2. Type I IFN inhibits the delivery of hydrolase activity-related proteins to phagosomes.

Proteomics data obtained by mass spectrometry of phagosomes and lysosomes isolated from (a) WT BMDMs and (b) IFITM3 KO BMDMs. Legend denotes relative protein abundance between IFNβ treatment and UT controls. Black box indicates undetected proteins. Data shown are means for at least n = 3 independent experiments. P value was calculated using two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3. Type I IFN suppresses ActA degradation in phagosomes and promotes Lm actin recruitment.

a Schematic of GFP reporter Lm strains developed to measure actA transcription activation. Representative images (b) and quantification (c) of actA:GFP reporter expression and ActA degradation of BMDMs infected for 4 h with indicated Lm strains measured by GFP fluorescence and antibody staining, with or without treatment of IFNβ, bafilomycin A or protease inhibitors. Representative images at 1.25 hr p.i. (d) and quantification (e) of WT Lm actin recruitment, measured by phalloidin staining, after phagosome escape over 4 h time course. f Measurement of WT Lm actin-based motility with phalloidin-positive comet tails 4 h p.i. Data shown are means ± s.d. for n = 3 independent experiments. P value was calculated using (c, e) two-way ANOVA and (f) two-tailed unpaired t-test. *P = 0.0285, **P = 0.001, ****P < 0.0001. Scale bars, 11 µm.

Fig. 4. Type I IFN suppresses Lm virulence factor degradation in phagosomes.

a Schematic of experimental protocol detailing methods to obtain secreted and bacterial lysate fractions from BMDMs infected with indicated Lm strains. Representative images (b) and quantification (c) of ActA and InlB on the bacteria surface measured by western blot in BMDMs infected with indicated Lm strains 4 h p.i. Cell wall-associated protein p60 used as loading control. **P = 0.002, ****P < 0.0001. Representative images (d) and quantification (e) of LLO measured by western blot in BMDMs infected with indicated Lm strains over 4 h. *P < 0.0295, **P < 0.0039. Data shown are means ± standard deviation (s.d.) for at least n = 3 independent experiments. P value was calculated using two-way ANOVA. ns not significant, P > 0.1301. Source data are provided as a Source Data file.

Fig. 5. IFITM3 suppresses antibacterial activity in phagosomes and promotes Lm cell-to-cell spread.

Time course of bacteria killing over 24 h of (a) Δhly Lm or (b) E. coli infected WT and IFITM3 KO BMDMs with or without IFNβ. Representative images (c) and quantification (d) of ActA degradation of IFITM3 WT and KO BMDMs infected for 4 h with indicated actA:GFP reporter Lm strains measured by antibody staining of surface ActA under treatment with or without IFNβ. Scale bar, 11 µm. Representative images (e) and quantification (f) of WT Lm actin recruitment after phagosome escape at 1.25 h p.i. in IFITM3 WT and KO BMDMs treated with or without IFNβ. Scale bar, 11 µm. Representative images (g) and quantification (h) of an infection focus assay to measure WT Lm cell-to-cell spread. WT and IFITM3 KO BMDMs were treated with or without IFNβ and infected for 18 h. Dotted lines delineate edge of the infection foci. Scale bar, 70 µm. Data shown are means ± s.d. for at least n = 3 independent experiments. P value was calculated using two-way ANOVA. *P = 0.0242, **P < 0.0041, ***P = 0.001, ****P < 0.0001.

Fig. 6. IFITM3 promotes Lm infection in vivo.

a 10-day survival rate of WT (n = 25), IFITM3 (n = 15), and IFNAR1 KO (n = 10) mice infected with WT Lm plotted using Kaplan–Meier function. b Bacteria load of liver, spleen and brain of WT (n ≥ 10), IFITM3 (n ≥ 8), and IFNAR1 KO (n ≥ 6) mice infected with Lm over 72 h. Representative images (c, e) and quantification (d, f) of infection foci taken from the liver of mice (n = 3) 72 h p.i. Tissues were stained with H&E (c, d) or Lm and DAPI (e, f). Dotted lines delineate edge of the infection foci. Representative images (g) and quantification (h) of cell surface ActA expression by Lm in liver taken from mice (n = 3) 72 h p.i. Data shown are medians (b, h) or means ± s.d. (d, f). P value was calculated using two-tailed Mann–Whitney test (b, f) and two-tailed unpaired t-test (d, f). ****P < 0.0001. Scale bars, 70 µm.

Fig. 7. IFITM3 modulates cytokine signaling and immune cell recruitment.

a mRNA expression of IFNβ from Lm-infected liver of mice (n ≥ 4) 72 h p.i. b IFNβ production measured by ELISA of WT and IFITM3 KO BMDMs infected with Lm over 24 h. UI uninfected. c–g mRNA expression of indicated cytokines from Lm-infected liver of mice (n ≥ 4) 72 h p.i. Representative images (h) and quantification of (i) lymphocyte recruitment (CD3e) and (j) macrophage recruitment (F/480) of liver taken from mice (n = 3) 72 h p.i. Dotted lines delineate edge of the infection foci. Data shown are means (a–g) or medians (i, j) ± s.d. for at least n = 3 independent experiments. P value was calculated using two-tailed unpaired t-test (a–g) and two-tailed Mann–Whitney test (i, j). *P < 0.05, ****P < 0.0001. ns not significant, P > 0.055. Scale bar, 70 µm.

Fig. 8. Model of type I IFN-mediated effects of IFITM3.

During viral infection, IFITM3 decreases proteolysis of virus capsid proteins, and restricts fusion and entry into the cytosol. During Lm infection, IFITM3-mediated suppression of proteolysis leads to increased LLO and ActA in the phagosome. After entering the cytosol, increased actin recruitment subsequently leads to an increase in cell-to-cell spread. Arrows indicate decrease (red) or increase (green) by IFITM3.