Direct Antimicrobial Activity of IFN-β

Abstract

Type I IFNs are a cytokine family essential for antiviral defense. More recently, type I IFNs were shown to be important during bacterial infections. In this article, we show that, in addition to known cytokine functions, IFN-β is antimicrobial. Parts of the IFN-β molecular surface (especially helix 4) are cationic and amphipathic, both classic characteristics of antimicrobial peptides, and we observed that IFN-β can directly kill Staphylococcus aureus Further, a mutant S. aureus that is more sensitive to antimicrobial peptides was killed more efficiently by IFN-β than was the wild-type S. aureus, and immunoblotting showed that IFN-β interacts with the bacterial cell surface. To determine whether specific parts of IFN-β are antimicrobial, we synthesized IFN-β helix 4 and found that it is sufficient to permeate model prokaryotic membranes using synchrotron x-ray diffraction and that it is sufficient to kill S. aureus These results suggest that, in addition to its well-known signaling activity, IFN-β may be directly antimicrobial and be part of a growing family of cytokines and chemokines, called kinocidins, that also have antimicrobial properties.

FIGURE 1. Mouse interferon-β (IFN-β) kills Staphylococcus aureus

(A) S. aureus were incubated with vehicle (VEH) or 500 units/ml recombinant mIFN-β. Bacteria and treatments were incubated 3 and 24 hours. Remaining bacteria were serially-diluted and plated for enumeration. (B) S. aureus were incubated with VEH or mIFN-β (50, 500, or 5000 units/ml which is equal to 0.0006, 0.006, or 0.06 μM). Bacteria were treated for 1 and 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (C) S. aureus or E. coli were incubated with VEH or mIFN-β at indicated concentrations for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (D) S. aureus were incubated with Sytox Green for 20 min. Bacteria were added to a 96-well plate pre-loaded with VEH, 1 mM nigericin (NIG), or 5×10³ units/ml mIFN-β. The plate was imaged at 485/520 nm. Relative fluorescent units (RFU) are shown. (E) S. aureus were incubated with VEH, 1 mM NIG, or 5×10³ units/ml mIFN-β for 10 min. Bacteria were stained with DAPI (blue) or Sytox (green) and imaged on a confocal microscope. Five separate images from each treatment were enumerated for blue versus green bacteria and results are plotted. Data are shown as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (unpaired, two-tailed t test).

FIGURE 2. IFN-β has properties resembling antimicrobial peptides

(A) S. aureus were incubated with VEH or indicated units of mIFN-β for 1 hour. Bacteria were washed 2X to remove any mIFN-β not adhered to bacterial surface. mIFN-β interacting with S. aureus cell wall were detected by immunoblotting and 500 units mIFN-β was used as a positive control. Detection of bacterial GAPDH was used to control for the loading of equal amounts of bacteria. (B) Wild-type (WT) or DLT-deficient (ΔDLT) S. aureus were incubated with VEH or mIFN-β at indicated concentrations for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (C) S. aureus were incubated with VEH, 5×10² units/ml mIFN-β (−), 5×10² units/ml mIFN-β incubated at 37°C for 60 min (37C), or 5×10² units/ml mIFN-β incubated with proteinase K for 60 min (Pro K). Bacteria were incubated with variably-treated mIFN-βs for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. Data are shown as mean ± SD, * p ≤ 0.05 (unpaired, two-tailed t test).

FIGURE 3. Human IFN-β kills S. aureus, but only at low pH

(A) S. aureus were incubated with VEH, mIFN-β, or recombinant hIFN-β at the same concentration of 5×10² units/ml for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (B) Mouse and human IFN-β were compared at the nucleic acid, amino acid and structural levels. Structural RMS is shown for overlay. (C) S. aureus were incubated with VEH) or 5×10³ units/ml hIFN-β in varying pHs for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (D) S. aureus were incubated with VEH, hIFN-β produced in E. coli (E. coli), or hIFN-β produced in CHO cells (CHO) at indicated concentrations. Bacteria were incubated with hIFN-β in neutral and low pH for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. Data are shown as % surviving bacteria based on initial CFU. Data are shown as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01 (unpaired, two-tailed t test).

FIGURE 4. Structural comparison of mouse and human IFN-β

(A) Mouse and human IFN-β ribbon structures are shown. N and C termini are labeled. The five α-helices are color coded from N to C terminus blue, green, yellow, orange, and red. (B) Mouse and human IFN-β electrostatic space filling models are shown for neutral (pH 7) and acidic (pH 5) pH. Regions of positive charge are shown in blue, regions of neutral charge are shown in white, and regions of negative charge are shown in red. (C) Mouse and human IFN-β amino acid sequences are shown. The α-helices are color-coded as in (A). (D) Charge was calculated for all five helices for mouse and human IFN-β. The high positive charge of helices 4 and 5 are highlighted in blue. (E) Mouse and human IFN-β amino acid plots were generated showing approximate charge of each amino acid residue. Values for neutral (blue) and acidic (red) pH are shown. Blue region shows helices 4 and 5.

FIGURE 5. Mouse and human IFN-β helix 4 are similar to antimicrobial peptides

(A) Sequences of both mouse and human IFN-β helix 4 plotted as helical wheel projections to illustrate their facially amphipathic character. Positively-charged hydrophilic residues are indicated in blue, uncharged hydrophilic residues in violet, and hydrophobic residues in green. (B) Mouse and human IFN-β helix 4 sequences; native sequences are shown alongside synthesized peptides. Residues are color-coded as in (A). (C) Relationship between positively-charged residues (N_K/(N_K + N_R)) and average peptide hydrophobicity for 1080 cationic AMPs in the antimicrobial peptide database (grey open circles). Mouse IFN-β helix 4 (red circle), human IFN-β helix 4 (blue triangle), human LL-37 (cyan square), and human IL-26 (magenta square). (D) The hydrophobicity of IFN-β helix 4 is comparable to that of antimicrobial peptides. Histogram depicting the distribution of average hydrophobicities among 1080 cationic AMPs in the antimicrobial peptide database (green bars) compared with the hydrophobicities of mouse IFN-β helix 4, human IFN-β helix 4, human LL-37, and human IL-26.

FIGURE 6. Mouse IFN-β helix 4 induces membrane curvature in bacterial membranes and kills S. aureus

(A) Model of negative Gaussian curvature (NGC) required for membrane destabilization processes such as pore formation by AMPs. (B) IFN-β helix 4 does not generate NGC in eukaryotic-like membranes. SAXS spectra for mouse and human IFN-β helix 4 with eukaryotic model membranes DOPG/DOPE/DOPC = 20/40/40 and DOPS/DOPC = 20/80 at P/L = 1/50 peptide to lipid (P/L) molar ratio. (C) IFN-β helix 4 generates NGC in prokaryotic-like membranes. SAXS spectra from prokaryotic model membrane, DOPG/DOPE = 20/80, vesicles incubated with either mouse or human IFN-β helix 4 at P/L = 1/50 molar ratio. Insets show expanded view of cubic phase reflections. (D) S. aureus were incubated with VEH or mIFN-β helix 4 at 0.006, 0.06, 0.6, and 6 μM. Bacteria and treatments were incubated for 1 or 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (E) S. aureus were incubated with VEH or mouse IFN-β helix 4 at 6.25, 12.5, 25, 50, 100, and 200 μM. Bacteria and treatments were incubated for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (F) Wild-type (WT) and mutant IFN-β helix 4 peptide were incubated with 1×10⁵ SA113/ml in buffer + 1%THB for 3 hours. Remaining bacteria were serially-diluted and plated for enumeration. (G–I) Bacteria (1×10⁵/ml) were incubated with vehicle or 6 μM mouse hexlix 4 peptide for 3 hours at 37°C. Remaining bacteria were serially-diluted and plated for enumeration. Data are shown as mean ± SD, * p ≤ 0.05,** p ≤ 0.01, ***p≤0.001 (unpaired, two-tailed t test). N.D.- below limit of detection.