Broad-Spectrum Robust Direct Bactericidal

Activity of Fish IFN φ1 Reveals an

This information is current as
of May 28, 2022.
Antimicrobial Peptide−like Function for
Type I IFNs in Vertebrates
Xun Xiao, Wentao Zhu, Yanqi Zhang, Zhiwei Liao,
Changsong Wu, Chunrong Yang, Yongan Zhang, Shaobo
Xiao and Jianguo Su
J Immunol 2021; 206:1337-1347; Prepublished online 10
February 2021;
doi: 10.4049/jimmunol.2000680

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http://www.jimmunol.org/content/206/6/1337

Supplementary http://www.jimmunol.org/content/suppl/2021/02/09/jimmunol.200068
Material 0.DCSupplemental
References This article cites 49 articles, 13 of which you can access for free at:
http://www.jimmunol.org/content/206/6/1337.full#ref-list-1

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 The Journal of Immunology

Broad-Spectrum Robust Direct Bactericidal Activity of Fish

IFNw1 Reveals an Antimicrobial Peptide–like Function for
Type I IFNs in Vertebrates

Xun Xiao,*,†,‡ Wentao Zhu,* Yanqi Zhang,* Zhiwei Liao,* Changsong Wu,*
Chunrong Yang,‡ Yongan Zhang,* Shaobo Xiao,‡ and Jianguo Su*,†
Type I IFNs (IFN-Is) play pivotal roles in host defense against viral infections but remain enigmatic against bacterial pathogens. In
this study, we recombinantly expressed and purified intact grass carp (Ctenopharyngodon idella) IFNw1 (gcIFNw1), a teleost IFN-I.
gcIFNw1 widely powerfully directly kills both Gram-negative and Gram-positive bacteria in a dose-dependent manner. gcIFNw1
binds to LPS or peptidoglycan and provokes bacterial membrane depolarization and disruption, resulting in bacterial death.
Furthermore, gcIFNw1 can efficiently protect zebrafish against Aeromonas hydrophila infection and significantly reduce the
bacterial loads in tissues by an infection model. In addition, we wonder whether antibacterial IFN-I members exist in other

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vertebrates. The amino acid compositions of representative IFN-Is with strong positive charges from Pisces, Amphibia, reptiles, Aves,
and Mammalia demonstrate high similarities with those of 2237 reported cationic antimicrobial peptides in antimicrobial peptide
database. Recombinant intact representative IFN-I members from the nonmammalian sect exhibit potent broad-spectrum robust
bactericidal activity through bacterial membrane depolarization; in contrast, the bactericidal activity is very weak from mammalian
IFN-Is. The findings display a broad-spectrum potent direct antimicrobial function for IFN-Is, to our knowledge previously unknown.
The results highlight that IFN-Is are important and robust in host defense against bacterial pathogens, and unify direct antibacterial
and indirect antiviral bifunction in nonmammalian jawed vertebrates. The Journal of Immunology, 2021, 206: 1337–1347.

I
nterferons are a subset of class 2 a-helical cytokines that
have existed in early chordates for ∼500 million years and
represent early elements in innate and adaptive immune
systems (1). Based on the distinctive patterns of structures,
functions, receptors, and signaling pathways, three types of IFNs
have been characterized as type I IFN (IFN-I), type II IFN (IFN-
II), and type III IFN (IFN-III) from cartilaginous fishes to mam-
mals (2, 3). So far, at least 20 (13 IFN-a subtypes, IFN-b, IFN-v,
IFN-ε, IFN-k, IFN-d, IFN-z, and IFN-t) and 3 (IFN-a, IFN-b, and
IFN-k) IFN-Is have been identified in mammals and Aves, respec-
tively (4, 5). However, only IFN-I and IFN-II are present in teleosts
(6, 7); in addition, different fish species contain different subgroups
and members of IFN-Is (8). Grass carp (Ctenopharyngodon idella),
*Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricul-
tural University, Wuhan 430070, China; †Laboratory for Marine Biology and Bio-
technology, Pilot National Laboratory for Marine Science and Technology, Qingdao
266237, China; and ‡College of Veterinary Medicine, Huazhong Agricultural Uni-
versity, Wuhan 430070, China
ORCID: 0000-0002-9083-0715 (J.S.).
Received for publication June 8, 2020. Accepted for publication January 11, 2021.
This work was supported by the National Key Research and Development Program of
China (2018YFD0900504), the National Natural Science Foundation of China
(31873044 and 31930114), and Fundamental Research Funds for the Central Uni-
versities (2662018PY062).
Address correspondence and reprint requests to Dr. Jianguo Su, College of Fisheries,
Huazhong Agricultural University, No. 1 Shizishan Street, Wuhan 430070, Hubei,
China. E-mail address: sujianguo@mail.hzau.edu.cn
The online version of this article contains supplemental material.
Abbreviations used in this article: afIFN2.3, African clawed frog IFN2.3; AFM,
atomic force microscopy; APD, antimicrobial peptide database; ATCC, American
Type Culture Collection; caIFN-a2, Chinese alligator IFN-a2; CCCP, carbonyl
cyanide m-chlorophenyl hydrazone; chIFN-b, chicken IFN-b; gcIFNw, grass carp
IFNw; IFN-I, type I IFN; IFN-II, type II IFN; LB, Luria–Bertani; MBC, minimal
bactericidal concentration; PGN, peptidoglycan; pI, isoelectric point.
Copyright Ó 2021 by The American Association of Immunologists, Inc. 0022-1767/21/$37.50
the highest-yield economic teleost fish with great commercial
value and worldwide distribution (9), has four IFN-Is (IFNw1–4)
and two IFN-IIs (IFN-g and IFN-g-rel), corresponding to those in
zebrafish (Danio rerio; a model organism) (10). In higher verte-
brates, such as mammals, IFN-Is (mainly IFN-a/b) are very im-
portant for protecting host against viral infections (11–13). By
contrast, IFN-II (IFN-g) is essential for host defense against some
bacterial and parasitic pathogens (14). IFN-IIIs (IFN-l1–4) have
antiviral functions similar to IFN-I family but with restricted ac-
tivity (15, 16). In lower vertebrates, such as Pisces, IFN-Is also
display powerful antiviral activity (17–20).
Apart from the antiviral function, IFN-Is are also induced by
bacterial pathogens (21). In mammals, germline-encoded pattern
recognition receptors (PRRs) recognize bacterial components and
initiate antibacterial inflammatory gene program, which leads to
the induction of IFN-Is (21). However, the role of IFN-Is in
bacterial infections is enigmatic and even detrimental to the host
in mammals (22). In infection with Listeria monocytogenes or
Mycobacterium tuberculosis, high concentrations of IFN-Is be-
come harmful to the host because they block B cell responses or
lead to the production of immunosuppressive molecules, and such
concentrations also reduce the responsiveness of macrophages to
IFN-g activation (13, 14, 23). In recent studies, mouse (Mus
musculus) and human (Homo sapiens) IFN-bs can directly kill
Staphylococcus aureus, not Escherichia coli, in vitro, even only
under acidic pH for human IFN-b (24). In Pisces, zebrafish IFNw1
was discovered to protect zebrafish from Streptococcus iniae in-
fection in 2009 (17). However, bactericidal function of IFN-Is was
not brought to the forefront till now.
Antimicrobial peptides are evolutionarily ancient potent weapons
throughout plant and animal kingdoms that directly kill a wide
range of microbes, including bacteria, fungi, viruses and protozoa
(25), and cancer cells (26). Antimicrobial peptides are an abundant
and diverse group of molecules that are produced by many tissues

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000680
1338 DIRECT BACTERICIDAL ACTIVITY OF IFN

and cell types (27). Generally, most natural antimicrobial peptides
possess cationic and amphipathic features that can selectively
disrupt bacterial membrane or/and inhibit bacterial metabolisms
(27). In addition to direct bactericidal activity, some antimicrobial
peptides also possess bacterial endotoxin-neutralizing effect to
inhibit the excessive release of proinflammatory cytokines in
response to LPS in vitro and in vivo (28, 29). Antimicrobial
peptides are less probable in inducing bacterial resistance than
conventional antibiotics, and diverse applications have been
demonstrated for antimicrobial peptides as anti-infective agents
(25, 30, 31).
In this study, we found that antimicrobial peptide–like IFN-Is
exist in Pisces, Amphibia, reptiles, and Aves, but not in mammals.
IFNw1, as a representative IFN-I in lowest vertebrate taxonomy
owning IFN (Pisces), exhibits broad-spectrum powerful direct
bactericidal activity by bacterial membrane depolarization and
disruption. Our results indicated that IFN-Is are important and
forceful in host defense against bacterial pathogens in nonmammalian
gnathostome vertebrates.
Materials and Methods
Ethics statement
Zebrafish were acquired from the China Zebrafish Resource Center
(Wuhan, China). All the experiments were performed in accordance with
the guidelines of the Laboratory Animal Center of Huazhong Agricultural
University. The protocols were approved by the Ethical Committee on
Animal Research at Huazhong Agricultural University (HZAUMO-2017-
009). All efforts were made to minimize animal suffering.
Bacterial isolates
E. coli (American Type Culture Collection [ATCC] 25922), Pseudomonas
aeruginosa (ATCC 9027), Aeromonas hydrophila (ATCC 7966), S. aureus
(ATCC 25923), S. agalactiae (ATCC 13813), and S. pneumoniae (ATCC
49619) were obtained from ATCC. The clinical isolate of A. hydrophila
1703 was isolated from grass carp intestine and identified by conventional
biochemical method and 16S rRNA sequencing in our laboratory.

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FIGURE 1. The sequence alignment, structures, and charge distributions of grass carp IFN-I. (A) Multiple sequence alignments, net charges, and pIs of
grass carp IFN-Is. Asterisks indicate fully conserved residue positions. Colons and periods indicate conservation positions with strongly or weakly similar
property, respectively. Conserved cysteine (C), positively charged lysine (K), and arginine (R) residues are shown in bold. (B) Ribbon diagrams of
gcIFNw1–4 protein are constructed by homology modeling. Six predicted a-helices designated aA–aF are presented by different colors. (C) Color-coded
electrostatic potentials are mapped on the surfaces of grass carp IFN-Is. Regions with positive charges are shown in blue, negative charges in red, and
hydrophobic residues in white.
The Journal of Immunology 1339

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FIGURE 2. Powerful bactericidal activity of gcIFNw1 in vitro. (A) SDS-PAGE analysis of purified intact gcIFNw1–3. (B) Growth of E. coli in culture
with increasing concentrations of gcIFNw1, gcIFNw2, and gcIFNw3. (C) Growth of S. aureus ATCC 25923, S. agalactiae ATCC 13813, A. hydrophila
ATCC 7966, P. aeruginosa ATCC 9027, and E. coli ATCC 25922 in the culture with increasing concentrations of gcIFNw1. (D) Bactericidal kinetics of
gcIFNw1 (1 mM) against E. coli, A. hydrophila, S. agalactiae, and S. aureus. The samples were serially diluted and plated for enumeration. All the ex-
periments were performed in triplicate. Data are shown as means 6 SD.

Zebrafish infection model
Adult zebrafish were challenged by i.p. injection with 1 3 10 CFU of3
A. hydrophila strain 1703 per fish and maintained in 25-l water tanks at 28˚C.
At 1 h after bacterial injection, the animals were i.p. injected with 5 ml of
grass carp IFNw (gcIFNw)1–3 at 10 ng, ampicillin at 50 ng (positive control),
or PBS (negative control) (n = 25 per group). The fish were monitored every
6 h for 7 d for clinical signs of disease and mortality. Spleen, intestine, and
blood were collected at 12 h after bacterial injection (n = 5 per group).
The spleen and intestine were homogenized. The homogenate and blood were
diluted using sterile PBS spread on Luria–Bertani (LB) plates for 12–16 h at
28˚C. A. hydrophila colonies were counted by two independent investigators.
Recombinant expression and purification of intact IFN-Is
A bacterial expression system consisting of pGEX-4T1 plasmid and E. coli
BL21(DE3)pLysS (Invitrogen) was used to produce IFN-Is (adding 6xHis
at C terminus for ELISA). The bacteria were incubated overnight in LB
medium containing 100 mg/ml ampicillin at 37˚C. Then, the culture
was transferred to 600 ml of LB medium with 100 mg/ml ampicillin at 37˚
C until OD600 reached 0.5–0.6. Expression was induced with 1.0 mM
isopropyl b-D-1-thiogalactopyranoside at 25˚C for 4 h. After that, the cells
were pelleted by centrifugation (5000 3 g) for 10 min at 4˚C. The IFN-Is
with GST tag were extracted and purified by affinity chromatography
(Glutathione Beads; Smart-Lifesciences) under denaturing conditions
(20 mM Tris-HCl, 1 M NaCl, and 10% glycerine [pH 7.4]) and then eluted
with 10 mM glutathione in 20 mM Tris-HCl, 0.2 M NaCl, and 10%
glycerine (pH 7.4). Then, we removed the GST tag by enterokinase and
purified the intact IFN-Is by ion exchange chromatography (SP Beads 6FF;
Huiyan Biotechnology) under denaturing conditions (20 mM Tris-HCl [pH
7.4]), except IFNw3 (Q Focurose 6FF). The IFN-Is were extensively
washed with 200 mM NaCl in 20 mM Tris-HCl and then eluted with
300 mM NaCl in 20 mM Tris-HCl (pH 7.4). After that, the IFN-Is were
desalted in 20 mM Tris-HCl (pH 7.4) by stepwise dialysis (3-kDa
molecular mass cutoff by Amicon Ultra centrifugal filter device;
MilliporeSigma) and then stored at 4˚C. The purified intact IFN-I proteins
were confirmed via SDS-PAGE and Coomassie Bright Blue staining.
Antimicrobial assay
Antimicrobial activities were evaluated by CFU assay (32). The bacteria
were cultured overnight in LB medium at 37˚C (28˚C for aquatic bacteria)

Table I. MBC90 determination of gcIFNw1, afIFN2.3, caIFN-a2, and chIFN-b antimicrobial activity against several microorganisms

Bacterial Strain gcIFNw1 (mM) afIFN2.3 (mM) caIFN-a2 (mM) chIFN-b (mM) G+/G2

A. hydrophila (ATCC 7966) 0.60 0.50 0.15 0.40 G2

P. aeruginosa (ATCC 9027) 0.62 0.60 0.10 0.60 G2
E. coli (ATCC 25922) 0.55 0.55 0.15 0.25 G2
S. pneumoniae (ATCC49619) 0.25 0.25 0.05 0.10 G+
S. agalactiae (ATCC 13813) 0.25 0.25 0.05 0.15 G+
S. aureus (ATCC 25923) 0.25 0.25 0.05 0.10 G+
MBC90 was measured by microbroth dilution method.
G+, Gram-positive bacterium; G2, Gram-negative bacterium.
1340
FIGURE 3. gcIFNw1 kills bacteria by targeting LPS and PGN. (A) The binding capacity of gcIFNw1 to LPS or PGN was assessed by ELISA. (B) E. coli
was incubated with buffer, 13MBC gcIFNw1, and 13MBC gcIFNw1 plus 1 mg/ml LPS or 20 mM MgCl2 for 1 h. The samples were serially diluted and
plated for enumeration. All the experiments were done in triplicate. Data are shown as means 6 SD. *p # 0.05, **p # 0.01.
and then subcultured to achieve midlogarithmic phase growth. The bacteria
were collected, washed with PBS, and then diluted to a final concentration
of 1 3 105 CFU/ml in an incubation buffer (10 mM Tris-HCl and 5 mM
glucose [pH 7.4]). A bacterial suspension (100 ml) was incubated with
100 ml of protein or peptide for 3 h at 37˚C (28˚C for aquatic bacteria).
After that, the suspension was spread on LB plates for 12–16 h. The
number of colonies was counted by two independent investigators.
Membrane potential assay
To investigate the membrane potential change, a total of 500 ml of bacteria
(1 3 107 CFU) was incubated with 1 mM IFN-I, 5 mM proton ionophore
carbonyl cyanide m-chlorophenyl hydrazone (CCCP), or buffer for 15 min.
Then, the bacteria were stained with the fluorescent membrane potential
indicator dye DiOC2 (3) (Invitrogen) according to the manufacturer’s
protocol. A total of 1 3 105 cells were analyzed on a flow cytometer using
488-nm excitation, 530/30-nm bandpass, and 650-nm-long pass filters.
SYTOX Green assays
The S. aureus (ATCC 25923) and E. coli (ATCC 25922) cells were
employed to detect the membrane integrity with SYTOX Green
(Invitrogen). The bacterial suspensions were grown in LB medium to
exponential phase. Bacterial suspensions (1 3 107 CFU/ml) were in-
cubated with 1 mM IFNw1–3 at indicated concentrations with shaking
at 37˚C for 15 min and collecting and washing cells. SYTOX Green
(1 mM) was incubated with the treated samples for 15 min in the dark.
The fluorescence intensity was measured at wavelength 485- and 520-nm
filters for excitation and emission. LL-37 and buffer were employed as
positive and negative controls, respectively.
Scanning electron microscopy
The S. aureus (ATCC 25923) and E. coli (ATCC 25922) were cultured at
37˚C overnight in LB medium and then subcultured to achieve mid-
logarithmic phase growth. A total of 400 ml of bacteria (5 3 107 CFU/ml)
were centrifuged at 4000 3 g for 10 min. The cells were washed three
times with PBS and incubated with gcIFNw1–3 or buffer (20 mM Tris-HCl
and 10 mM NaCl [pH 7.4]) at 37˚C for 15 min. Then, the cells were fixed
with 2.5% glutaraldehyde at 4˚C overnight. The cells were dehydrated by
10–100% ethanol series (10 min per step) and coated with gold. Finally,
the cells were examined by a scanning electron microscope (JSM-840).
Atomic force microscopy
E. coli or S. aureus was prepared and treated as above-mentioned scanning
electron microscopy samples. After the cells were incubated with gcIFNw1
or buffer (20 mM Tris-HCl and 10 mM NaCl [pH 7.4]) at 37˚C for 15 min.
Table II. gcIFNw1 binds to the bacterial cell wall components LPS and PGN
Protein gcIFNw1 gcIFNw1
Ligand LPS-1 LPS-2
Kd (nM) 83.4 6 3.5 51.5 6 6.1
Kd of gcIFNw1, gcIFNw2, and LL-37 binding to LPS or PGN, measured by ELISA. LPS-1 is from E. coli O111:B4 and LPS-2 from E. coli O55:B5.
NI, no interaction.
DIRECT BACTERICIDAL ACTIVITY OF IFN
The bacteria were centrifuged at 4000 3 g for 10 min, and the cells were
washed three times by deionized water. The cells were transferred to
mica disks and dried overnight at 25˚C before imaging. The atomic force
micrographs were recorded on a Veeco multimode atomic force micro-
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scope with NanoScope III controller operating in contact mode. The data
were analyzed with NanoScope Analysis software v.1.40 (Veeco). Scans
were acquired at 25˚C at the rates of 1.0 Hz and 256 samples per line
resolution.
ELISA binding assay
The activities of IFNw1 binding LPS or peptidoglycan (PGN) were mea-
sured by ELISA method as we previously described (33). In brief, 96-well
microtiter plates were coated with LPS-1 (from E. coli O111:B4, L2630;
Sigma-Aldrich), LPS-2 (from E. coli O55:B5, L2880; Sigma-Aldrich), or
PGN (69,554; Sigma-Aldrich) (10 mg/well). The plates were washed with
TBST (0.1% Tween 20) and blocked with 1% BSA. One hundred microliters
of gcIFNw1–2 proteins and LL-37 with different concentrations were added
to the corresponding wells for 3 h at room temperature. The plates were
washed three times, and 100 ml of anti-6xHis (1:1000; Abcam) or LL-37
(1:1000; ABclonal) was added as the first Ab. After incubation at 37˚C
for 1 h, the plates were washed, and 100 ml of goat anti-mouse IgG-HRP
conjugate (polyclonal Ab, 1:3000, A0216; Beyotime Biotechnology)
was added for 1 h. The plates were washed, and 100 ml of 3,39,5,59-
tetramethylbenzidine solution (TIAGEN Biotech) was added to each
well. These mixtures were incubated at room temperature in the dark for
15 min. The reaction was stopped by adding 50 ml of 2 M H2SO4.
The plates were examined on a Synergy 4 Hybrid Microplate Reader
(BioTek Instruments, Winooski, VT) at 450 nm.
Compositional comparison of IFN-Is with
antimicrobial peptides
We compared the amino acid compositions of human IL-26 and repre-
sentative IFN-Is with known antimicrobial peptides for generating mem-
brane curvature based on the geometry of membrane destabilization (34). A
set of 2237 cationic antimicrobial peptides sequences were sourced from
the antimicrobial peptide database (APD) (35). The minimum and
maximum hydrophobicity values within the set of antimicrobial peptide
sequences were defined as the hydrophobicity range. This range was
divided into 100 equal bins, into which the peptides were partitioned.
NK/(NK + NR) represents the ratio of the lysines/lysines plus arginines. For
each bin, NK/(NK + NR) versus hydrophobicity was plotted using MATLAB.
To compare the hydrophobicity, the hydrophobicity histogram values for the
set of antimicrobial peptides were constructed with 50 bins using MATLAB.
The average hydrophobicities of IL-26 and IFN-Is were superimposed over
the antimicrobial peptides histogram.
gcIFNw1 LL-37 gcIFNw2 gcIFNw
PGN LPS-1 LPS-1 PGN
26.1 6 2.3 6.35 6 0.4 NI NI
The Journal of Immunology 1341

Protein modeling and charge analysis Results

Protein modeling was based on the SWISS-MODEL Template Library
(https://www.swissmodel.expasy.org/). The predicted protein structures
were downloaded as Protein Data Bank files and then imported into
PyMOL (PyMOL Molecular Graphics System, Version 1.3, 2011;
Schrödinger) to generate the protein models. The structural homology
of gcIFNw1 and zebrafish IFNw1 was also carried out by PyMOL.
Further net charge distribution was performed by APBS software plug-
in (https://www.easycounter.com/report/poissonboltzmann.org). The
net charge analyses of IFN-Is use the online software (http://www.
novopro.cn/tools/protein_iep.html).
Statistical analysis
Statistical analyses were performed using the two-tailed unpaired Student
t test in in vitro experiments and the two-tailed unpaired nonparametric
Mann–Whitney U test in in vivo experiments. The p values were the
following: *p # 0.05, **p # 0.01, and ***p # 0.001.
Sequence and structural analyses of grass carp IFN-Is
The multiple sequence alignment of the mature proteins of grass
carp IFN-Is (IFNw1, -2, -3, and -4) indicates that gcIFNw1 has
low sequence homology with other IFN-Is (,25%) (Fig. 1A).
In comparison with IFNw2 (net charge +4.3, isoelectric point
[pI] = 8.72), IFNw3 (net charge 29.0, pI = 4.57), and IFNw4 (net
charge +0.8, pI = 7.49), IFNw1 possesses unusual cationicity (net
charge +12.6, pI = 10.13) (Fig. 1A). For IFNw1, the majority of
cationic charges concentrate in two of the six predicted helices,
helices E and F, which contain four arginines (R) and nine lysines
(K) (Fig. 1A). gcIFNw1 shares remarkable structural similarity
with zebrafish (model animal) IFNw1 (Supplemental Fig. 1), whose
crystal structure has been disclosed (36). The three-dimensional

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FIGURE 4. gcIFNw1 kills Gram-negative bacteria by membrane depolarization and disruption mechanisms. (A) Membrane potential of E. coli. Red
fluorescence aggregates at polarized membrane. Loss of red fluorescence indicates membrane depolarization. CCCP was used as the positive control. (B)
E. coli was cultured for 15 min with or without 1 mM gcIFNw1 and visualized by scanning electron microscopy. (C) E. coli was incubated for 15 min with
or without 1 mM gcIFNw1 and visualized by AFM.
1342
modeling structure for gcIFNw1, based on the zebrafish IFNw1,
shows that the helices E and F are adjacent, which generates
cluster formation and surface exposure of the cationic residues
(Fig. 1B, 1C). On the opposite side of this cationic cluster, we
observed a hydrophobic patch (helix D) consisting of nine hydro-
phobic residues (Fig. 1B, Supplemental Fig. 1B). The predominant
polar residues and the hydrophobic amino acids locate on the op-
posite sides, which indicates that gcIFNw1 is an amphipathic cy-
tokine. The cationic and amphipathic features of gcIFNw1 accord
with the natural antimicrobial peptides (37). By contrast, another
IFN-I (IFNw3) in grass carp has negative net charges (anionic).
Although the other IFN-Is (IFNw2 and IFNw4) in grass carp contain
cationic patches, they are weak and dispersive (Fig. 1C). The results
indicate that gcIFNw1 is a highly cationic and amphipathic
a-helical protein.
gcIFNw1 is a broad-spectrum potent bactericidal cytokine that
targets both Gram-negative and Gram-positive bacteria
gcIFNw1 has been extensively studied in regulating antiviral im-
mune signaling. Finding cationic and amphipathic features,
which are hallmarks of naturally occurring antimicrobial proteins (37),
we investigated the antimicrobial property of gcIFNw1. We expressed
the recombinant gcIFNw1–3 protein in E. coli BL21(DE3)pLysS.
The purified gcIFNw1–3 proteins were examined by SDS-PAGE
FIGURE 5. Antimicrobial activity of gcIFNw1 in vivo. (A) In vivo protection against A. hydrophila infection was examined with a zebrafish infection
model (n = 25). (B–D) Bacterial loads in the zebrafish intestine, blood, and spleen at 12 h after bacterial injection (n = 5). All the experiments were done in
triplicate. Data are shown as means 6 SD. **p # 0.01, ***p # 0.001.
DIRECT BACTERICIDAL ACTIVITY OF IFN
(Fig. 2A). Then, we examined the antimicrobial activity of
gcIFNw1–3 against bacteria by CFU assay. We found that only
gcIFNw1, but not gcIFNw2–3, showed bactericidal activity
in vitro (Fig. 2B). gcIFNw1 displays a fairly wide-spectrum and
forceful microbicidal activity against typical pathogens, not only
Gram-positive S. aureus and S. agalactiae but also Gram-negative
E. coli, P. aeruginosa, and A. hydrophila (Fig. 2C). The minimum
bactericidal concentration for 90% (minimal bactericidal concen-
tration [MBC]90) values of gcIFNw1 against some bacterial strains
were examined and listed in Table I. Further study on the bacteri-
cidal kinetics shows that the number of bacteria treated with 1 mM
gcIFNw1 decreases over 90% within 10–50 min (Fig. 2D). To-
gether, these data potently support that gcIFNw1 is a direct anti-
microbial cytokine with extensive and forceful bactericidal ability.
gcIFNw1 directly kills bacteria through cytoderm binding,
membrane depolarization, and disruption
Nature antimicrobial peptides are commonly characterized by
positive charges and amphipathicity. The positive charges pro-
mote the electrostatic attraction to the various negative membrane
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components of bacteria (38). To explore the possible molecular
basis for gcIFNw1 bactericidal feature, we incubated LPS and
PGN with gcIFNw1–2 for ELISA. The result shows that only
gcIFNw1 has the ability to bind LPS and PGN (Fig. 3A, Table II).
The Journal of Immunology 1343

In addition, the antimicrobial activity of gcIFNw1 is significantly
weakened when bacteria grow in the presence of LPS or a high
concentration of Mg2+ (Fig. 3B). The cell wall of the bacteria
contains certain Mg2+ to keep the structure stable (39). gcIFNw1
may bind to LPS or PGN by substituting Mg2+ on the bacterial
surface, thus adsorbing a large amount on the bacterial outer
membrane surface.
To further explore the bactericidal mechanisms, we confirmed
the depolarization of the bacterial membrane by flow cytometry
(Fig. 4A, Supplemental Fig. 2A). The proportion of depolarized E.
coli and S. aureus were 81.1 and 56.4%, respectively. We observed
an obvious increase in the fluorescent signal when bacteria were
exposed to gcIFNw1 and LL-37, but not to gcIFNw2 or gcIFNw3
(Supplemental Fig. 3A), suggesting that gcIFNw1 disrupts the
bacterial membrane. Then, scanning electron micrographs reveal
bacterial surface disruption as presented by sunken and viscid
cells (Fig. 4B, Supplemental Fig. 3B). Furthermore, the surface
disruption is shown more clearly by atomic force microscopy
(AFM) (Fig. 4C, Supplemental Fig. 2B). The results show that
gcIFNw1 kills bacteria through bacterial membrane depolarization
gcIFNw1 can efficiently protect zebrafish against
A. hydrophila infection
Furthermore, we tested the antimicrobial activity of gcIFNw1
in vivo using a zebrafish A. hydrophila infection model. Zebrafish
cured with gcIFNw1 protein show a substantially higher survival
rate (60%) than those in control groups (40% for ampicillin
and 0% for PBS) (Fig. 5A). In addition, bacterial titers in
intestine, blood, and spleen are extremely significantly re-
duced in the gcIFNw1–treated group (Fig. 5B–D). However,
gcIFNw2–3 did not show significant antibacterial activity in
zebrafish (Supplemental Fig. 4A, 4B). The results demonstrate
that gcIFNw1 exerts powerful bactericidal activity in vivo.
Representative cationic IFN-Is from different vertebrates
possess similar amino compositions with those in
antimicrobial peptides
gcIFNw1 protects hosts against A. hydrophila infection (Gram-
negative bacteria) in the above study. Zebrafish IFNw1 protects
host against S. iniae infection (Gram-positive bacteria) in the
previous study (17). Human and mouse IFN-bs kill S. aureus, not

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and disruption, causing cytoplasm outflow. These findings indicate E. coli, in vitro, only under acidic pH for human IFN-b (24).
that gcIFNw1 kills bacteria through cytoderm binding, membrane Given that Pisces represents the low vertebrate taxonomy that
depolarization, and disruption. contains IFN and Mammalia represents the highest vertebrates, we

FIGURE 6. Charge distributions and amino acid compositions of representative cationic IFN-Is from different taxonomic vertebrates. (A) Charge
distributions on the surfaces of the representative positively charged IFN-Is. Positive and negative charges are in blue and red, respectively. Hydrophobic
residues are in white. The calculated net charges are marked on the right. (B) Relationship between electropositive amino acid residues (NK/(NK + NR))
and average peptide hydrophobicity for 2237 cationic antimicrobial peptides from the APD (gray circles). Human IL-26 is displayed as reference. (C)
The hydrophobicity comparisons between IFN-Is and antimicrobial peptides. Histograms indicate the distribution of average hydrophobicity among 2237
cationic antimicrobial peptides in the APD (gray bars).
1344 DIRECT BACTERICIDAL ACTIVITY OF IFN

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FIGURE 7. Antimicrobial activity and mechanism of representative IFN-Is from Amphibia, reptiles, and Aves. (A–C) Growth of S. aureus,
S. agalactiae, A. hydrophila, P. aeruginosa, and E. coli in culture at increasing concentrations of afIFN2.3, caIFN-a2, and chIFN-b (n = 3). Data are
shown as means 6 SD. All species are the same as those in Fig. 2. (D) Membrane potentials of E. coli (25922; ATCC). Red fluorescence indicates the
polarized membrane. A loss of red fluorescence indicates membrane depolarization. CCCP was used as the positive control. All the experiments were
performed three times.

wondered whether the powerful bactericidal members of IFN-Is
emerged as a unique trait in Pisces or exist widely in other animal
taxonomic categories, and whether they also kill Gram-negative
bacteria in addition to Gram-positive bacteria. Generally, antimi-
crobial cytokines or chemokines possess high positive charges
(25). Hence, we first calculated the total net charges of IFN-Is in
representative species, including grass carp (Pisces), African
clawed frog (Xenopus laevis; Amphibia), Chinese alligator (Alli-
gator sinensis; reptile), chicken (Gallus gallus domesticus; Aves),
and human (Mammalia). We found out some IFN-Is with the
strong positive charges (calculated charge $+11.0 [pH 7]) in
Pisces, Amphibia, reptiles, and Aves, except for Mammalia
(Fig. 6A). Although two cationic IFN-Is, IFN-b and IFN-k, exist
in human, the positive charges are evidently low (calculated
charge +4.3 [pH 7]). We further analyzed the amino acid com-
positions in these IFN-Is between NK/(NK + NR) (the ratio of the
number of lysines/total number of lysines and arginines) and av-
erage peptide hydrophobicity based on the Eisenberg consensus
scale of 2237 reported cationic antimicrobial peptides in database
(34, 35), which are similar to those in cationic antimicrobial pep-
tides (Fig. 6B). Moreover, we evaluated the hydrophobicities in
these IFN-Is, which fall into the range of the hydrophobicity in
the antimicrobial peptides (Fig. 6C). Altogether, representative
cationic IFN-Is from different vertebrate taxonomy share similar
amino compositions with those in nature APD3.
Extensively and forcefully bactericidal IFN-I members also
exist in Amphibia, reptiles, and Aves
To determine the antimicrobial activity of these IFN-Is, we
employed African clawed frog IFN2.3 (afIFN2.3; calculated
charge +11.0 [pH 7]), Chinese alligator IFN-a2 (caIFN-a2; cal-
culated charge +19.2 [pH 7]), and chicken IFN-b (chIFN-b; cal-
culated charge +15.1 [pH 7]) as representatives. We expressed and
obtained the intact IFN-I proteins as above gcIFNw1. All the three
representative IFN-Is display strong microbicidal activity against
typical pathogens, not only Gram-positive bacteria (S. aureus
and S. agalactiae) but also Gram-negative bacteria (E. coli,
P. aeruginosa, and A. hydrophila) (Fig. 7A–C, Table I). In ad-
dition, the bactericidal mechanism of these IFN-Is was explored,
which, in all of them, induces the depolarization of the bacterial
membrane (Fig. 7D). These data indicate that forceful bacteri-
cidal IFN-Is widely exist in jawed vertebrates except Mammalia.
The Journal of Immunology 1345

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FIGURE 8. The functional schematic diagram of antimicrobial peptide–like IFN-Is. Some IFN-Is possess antimicrobial peptide–like features in addition
to the well-known antiviral activity. In the context of bacterial infections, the nonmammalian antimicrobial peptide–like IFN-Is efficiently promote mi-
crobial killing by targeting the bacterial membrane. In contrast, the mammalian antimicrobial peptide–like IFN-Is lose their original antimicrobial activity,
becoming “pure cytokines” during bacterial infections.

Antimicrobial peptide–like IFN-Is are the efficient effector
molecules to combat bacterial infections in nonmammalian jawed
vertebrates (Fig. 8).
Discussion
IFN-Is can be produced by almost every cell type, including
leukocytes, fibroblasts, and endothelial cells (21), which are in-
duced by viruses and trigger strong antiviral responses in jawed
vertebrates (6, 40). Although IFN-Is are mainly induced by viral
pathogens, IFN-Is could also be induced by most, if not all,
bacterial pathogens (41). What is confusing is that these bacteria-
induced IFN-Is seemed unable to contribute to a prominent pro-
tective role in bacterial infections in mammals (13, 22, 42). All the
IFN-Is are not expressed by the same cell types in the same way.
In 2009, scientists found that zebrafish IFNw1 protects host
against S. iniae infection (Gram-positive bacteria) (17). Do piscine
IFN-Is kill bacteria directly or through signaling pathway? Can
piscine IFN-Is kill Gram-negative bacteria as well as Gram-
positive bacteria in vitro and in vivo? How is the antimicrobial
spectrum? Is the bactericidal ability strong or weak? What is the
1346
mechanism? In the current study, we demonstrated that gcIFNw1,
an ortholog of the zebrafish IFNw1 (Supplemental Fig. 1), is a
strong cationic and amphiphilic a-helical cytokine that is different
from the other grass carp IFN-Is (Fig. 1). gcIFNw1 exerts forceful,
wide-spectrum, direct, bactericidal activity to not only Gram-
positive bacteria but also Gram-negative bacteria in vitro and
in vivo (Figs. 2, 5, Table I). Human chemokine CXCL9 is a cy-
tokine with potent antibacterial activity that could kill 100%
Citrobacter rodentium at ∼4 mg/ml (43). This concentration is
biologically relevant, as CXCL9 levels in rectal perfusions from
the inflamed human intestine can reach up to 2 mg/ml (138 nM)
(44). Our data demonstrated that recombinant gcIFNw1 resulted
in 100% killing for E. coli at 8 mg/ml (500 nM) (Fig. 2C), which
meant that gcIFNw1 possessed comparable bactericidal activ-
ity with CXCL9. Furthermore, recombinant gcIFNw1 reduced
tissue bacterial loads to 100-fold at a concentration of 10 ng/fish
(∼40 ng/g body weight), and survival rate improved 60% (Fig. 5).
Natural IL-26 has an antimicrobial potency 80-fold to 120-fold
greater than that of rIL-26 (45). Considering that IL-26 and
gcIFNw1 share high similarity in structural features (6 a-helical
elements and potential distribution), we deduced that natural
gcIFNw1 may possess more powerful bactericidal activity in vivo.
Like many cationic and amphiphilic antimicrobial peptides or
proteins, gcIFNw1 could accumulate on and interact with the
negative LPS or PGN in bacterial cell wall (Fig. 3, Table II)
and then insert into the bacterial membrane, which results in
the depolarization and leakage of bacterial contents (Fig. 4,
Supplemental Figs. 2, 3). Together, our findings indicate that
piscine IFNw1 is a broad-spectrum potent bactericidal a-helical
cytokine.
In 2017, scientists found that human and mouse IFN-bs kill
S. aureus (Gram-positive bacteria), not E. coli (Gram-negative
bacteria), in vitro, just under acidic pH for human IFN-b (24).
How does the bactericidal activity degenerate from gcIFNw1 to
human or mouse IFN-b? Natural antimicrobial peptides and other
antimicrobial cytokines, such as IL-26, CXCL14, and LL-37,
share the cationic and amphipathic features (25, 32, 45, 46).
Antimicrobial activity of IFN-Is also closely relates such struc-
tural characterizations (Figs. 1, 6). We calculated the net charges
of all the IFN-I members in human and found that IFN-b and
IFN-k have the highest positive charges (+4.3); however, IFN-Is
with the net charges $+11.0 exist in all the taxonomic classes in
the nonmammalian jawed vertebrates (Fig. 6A). Previous studies
found that mouse and human IFN-bs can only kill S. aureus, not
E. coli, in vitro (24), which is in accord with the low positive
charges (Fig. 6A). Meanwhile, caIFN-a2 exhibits the strongest
bactericidal activity in the representative IFN-Is (Fig. 7, Table I),
which is also consistent with the highest positive charges
(+19.2). The amino acid compositions (Fig. 6B, 6C) accord with
“saddle-splay curvature” model based on geometry of membrane
destabilization (34). The bactericidal activity of antimicrobial
peptide–like IFN-Is is confirmed by the experiments in vitro and
in vivo (Figs. 2C, 2D, 5, 7A–C). They bind bacterial LPS and
PGN (negative charges) (Fig. 3A, Table II) and cause membrane
depolarization (Figs. 4A, 7D, Supplemental Figs. 2A, 3A), dis-
ruption, and cytoplasm leakage (Fig. 4B, 4C, Supplemental
Figs. 2B, 3B), which affects metabolisms and survival. We
speculated that the potent antimicrobial activity of IFN-I mem-
bers is beneficial in nonmammalian vertebrates. Host infections
often aggravate at the late stage of viral infections accompanied
by the bacterial infections. When the infections become serious,
a large amount of IFN-Is are released for viral and bacterial
clearance. IFN-Is induced by the bacterial infections may be
divided into two categories: antimicrobial peptide–like IFN-Is
DIRECT BACTERICIDAL ACTIVITY OF IFN
and common IFN-Is (Fig. 8). During bacterial infections, both
category IFN-Is may be released at the early stage to initiate
necessary cell-mediated immune responses (47). But at the late
stage, high concentrations of antimicrobial peptide–like IFN-Is
kill pathogenic bacteria.
In general, viruses, bacteria, and fungi can and will develop
resistance to any conceivable substance. However, antimicrobial
peptides target a previously underappreciated “microbial Achilles
heel,” a feature of the microbial cellular membrane that distin-
guishes broad species of microbes from multicellular plants and
animals (25). The innate immune system largely depends on an-
timicrobial peptides, providing the first line of defense against
invading microbes (25, 48, 49). These powerful antimicrobial
peptide–like IFN-Is play crucial roles in the host defense against
bacterial infections in nonmammalian jawed vertebrates, which
marks an important role of IFN-Is in antibacterial infections in
immune system evolution.
In summary, the present study gives insight into the direct robust
bactericidal function of piscine IFNw1, which is different from the
indirect antiviral function through signaling pathway. IFNw1 di-
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rectly kills bacteria by cytoderm interaction, membrane depolar-
ization, and disruption mechanisms. Antimicrobial peptide–like
IFN-Is belong to natural antimicrobial peptides with broad-
spectrum powerful direct antimicrobial activity and may also ex-
ist in nonmammalian jawed vertebrates. The results will deepen
our understanding of the antimicrobial function and distinguish
various roles in bacterial infections between mammals and non-
mammals for IFN-Is.
Acknowledgments
We thank Dr. Yupeng Zhao (Shanghai Jiao Tong University) for scanning
electron microscopy; Dr. Chenchen Qu for AFM; and Prof. Zhen Xu,
Dr. Xiaoling Liu, Dr. Gailing Yuan, Dr. Xujie Zhang, and Dr. Jiagang
Tu for helpful discussions and precious advice. We also thank Dr. Quanyuan
Wan, Dr. Youliang Rao, Xueying Shang, Rui Jiang, and Jianfei Ji for assis-
tance in experiments.
Disclosures
The authors have no financial conflicts of interest.
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