https://orcid.org/0000-0002-5805-7208
Figures
Abstract
Type I interferons (IFNs) are indispensable antiviral cytokines in nonspecific immunity, yet they play dual roles in bacterial infections in mammals. Recent studies have revealed a subset of strongly cationic type I IFNs possessing potent antimicrobial properties across nonmammalian vertebrates. In this study, we identified a type I IFN gene, CaIFNi, from Cromileptes altivelis that is characterized by a unique triple-disulfide bond architecture. In Vibrio harveyi-challenged models, overexpression of CaIFNi potentiated bacterial clearance capacity in tissues, whereas its knockdown exacerbated bacterial colonization, highlighting its ability to protect the host against bacterial infection in vivo. In vitro assays further confirmed that CaIFNi directly binds to and kills both gram-negative (G-) and gram-positive (G+) bacteria, which first revealed the antibacterial function of new subgroup IFNi within teleost type I IFNs. Furthermore, the α-helical peptide CaIFNi-18 derived from CaIFNi was identified as a novel antimicrobial peptide (AMP) that has broad-spectrum antibacterial efficacy against G- and G+ bacteria and membrane-targeting ability. Further mechanistic studies revealed that CaIFNi has bactericidal effects on both G- and G+ bacteria through membrane depolarization and disruption, alteration of the bacterial ultrastructure, and in vitro binding to genomic DNA. In addition, CaIFNi-18 also has significant in vivo therapeutic efficacy against bacterial infection, highlighting its great potential as an antibacterial agent. Encouragingly, the loss of antibacterial activity in the truncation mutant (rCaIFNiΔ148-165) lacking the CaIFNi-18 segment suggests that this region is essential for the bactericidal function of the full-length protein and likely acts as its core domain. Further computational simulations revealed that the deletion of the CaIFNi-18 region attenuated the interaction between the protein and the bacterial membrane. These findings not only expand the functional scope of type I IFNs beyond their canonical antiviral role but also identify their derivative CaIFNi-18 as both a promising antimicrobial candidate and the essential bactericidal domain of CaIFNi, thereby offering novel therapeutic strategies against bacterial infections in the aquaculture industry and beyond.
Author summary
Interferons are classically recognized as key immune cytokines that defend hosts against viral infections. However, the pathogen-rich aquatic environment has driven teleost fish to evolve uniquely versatile immune defenses. In this study, we characterized a specific type I interferon (CaIFNi) from the humpback grouper (Cromileptes altivelis) and uncovered its surprising ability to directly kill both Gram-negative and Gram-positive bacteria. We demonstrate that this noncanonical antibacterial function is governed by a small, positively charged segment located at the protein’s C-terminus, designated CaIFNi-18. This short peptide functions as a potent antimicrobial agent by directly targeting and disrupting bacterial membranes. Notably, deleting this segment completely abolishes the full-length interferon’s capacity to bind to and kill bacteria. Our findings reveal that a classic antiviral molecule harbors a critical domain that mediates both direct bactericidal activity and robust therapeutic protection against bacterial infections, which uncovers a new dimension of innate immunity in teleosts and highlights its potential for anti-infective drug design.
Citation: Zhang H, Wu Y, Zheng X, Wang Z, Zhang C, Cao Z, et al. (2026) Noncanonical bactericidal activity of teleost type I interferon is conferred by a membrane-targeting C-terminal peptide. PLoS Pathog 22(7): e1014419. https://doi.org/10.1371/journal.ppat.1014419
Editor: Nuno M. S. dos Santos, Universidade do Porto Instituto de Investigacao e Inovacao em Saude, PORTUGAL
Received: December 15, 2025; Accepted: June 25, 2026; Published: July 28, 2026
Copyright: © 2026 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data generated and analyzed in this study are included in the manuscript and Supporting information.
Funding: This work was supported by the National Natural Science Foundation of China (Grant No. U22A20534 to Y.S.), the Hainan Provincial Natural Science Foundation of China (Grant No. 326JCQN0967 to Y.S.), and the Innovational Fund for Scientific and Technological Personnel of Hainan Province (Grant No. KJRC2023B22 to Y.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Interferons (IFNs), classified among class II alpha-helical cytokines, serve as indispensable components of both adaptive and innate immune responses [1,2]. IFNs are categorized into four types: IFN-I, IFN-II, IFN-III, and IFN-IV [3]. In teleosts, type I, II, and IV IFNs have been identified [4]. Type I IFNs are highly diversified across teleosts and can be divided into two distinct groups: Group I, which are characterized by two cysteines and are further subdivided into Groups a, d, e, and h; and Group II, which contain four or six cysteines and are subdivided into Groups b, c, f, and i [5]. In contrast to the ubiquitous presence of Group I IFNs in all teleosts, Group II IFNs exhibit a more constrained distribution and are found only in specific lineages, such as cyprinids, salmonids and perciformes [5,6].
While type I IFNs in mammals are known primarily for their central role in antiviral defense, their functional repertoire in teleosts has undergone remarkable expansion, encompassing not only antiviral immunity but also direct antibacterial activity. As a case in point, IFNd in large yellow croaker can elicit an antiviral response by upregulating ISG expression and suppressing the replication of giant salamander iridovirus in the GS and LYCK cell lines [7]. Similarly, the recombinant protein On-IFNc efficiently reduces the ISKNV viral load in MFF-1 cells [8]. Beyond their canonical antiviral functions, accumulating evidence has demonstrated that teleost type I IFNs also exert considerable direct antibacterial effects. Akin to antimicrobial peptides (AMPs), the cationic recombinant protein rgcIFNφ1 from grass carp not only possesses broad-spectrum antimicrobial activity against both gram-negative (G-) and gram-positive (G+) bacteria in vitro but also confers potent protection in zebrafish against A. hydrophila infection in vivo [9]. Furthermore, a recent report revealed that in Chinese sturgeon, the Group II IFN protein rAsIFNf, which has a positive charge, also displays wide-spectrum bactericidal properties in vitro [10]. Thus, type I IFNs in teleosts function as direct bactericidal agents because of their strong positive charge—an AMP-like trait.
AMPs are small molecular polypeptides (typically 12–50 amino acids) that are ubiquitously found across natural organisms and act as key effector molecules of innate immunity [11]. Unlike conventional antibiotics that target specific biosynthetic pathways, their cationic and amphipathic nature facilitates interactions with anionic phospholipids of the bacterial membrane and in turn triggers bacterial membrane disruption, which endows them with broad-spectrum antimicrobial activity [12]. Furthermore, AMPs exert antimicrobial effects through diverse modes of action, such as inhibiting biofilm formation, blocking nucleic acid replication, tuning immune responses and impairing core metabolic functions in microbes [11,13]. These multifaceted mechanisms are conducive to alleviating resistance development in bacteria and make them promising candidates for addressing antibacterial infections [14].
Humpback grouper (Cromileptes altivelis), a high-value mariculture species in the South China Sea, sustains diverse aquaculture stressors, culminating in disease outbreaks and substantial economic losses in the aquaculture industry [15]. In particular, bacterial infections pose a severe threat to humpback grouper aquaculture, among which V. harveyi emerges as a highly virulent pathogen. To combat these infections, antibiotic use constitutes the prevailing strategy, yet its indiscriminate application has exacerbated bacterial resistance and environmental contamination [16]. Therefore, the identification and development of ecologically sustainable antimicrobial agents is critical. IFNi, a new subgroup of type I IFNs first identified in Larimichthys crocea, was previously assigned to the IFNc subtypes of group II IFNs in light of phylogenetic analysis and four highly conserved cysteines [17]. Nevertheless, a recent study revealed that compared with IFNc, IFNi possesses two additional cysteine residues, leading to the formation of three disulfide bonds, which distinguishes it from other type I IFNs [5]. Phylogenetically, IFNi forms a distinct evolutionary clade within teleost group II IFNs. To date, functional characterization of IFNi has been limited primarily to L. crocea, where its expression is robustly induced by Aeromonas hydrophila and poly I:C, resulting in potent antiviral activity in vitro [17]. However, its antimicrobial activity and modes of action in bacterial infections are still poorly defined.
In the present research, we identified a Group II type I IFN (CaIFNi) in C. altivelis and revealed for the first time its direct antibacterial activity both in vitro and in vivo. Furthermore, the C-terminal-derived cationic peptide CaIFNi-18 was identified as a novel, membrane-targeting AMP with broad-spectrum antibacterial activity and high biocompatibility. More importantly, by employing a truncation mutant (rCaIFNiΔ148-165) coupled with computational simulations, we demonstrated that the CaIFNi-18 peptide region is essential for the antibacterial activity and membrane interaction of the full-length protein, as its deletion abrogated bioactivity. Therefore, our work not only reveals a noncanonical antibacterial function for teleost type I IFNs but also precisely identifies its structural basis for functionality, highlighting CaIFNi-18 as a promising therapeutic candidate and paving the way for novel antimicrobial strategies in aquaculture.
Results
Identification and bioinformatics analysis of CaIFNi
The CaIFNi gene, comprising a 567 bp ORF, encodes a 188-amino acid precursor with a putative signal peptide of 22 residues. As presented in Fig 1A and 1C, the mature peptide of CaIFNi had a prototypical type I IFN tertiary structure characterized by six α-helices and contained six highly conserved cysteine residues that formed three pairs of disulfide bridges. To further elucidate the evolutionary relationship of CaIFNi, a phylogenetic analysis based on multiple type I IFN subgroups in fish was performed, and we found that CaIFNi clustered with LcIFNi, ArIFNc, ScIFNc, SmIFN1, PoIFN3 and SpIFNc (Fig 1B), forming a novel clade designated previously as the IFNi group [5]. Computational prediction by ExPASy revealed that CaIFNi possessed a molecular weight of 18.78 kDa and an isoelectric point (pI) of 6.78. Notably, electrostatic surface analysis revealed a localized accumulation of positive charge within the C-terminal region of CaIFNi, in comparison with a mild negative overall net charge of − 0.3 (Fig 1D).
(A) Multiple sequence alignment of the mature peptide sequence of CaIFNi and fish homologs using the ESPript 3.0 server. Six conserved cysteine residues (C1–C6), which are essential for the formation of intramolecular disulfide bonds, are annotated. (B) Phylogenetic analysis of CaIFNi and other teleost type I IFN amino acid sequences constructed by the neighbor-joining method using MEGA 9.0 software. The GenBank accession numbers of the amino acid sequences used for phylogenetic tree construction are provided in Table S2. (C) The 3D structure of CaIFNi predicted by AlphaFold3. Disulfide bonds are highlighted in yellow. (D) Electrostatic surface potential mapping of CaIFNi visualized via PyMOL. The hydrophobic, negatively charged and positively charged regions are colored white, red and blue, respectively.
Bacterial infection rapidly induces CaIFNi expression
To determine the tissue distribution pattern of CaIFNi, we carried out a qRT‒PCR analysis and revealed that it was ubiquitously expressed in all the collected tissues, with the highest mRNA levels detected in blood and the lowest in the head kidney (Fig 2A). Following V. harveyi stimulation, the expression of CaIFNi increased significantly in the major immune tissues, peaking at 12 hpi in the liver (Fig 2B) and spleen (Fig 2C), whereas the maximum expression occurred in the head kidney at 24 hpi (Fig 2D). These results indicate that bacterial infection triggers rapid and robust transcriptional activation of CaIFNi in immune-related tissues.
(A) Tissue distribution of CaIFNi among humpback grouper. (B-D) Expression profiles of CaIFNi in the liver (B), spleen (C) and head kidney (D) of humpback grouper at 6, 12, 24, and 48 h post infection. The housekeeping gene RPL13 was used as the internal control. Data are shown as the mean ± SD from one representative experiment (n = 3 biological replicates). Each replicate (n) represents a composite sample consisting of pooled tissues from five individual fish. (B–D) Statistical significance relative to the uninfected control at the corresponding time point was evaluated using an unpaired Student’s t test. *p < 0.05; **p < 0.01.
CaIFNi protects C. altivelis from V. harveyi infection
To explore its resistance to bacterial infection, CaIFNi was overexpressed in groupers via intramuscular injection with pCaIFNi plasmids (Fig 3A). The successful overexpression of CaIFNi at 5 dpi was confirmed at both the mRNA and protein levels in the spleen and head kidney via qRT‒PCR (Fig 3B) and Western blot analysis (Fig 3D), respectively. Subsequently, quantification of the bacterial burden in tissues revealed that the degree of bacterial colonization in the spleen and head kidney was significantly attenuated in the pCaIFNi group compared with the control group at 9 and 12 hpi (Fig 3E). Notably, compared with those in the control group, the bacterial loads in the head kidney in the pCaIFNi group were suppressed as early as 6 hpi, whereas those in the spleen were not significantly different (Fig 3E).
(A) Schematic diagram of the in vivo experimental design. (B-C) The transcript level of CaIFNi in the immune tissues of groupers after injection with overexpression plasmids at 120 hpi (B) or siRNA at 12 hpi (C). RPL13 was employed as the internal control. (D) Western blot analysis of CaIFNi protein expression in the spleens and head kidneys of groupers injected with overexpression plasmids at 120 hpi using an anti-His antibody. β-actin was used as the control. (E–F) The groupers were injected i.p. with 5 × 104 CFU of V. harveyi per fish. The bacterial burden in the spleen and head kidney was quantified at 6, 9, and 12 h post challenge under conditions of CaIFNi overexpression (E) and knockdown (F). All the data are presented as the mean ± SD from one representative experiment (n = 3 biological replicates for B-C; n = 5 biological replicates for E-F). Statistical significance was evaluated using an unpaired Student’s t test for (B-C) and the Mann‒Whitney U test for (E-F). *p < 0.05; **p < 0.01.
siRNA technology was used to interfere with the expression of CaIFNi to further elucidate its role in antibacterial infection (Fig 3A). Similarly, qRT‒PCR analysis revealed a significant reduction in the mRNA expression of CaIFNi among the examined tissues at 12 h after siRNA administration (Fig 3C). As illustrated in Fig 3F, following V. harveyi challenge, the bacterial colonies in the SiCaIFNi-administration group were significantly greater in the spleen at 6, 9, and 12 hpi and in the head kidney at 6 and 9 hpi than those in the control group. These results suggest that CaIFNi plays a pivotal role in resistance to bacterial infection in vivo.
CaIFNi binds to diverse bacteria and kills both gram-negative and gram-positive bacteria in vitro
In addition to its anti-infection functions in vivo, we wondered whether CaIFNi exhibits direct antibacterial activity in vitro. To this end, the recombinant protein rCaIFNi, containing Trx and His tags, was expressed and purified to explore its functions in vitro. In light of the results of the SDS‒PAGE analysis presented in Fig 4A, the protein’s molecular weight (38.76 kDa) aligned with the predicted value. ELISA was performed to first investigate the binding activity of rCaIFNi to various bacterial pathogens. As shown in Fig 4B, rCaIFNi bound to both G- bacteria (V. harveyi and V. parahaemolyticus) and G+ bacteria (S. agalactiae and S. iniae). A plate counting assay was subsequently conducted to further confirm its antibacterial activity in vitro. The results revealed that rCaIFNi possessed significant antibacterial efficacy against V. harveyi (45.25%), S. agalactiae (53.57%) and S. iniae (39.34%), in contrast to the marginal bactericidal effects on V. parahaemolyticus (78.03%) (Fig 4C). These results reveal that rCaIFNi binds to bacteria and has direct antimicrobial potency in vitro.
(A) Analysis of recombinant CaIFNi protein purified from E. coli by SDS‒PAGE. Lanes 1 and 2 represent the purified rTrx and rCaIFNi proteins, respectively. (B) ELISA of rCaIFNi binding to various aquatic bacterial pathogens. (C) Antibacterial effect of rCaIFNi against bacterial pathogens determined via plate counting. PBS and rTrx treatments served as the control and tag-matched control, respectively. All the data are presented as the mean ± SD from one representative experiment (n = 3 independent samples). Statistical significance was evaluated using two-way ANOVA for (B) and one-way ANOVA for (C). Significant differences (p < 0.05) are denoted by different letters. For visual clarity in the ELISA curves (B), significance is marked exclusively at the highest concentration.
CaIFNi-18 is a novel cationic α-helical peptide localized in the C-terminal region of CaIFNi
Considering the aforementioned electrostatic surface of CaIFNi (Fig 1D), we speculated that a cationic domain localized to its C-terminal region is likely responsible for the antibacterial activity of CaIFNi. To validate this, the C-terminal short peptide CaIFNi-18 was screened via AMPA and CAMPR4 servers, both of which predicted significant antibacterial potential, and then synthesized for follow-up assays. Additionally, our predictions revealed potential protease cleavage sites at positions 147 (Fig 5A), suggesting that the functional motif represented by CaIFNi-18 may be naturally released in vivo. The helical wheel projection (Fig 5B) and electrostatic surface distribution (Fig 5D) revealed a cationic but nonamphiphilic feature in CaIFNi-18, whereas the tertiary structure modeled by I-TASSER indicated that CaIFNi-18 contained random coil regions and α-helix structures (Fig 5C). Furthermore, CD spectroscopy was utilized to further measure the secondary structures of CaIFNi-18. As displayed in Fig 5E, CaIFNi-18 predominantly adopted the random coil conformation in PBS, whereas its helix content increased markedly both in 30 mM SDS (simulating anionic prokaryotic membrane) and 50% TFE (modeling hydrophobic membrane), suggesting that CaIFNi-18 might undergo a coil-to-helix transition upon interaction with the bacterial membrane.
(A) Predicted protease cleavage sites of CaIFNi-18 generated by ExPASy PeptideCutter (B) Helical wheel projection of CaIFNi-18 generated by Heliquest. (C) The 3D structure of CaIFNi-18 predicted by I-TASSER. PyMOL was utilized for visualization, resulting in a yellow, hydrophobic and blue, hydrophilic surface. (D) Electrostatic surface potential mapping of CaIFNi-18 visualized via PyMOL. The hydrophobic, negatively charged and positively charged regions are colored white, red and blue, respectively. (E) Analysis of the CaIFNi-18 secondary structure by CD spectroscopy.
CaIFNi-18 interacts with bacterial membranes by targeting LPS or LTA
Natural AMPs or antimicrobial cytokines, such as IL-26 and gcCXCL20a, are typically cationic and amphipathic, enabling electrostatic attraction to negatively charged bacterial membranes [18,19]. Most of them consistently appear to exert their functions starting from membrane interactions. Hence, we first employed all-atom MD simulations to characterize the binding of CaIFNi-18 to G- (E. coli) and G+ (S. aureus) bacterial membrane systems. As clearly presented in Fig 6A and 6B, CaIFNi-18 was found to associate with the membranes of E. coli and S. aureus, followed by partial embedding into the membrane systems. However, during the simulation processes, the α-helical conformation of CaIFNi-18 destabilized and transitioned to a random coil, which aligned with the significant RMSD fluctuations of the peptide (Fig 6C). Consistently low Rg fluctuations manifested conformational compactness within the peptide (Fig 6D). Critically, decreasing COM distances (Fig 6E) coupled with increasing numbers of hydrogen bonds (Fig 6F) also signified peptide‒membrane complex formation. To further validate the theoretical predictions, we performed ITC to investigate the interactions between CaIFNi-18 and bacterial membrane components (LPS and LTA). Similarly, the results revealed robust interactions between CaIFNi-18 and LPS (Kd = 137 nM) or LTA (Kd = 461 nM) (Fig 6G–6H). Taken together, these results reveal that CaIFNi-18 is capable of binding to both G- and G+ bacterial membranes and targeting LPS and LTA.
(A-B) MD simulation snapshots of the interaction between CaIFNi-18 and the G- outer membrane (A) or the G+ membrane (B) at 0, 50, and 100 ns. LPS is shown on the top leaflet of the G- outer membrane, whereas the phosphorus atoms of lipid A are shown as tan spheres. (C-D) Variation in RMSD (C) and Rg (D) values for CaIFNi-18 during MD simulations. (E) Distance between the COM of CaIFNi-18 and the bacterial membrane during MD simulations. (F) Hydrogen bonds of peptide‒membrane interaction systems during MD simulations. (G–H) ITC assays for determining the binding of CaIFNi-18 to LPS (G) or LTA (H). The right and left plots display the integrated heat measurements and corrected titration data, respectively.
CaIFNi-18 demonstrates potent broad-spectrum antimicrobial activity and excellent biocompatibility in vitro
The antibacterial efficacy of CaIFNi-18 was initially determined by a disc diffusion assay. As clearly shown in Fig 7A, discs impregnated with CaIFNi-18 exhibited distinct inhibition zones against all eight bacterial strains, in stark contrast with the negative control, in which no inhibitory zone was observed, revealing its wide antimicrobial spectrum. To further assess its antibacterial potency, the MIC and MBC values for CaIFNi-18 against the tested bacterial strains were measured. As summarized in Fig 7B, CaIFNi-18 demonstrated potent antibacterial activity against both G- and G+ bacteria, with MIC values ranging from 9.2 to 74.0 μg/mL and MBC values ranging from 18.5 to 148.0 μg/mL. Notably, the growth of two G- (V. harveyi and V. parahaemolyticus) and two G+ bacteria (S. agalactiae and S. iniae) was markedly suppressed under sub-MIC concentrations of CaIFNi-18 (Fig 7C).
(A) Assessment of CaIFNi-18 inhibitory zones against eight bacterial pathogens. Paper disks a, b, and c correspond to treatments with 1 mg/mL CaIFNi-18, PBS buffer and antibiotics, respectively. (B) Determination of the MIC and MBC values of CaIFNi-18 against a spectrum of bacterial pathogens. (C) Inhibition curves of CaIFNi-18 at MIC-fold concentrations across eight bacterial strains. (D–E) Cytotoxicity of CaIFNi-18 in CAK and RAW 264.7 cells, as determined by a CCK-8 assay. (F) Hemolytic activity of CaIFNi-18 toward MRBCs. (C–F) All the data are presented as the mean ± SD from one representative experiment (n = 3 independent samples). The statistical significance of the inhibition curves in (C) was evaluated using two-way ANOVA. Significant differences (p < 0.05) are denoted by different letters. For visual clarity in (C), significance is marked exclusively at the final time points.
Next, we evaluated the cytotoxicity of CaIFNi-18 toward RAW 264.7 and CAK cells. As shown in Fig 7D, the derived peptide CaIFNi-18 had a negligible effect on the viability of mammalian cells. Even at concentrations as high as 128 μM, the viability remained 86.25% in CAK cells and 92.00% in RAW 264.7 cells. Similarly, at a concentration of 128 μM, CaIFNi-18 exhibited slight hemolytic activity (3.58%) toward MRBCs (Fig 7E). In summary, CaIFNi-18 possesses potent broad-spectrum antibacterial efficacy and exceptional biocompatibility in vitro.
CaIFNi-18 exerts bactericidal activity through inducing membrane depolarization and disrupting membrane integrity, accompanied by an in vitro DNA-binding capacity
To elucidate the antibacterial mechanisms of CaIFNi-18, we first employed the voltage-sensitive probe DiSC3-5 to assess its ability to elicit bacterial membrane depolarization. As shown in Fig 8A, compared with the control, the addition of CaIFNi-18 to bacterial cultures in 96-well plates triggered a dose-dependent increase in fluorescence intensity, indicating its ability to induce bacterial membrane depolarization. A PI uptake assay was subsequently performed to determine the effects of CaIFNi-18 on the membrane integrity of these pathogens. As shown in Fig 8B, CaIFNi-18 caused substantial disruption of bacterial membrane integrity, facilitating the penetration of PI dye into the cytoplasm, and the effects on membrane permeabilization were concentration dependent. In addition, the effect of CaIFNi-18 on membrane integrity was verified by SEM. The control bacteria maintained an intact cellular architecture with smooth surfaces, whereas those treated with CaIFNi-18 displayed visible ultrastructural alterations, such as pore formation, shrinkage with cytoplasmic extrusion and cellular lysis (Fig 8C). Afterward, we investigated whether CaIFNi-18 was able to interact with bacterial genomic DNA after entry into the cytoplasm. The results revealed a concentration-dependent binding ability of CaIFNi-18 to both G- and G+ bacterial DNA in vitro (Fig 8D). These findings suggest that CaIFNi-18 exerts its antibacterial function primarily via alterations in membrane potential, integrity and ultrastructure coupled with in vitro DNA binding.
(A) Detection of bacterial membrane depolarization induced by CaIFNi-18 via DiSC3-5 staining. (B) Changes in membrane permeability induced by CaIFNi-18 through flow cytometric analysis. The percentage of PI-positive cells is shown in the upper right corner. (C) Alteration of bacterial morphology after treatment with 5 × MIC CaIFNi-18 for 1 h via SEM observation. The scale bar represents 1 μm. (D) Binding activity of CaIFNi-18 to bacterial genomic DNA. The bacterial DNA was mixed with different concentrations of peptide at 37 °C for 30 min. Lane 1: 32 μM BSA; Lanes 2–8: 32, 16, 8, 4, 2, 1, 0.5 μM CaIFNi-18. Data are presented as the mean ± SD from one representative experiment (n = 3 independent samples). Statistical significance for (A) was evaluated using two-way ANOVA. Significant differences (p < 0.05) are denoted by different letters. For visual clarity in (A), significance is marked exclusively at the final time points.
CaIFNi-18 has efficient therapeutic effects on bacterial infection in vivo
To evaluate the therapeutic efficacy of CaIFNi-18 in vivo, groupers were challenged via i.p. injection with 100 μL of V. harveyi, followed by the administration of CaIFNi-18 at 1 hpi (Fig 9A). Initially, we measured the bacterial loads in the liver, spleen and head kidney at 6 h (Fig 9B), 9 (Fig 9C) and 12 hpi (Fig 9D). Strikingly, compared with those in the immune tissues of the control groupers, the bacterial loads in the immune tissues of the groupers treated with CaIFNi-18 were much lower. Moreover, we monitored the survival rates over one week and observed that compared with the control treatment, CaIFNi-18 effectively increased the survival rate by 40% (Fig 9E). Together, these results demonstrate that CaIFNi-18 has potent in vivo therapeutic efficacy against V. harveyi infection.
(A) Schematic diagram of the in vivo experimental design. (B–D) The groupers were injected i.p. with 5 × 104 CFU of V. harveyi per fish and subsequently injected with CaIFNi-18 at 1 hpi. The bacterial burden in the immune tissues was quantified at 6 (B), 9 (C), and 12 h (D) post challenge. (E) Therapeutic efficacy of CaIFNi-18 on survival rates among groupers following bacterial challenge. (B–D) Data are presented as the mean ± SD from one representative experiment (n = 5 biological replicates). (E) Thirty fish were used per treatment group (n = 30). Statistical significance was evaluated using the Mann‒Whitney U test for (B–D) and the log-rank (Mantel‒Cox) test for (E). *p < 0.05; **p < 0.01; ns: not significant.
CaIFNi-18 is a critical domain for the antibacterial bioactivity of CaIFNi
To further investigate whether the antimicrobial activity of the CaIFNi-18 peptide is functionally relevant within the full-length CaIFNi protein, a truncation mutant devoid of this segment, designated CaIFNiΔ148-165, was successfully constructed, expressed, and purified (Fig 10A). As shown in Fig 10B, an ELISA revealed that compared with wild-type rCaIFNi, rCaIFNiΔ148-165 almost completely lost the ability to bind to bacterial pathogens. Similarly, in contrast with rCaIFNi, the truncation abolished the antibacterial efficacy of rCaIFNiΔ148-165 against V. harveyi, V. parahaemolyticus, S. agalactiae, and S. iniae (Fig 10C). Furthermore, we explored the antibacterial activity of rCaIFNi and rCaIFNiΔ148-165 in vivo using a grouper V. harveyi infection model (Fig 10D). In line with the in vitro results, compared with the control or rCaIFNiΔ148-165 treatments, rCaIFNi significantly potentiated bacterial clearance in tissues, whereas no significant difference in bacterial load was observed between the rCaIFNiΔ148-165 group and the control group (Fig 10E). Collectively, these results suggest that the CaIFNi-18 region is critical for the antibacterial effects of CaIFNi, suggesting its potential role as the functional core of the full-length protein.
(A) Analysis of recombinant CaIFNi△148-165 protein purification from E. coli by SDS‒PAGE. Lanes 1 to 3 represent the purified rTrx, rCaIFNi and rCaIFNi△148-165 proteins, respectively. (B) ELISA of rCaIFNi△148-165 binding to various aquatic bacterial pathogens. (C) Antibacterial effect of rCaIFNi△148-165 against bacterial pathogens determined by plate counting. (D) Schematic diagram of the in vivo experimental design. (E) The groupers were injected i.p. with 5 × 104 CFU of V. harveyi per fish and subsequently injected with recombinant protein (rTrx, rCaIFNi or rCaIFNi△148-165) or PBS at 1 hpi. The bacterial burden in the immune tissues was quantified at 6, 9, and 12 h post challenge. PBS and rTrx treatments served as the control and tag-matched control, respectively. All the data are presented as the mean ± SD from one representative experiment (n = 3 independent samples for B-C; n = 5 biological replicates for E). Statistical significance was evaluated using two-way ANOVA for (B), one-way ANOVA for (C) and the Mann‒Whitney U test for (E). Significant differences (p < 0.05) are denoted by different letters in (B–C). For visual clarity in (B), significance is marked exclusively at the highest concentration. *p < 0.05; **p < 0.01; ns: not significant.
Deletion of the CaIFNi-18 segment impairs the interaction of CaIFNi with bacterial membranes
To further determine the structural basis for the membrane binding capacity of CaIFNi and the functional significance of the CaIFNi-18 region, all-atom MD simulations were conducted using full-length CaIFNi and the truncation mutant CaIFNiΔ148-165. As illustrated in Fig 11A and 11B, full-length CaIFNi successfully approached both G- and G+ bacterial membranes, with its C-terminal region clearly embedding into the membrane system. Conversely, CaIFNiΔ148-165 exhibited a markedly reduced interaction with the membrane and failed to establish a stable insertion throughout the 100 ns simulation. Notably, this impairment was evidenced by distinctly fewer hydrogen bonds (Fig 11C) and a consistently greater COM distance from the bacterial membranes for CaIFNiΔ148-165 than for the full-length protein (Fig 11D). Collectively, our computational results demonstrate that CaIFNi-18 is the critical region mediating the binding and anchoring of CaIFNi to bacterial membranes.
(A-B) MD simulation snapshots of the interaction between CaIFNi or CaIFNi△148-165 and the G- outer membrane (A) or G+ membrane (B) at 0, 50, and 100 ns. Within the full-length CaIFNi, the CaIFNi-18 region is colored yellow. LPS is shown on the top leaflet of the G- outer membrane, whereas the phosphorus atoms of lipid A are shown as tan spheres. (C) Hydrogen bonds of protein‒membrane interaction systems during MD simulations. (D) Distance between the COM of CaIFNi or CaIFNi△148-165 and the bacterial membrane.
Discussion
As central mediators of innate immunity, type I IFNs rapidly induce the expression of hundreds of interferon-stimulated genes, resulting in cell-intrinsic antiviral states [4,20,21]. This antiviral function of type I IFNs is highly conserved across all vertebrates [22,23]. During bacterial infections, however, type I IFNs play disparate roles in host immunity, which hinges upon many elements, such as the site of the infection, virulence factors, and replication mechanisms [24]. Notably, mammalian (human and mouse) IFN-βs have been reported to possess limited bactericidal capacity in acidic environments, whereas strongly cationic type I IFNs in other jawed vertebrates exhibit broad-spectrum antimicrobial potency [9,25]. Among teleosts, both Group I and Group II IFNs exhibit classical antiviral functions. For instance, IFNc, IFNd and IFNh from Lateolabrax japonicus have potent antiviral activities against RGNNV [26]. Similarly, IFNi from L. crocea acts as an antiviral cytokine against SGIV, whereas IFNb from Mylopharyngodon piceus has strong antiviral activity against GCRV and SVCV [17,27]. In contrast to their extensive antiviral properties, only grass carp gcIFNφ1 (gcIFNa) and Chinese sturgeon AsIFNf have broad-spectrum antibacterial functions in vitro [9,10]. The IFNi subgroup, a novel member of the complex type I IFN system in teleosts, has the unprecedented characteristic of three conserved disulfide bonds, with antiviral roles reported only in L. crocea thus far [5]. Nevertheless, its antibacterial functions remain unexplored. In this study, for the first time, we identified an interferon gene (CaIFNi) from C. altivelis, which is a Group II type I IFN, and revealed its antimicrobial activity both in vitro and in vivo. Importantly, a cationic peptide derived from CaIFNi, designated CaIFNi-18, was identified and synthesized. We further examined its interaction with the bacterial membrane, antimicrobial activity, biosafety, and antibacterial mechanisms and ultimately evaluated its therapeutic efficacy. In addition, through the construction of the truncation mutant rCaIFNiΔ148-165, we further explored the functional link between the CaIFNi-18 peptide sequence and the full-length CaIFNi protein.
Phylogenetic analysis revealed that CaIFNi belongs to Group II IFNs, which cluster with LcIFNi, ArIFNc, ScIFNc, SmIFN1, PoIFN3 and SpIFNc to form a distinct clade designated the IFNi group. Notably, structural modeling indicated that CaIFNi possesses three unique pairs of disulfide bonds, distinguishing it from canonical Group I and Group II IFNs, which contain one and two pairs, respectively. The distinct structural architecture of CaIFNi aligns with that recently described for the IFNi of L. crocea [5]. Furthermore, following bacterial challenge, CaIFNi expression increased significantly in the immune tissues (liver, head kidney and spleen) of C. altivelis, indicating its crucial role in antibacterial immunity. Although primarily triggered by viruses, type I IFNs can also be elicited by the majority of bacteria [28]. In rock bream, after being stimulated with LPS or E. tarda, the transcript levels of two IFNd genes were significantly upregulated in the head kidney and blood [29]. Pereiro et al. reported that the expression of turbot IFN2, a Group I IFN, was markedly elevated in the head kidney following exposure to A. salmonicida [30]. To further validate its role in antibacterial infection, the overexpression and knockdown of CaIFNi were carried out prior to bacterial challenge. Our results revealed that CaIFNi overexpression markedly decreased bacterial loads within immune tissues, whereas CaIFNi silencing led to a substantial increase, underscoring its essential role in antibacterial infection in vivo. Similarly, IFNφ1 from zebrafish and IFN1 from grass carp provide robust protection against S. iniae and A. hydrophila infections in zebrafish, respectively [9,31]. In addition to their anti-infection activity in vivo, some type I IFNs with strong positive charges across jawed vertebrates have direct antibacterial functions in vitro [9,10,25]. Intriguingly, in the current study, while CaIFNi has a net charge of − 0.3, its recombinant protein rCaIFNi could still bind to bacteria and exhibit potent antimicrobial effects on both G- and G+ bacteria. A universal mechanism among antimicrobial cytokines or peptides is their initial electrostatic attraction to lipid layers, facilitated by their characteristic high positive charges [32,33]. Therefore, on the basis of the analysis of the electrostatic surface, we speculated that the antibacterial activity of CaIFNi was principally attributed to its positively charged C-terminal domain.
On this basis, we synthesized CaIFNi-18, a short peptide representing the C-terminal functional motif predicted to be released naturally by proteolysis and have potent antimicrobial activity. Here, we observed that CaIFNi-18 was an α-helical cationic peptide according to the results of structural modeling and CD spectroscopy, in line with the characteristics of AMPs. As the most abundant and well-studied class of antimicrobial peptides, α-helical AMPs rely on their α-helix conformation to mediate their lipid interactions with the bacterial membrane and subsequent permeation [34]. Given that the primary target of AMPs is the bacterial membrane, we first utilized MD simulations to evaluate the interaction between CaIFNi-18 and bacterial membranes. As a versatile computational approach, MD simulations enable in-depth exploration of peptide‒membrane interactions, revealing mechanistic insights into antimicrobial peptide functionality [35]. During the simulation processes, CaIFNi-18 progressively approached both the G+ and G- bacterial membrane systems, followed by insertion into them, which was consistent with a steady increase in the number of hydrogen bonds and a gradual reduction in the COM distance. Interestingly, despite retaining >30% helical content in CD spectroscopy, the α-helix of CaIFNi-18 underwent unfolding and transitioned to a random coil conformation during the initial simulation phase. This discrepancy suggests differential conformational stability under experimental versus simulated conditions—a phenomenon similarly observed in the lead peptide sC184b [36]. Furthermore, we performed ITC assays to clarify the interaction between CaIFNi-18 and bacterial membrane components. LPS, a core structural component of G- bacterial outer membranes, functions as a primary biophysical barrier against antimicrobial agents and represents a strategic target for the development of antimicrobial compounds [37]. LTA, a membrane-anchored amphiphilic polymer ubiquitous in G+ bacteria, facilitates the adherence of cationic AMPs to bacterial surfaces with anionic phosphate moieties [38]. In this study, ITC analysis revealed high-affinity binding between CaIFNi-18 and LPS (Kd = 137 nM) or LTA (Kd = 461 nM). This observation aligns with previous findings that gcIFN-20 (derived from the fifth helical region of gcIFN1) strongly interacts with LPS (Kd = 13 nM) [39], whereas intestinalin (P30), a derivative from the LysC N-terminal region, can directly bind to LTA through hydrophobic interactions [40]. Collectively, these findings indicate that CaIFNi-18 may exert its potential antimicrobial function by targeting LTA in G+ bacteria or LPS in G- bacteria and in turn interacting with bacterial membranes.
To date, only a limited subset of type I IFN derivatives in fish, such as grass carp gcIFN-20, zebrafish AMP-Z1, AMP-Z2 and Chinese sturgeon AsIFNf-α4, have been experimentally confirmed to exhibit wide-spectrum antibacterial functions in vitro [6,9,25,39]. Similarly, as a novel AMP, CaIFNi-18 also demonstrated potent broad-spectrum antimicrobial activity against diverse G- and G+ bacterial strains. Nevertheless, the clinical translation of AMPs remains hindered by their potential cytotoxicity [41]; as exemplified by arenicins, Hecate-βCG and melittin, these peptides inflict significant cytotoxicity on host cells despite their extraordinary antimicrobial efficacy [42–44]. Thus, we evaluated the biosafety of CaIFNi-18 and reported that CaIFNi-18 exhibited negligible cytotoxicity and low hemolytic activity at the tested concentrations, suggesting its extraordinary biocompatibility.
Cationic AMPs employ diverse modes of action against bacterial pathogens, principally by exerting bactericidal effects via membrane disruption following initial electrostatic adsorption [45]. Given this, we initially assessed its effects on the bacterial cell membrane to further elucidate the antibacterial mechanisms of CaIFNi-18 in vitro. The results revealed that CaIFNi-18 provoked bacterial membrane depolarization and altered membrane permeabilization and cellular ultrastructure in both G- and G+ bacteria, which is consistent with the effects of certain AMPs, such as Larimicin78–102 [14] and Sp-LECin [46]. In addition to membrane disruption, some AMPs penetrate bacterial membranes via transient pore formation or receptor-mediated transport, subsequently binding to intracellular targets such as nucleic acids and metabolic enzymes to induce bactericidal effects [47,48]. In line with this, we also observed that CaIFNi-18 can bind to bacterial DNA in vitro. Therefore, we concluded that CaIFNi-18 may exert its antibacterial effects principally by disrupting bacterial membrane integrity and binding to bacterial DNA. As a typical cationic α-helical AMP, CaIFNi-18 resembles classical AMPs such as LL-37 and magainins in that it binds electrostatically to negatively charged bacterial membranes to facilitate insertion and disruption [49]. Nevertheless, unlike certain potent AMPs, such as arenicins and melittin, whose application is often hindered by severe host cell cytotoxicity, CaIFNi-18 is highly specific for membrane targeting, achieving broad-spectrum efficacy and excellent biocompatibility [50].
Despite their potent bactericidal activity in vitro, most AMPs fail to achieve the desired therapeutic efficacy in vivo owing to their physiological complexity [51]. Here, we further evaluated the therapeutic efficacy of CaIFNi-18 against V. harveyi infection in vivo. V. harveyi, a G- marine pathogen, has recently been identified as the primary causative agent of vibriosis outbreaks in grouper aquaculture [52,53]. In this study, injection with CaIFNi-18 significantly reduced bacterial loads in grouper immune tissues and increased survival rates by 40% compared with those in the control group after one week, indicating its great potential as a therapeutic candidate for antibacterial applications. Although its efficacy in vitro was relatively modest compared with that of potent AMPs, the strong in vivo protection provided by CaIFNi-18 strongly implies that CaIFNi has additional immunomodulatory effects that are similar to those of other IFN-derived peptides, such as LPS neutralization [39]. Consistent with our results, CXCL20a and hepcidin have been shown to confer therapeutic efficacy in fish against A. hydrophila infection [54,55].
Although the CaIFNi-18 peptide demonstrates exceptional in vivo and in vitro antimicrobial activity and biosafety, whether it truly represents the critical functional region of the full-length CaIFNi protein remains unclear. Therefore, the truncation mutant rCaIFNiΔ148-165, which lacks the CaIFNi-18 segment, was constructed. Encouragingly, the deletion of this peptide domain completely abrogated the protein’s ability to bind to and kill bacteria in vitro as well as its therapeutic efficacy against bacterial infection in vivo, indicating that the CaIFNi-18 region is likely a critical functional domain conferring direct bactericidal properties to CaIFNi. Furthermore, MD simulations revealed that the deletion of the CaIFNi-18 segment attenuated the interaction between the protein and bacterial membranes, as evidenced by decreased hydrogen bonding and increased COM distance. These in silico findings strongly corroborate our experimental results, indicating that CaIFNi-18 is critical for membrane interaction. Similarly, in Chinese sturgeon, while the type I IFN AsIFNf protein and its derivative AsIFNf-α4 peptide possessed antimicrobial activity and membrane-disrupting capacity, deletion of the α4 domain abolished these functions in the AsIFNf-Δα4 protein [10].
In summary, we identified and characterized a Group II type I IFN gene, CaIFNi, in C. altivelis with a distinctive triple-disulfide bond architecture. Through in vivo and in vitro assays, we first revealed the antibacterial potency of the new subgroup of IFNi from fish type I IFNs. Moreover, a novel α-helical cationic AMP, CaIFNi-18, derived from the C-terminal domain of CaIFNi, was identified and found to have membrane-targeting ability, broad-spectrum antibacterial activity against both G- and G+ bacteria in vitro and extraordinary biocompatibility. Further mechanistic investigations revealed that the peptides first target LPS or LTA of the bacterial membrane and then interact with and disrupt them, potentially followed by entry into the cytoplasm and binding to genomic DNA. Notably, CaIFNi-18 has potent therapeutic efficacy against V. harveyi infection in vivo. More importantly, using a truncation mutant (rCaIFNiΔ148-165), we found that CaIFNi-18 constituted the critical bactericidal domain of the full-length protein, as its deletion not only abolished both antibacterial activity in vitro and therapeutic efficacy in vivo but also critically impaired membrane-targeting ability, as revealed by MD simulations. This study demonstrates for the first time the direct antibacterial activity of teleost IFNi and defines its derived peptide, CaIFNi-18, as a pivotal region mediating this function, elucidating the antimicrobial activity and mechanism of the peptide. These findings expand the functional repertoire of type I IFNs and their derivatives and identify highly promising candidate molecules for antimicrobial drug development in aquaculture and beyond.
Materials and methods
Ethics statement
All the healthy C. altivelis used in this study were purchased from a mariculture farm situated in Wenchang city (Hainan, China). Prior to experimentation, the groupers were maintained in a recirculating sea water system for at least one week for acclimation. To exclude potential interference from preexisting bacterial infections, five fish were randomly selected for aseptic tissue sampling followed by spread-plate culture analysis as previously described [56]. All the experiments involving animals were in strict compliance with the protocols approved by the animal research ethics committee of Hainan University (HNUAUCC-2024-00140).
Pathogenic bacteria and cell lines
Edwardsiella tarda (ETA1), V. alginolyticus (HN08155), V. harveyi (QT520), and Streptococcus agalactiae (LFY-5) were isolated from diseased marine fish in Hainan Province and preserved in our laboratory. S. iniae (29177), V. parahaemolyticus (ATCC17802), Escherichia coli (ATCC8739) and Staphylococcus aureus (ATCC6538) were purchased from the China General Microbiological Culture Collection Center. Among them, V. harveyi is resistant to ampicillin, while E. tarda and S. agalactiae are kanamycin resistant. The optimized conditions for culturing diverse pathogenic bacteria are as follows: S. agalactiae and S. iniae in BHI medium at 30 °C; V. alginolyticus, V. harveyi, V. parahaemolyticus and E. tarda in LB medium at 30 °C; and E. coli and S. aureus in LB medium at 37 °C. All the bacteria were cultivated to the mid-exponential phase prior to the initiation of formal experiments.
This research utilized two types of cell lines: mouse macrophages (RAW 264.7) and C. altivelis head kidney cells (CAK). RAW 264.7 cells, maintained in our laboratory, were cultivated in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2. CAK cells, previously established by our laboratory [57], were grown in L-15 medium (Gibco, USA) supplemented with 15% FBS at 26 °C.
Molecular cloning and identification of CaIFNi
The open reading frame (ORF) for the IFNi gene from C. altivelis, named CaIFNi, was obtained from our transcriptome library and amplified from cDNA of the spleen tissues with the specific primers CaIFNi-F and CaIFNi-R (S1 Table). The purified DNA products were subsequently ligated into the pEASY-T1 vector (TransGen, China) and subsequently transformed into Escherichia coli DH5α competent cells, among which positive clones were screened for sequencing.
The prediction for the three-dimensional (3D) structure model of CaIFNi was conducted via AlphaFold3. PyMOL (version 2.6, Schrödinger, LLC) was used to visualize the net charge distribution and the predicted 3D structure. The ExPASy server was used to assess the physical and chemical parameters of the CaIFNi protein. Additionally, multiple sequence alignment between CaIFNi and other homologs was carried out by using the ESPript 3.0 server with a CaIFNi 3D structure file. Phylogenetic tree analysis was performed with the neighbor-joining algorithm in MEGA 9.0 software.
Expression analysis of CaIFNi in humpback grouper under normal conditions and after V. harveyi stimulation
To analyze the tissue distribution of CaIFNi, a variety of tissues (blood, liver, skin, muscle, intestine, heart, gill, spleen, brain, stomach and head kidney) were dissected from 15 pathogen-free humpback groupers, with five identical tissues pooled into one composite sample. The samples were subsequently stored in RNAstore reagent (Tiangen, China) for subsequent RNA extraction and cDNA synthesis. The mRNA expression level of CaIFNi was measured by quantitative reverse transcription–PCR (qRT‒PCR) using the housekeeping gene RPL13 as an internal reference [58]. The qRT‒PCR primer sequences are listed in S1 Table, and the relative expression level was analyzed via the 2-ΔΔCt method.
To investigate the modulation of CaIFNi expression following V. harveyi infection, C. altivelis was divided into two groups, with each group containing 15 individuals. In the control group, C. altivelis received an intraperitoneal (i.p.) injection of 100 μL of PBS, whereas the challenged group was injected i.p. with an equal volume of V. harveyi suspension (2 × 106 CFU/mL). Immune tissues (spleen, liver and head kidney) were collected at 6, 12, 24, and 48 h postinjection (hpi), preserved in RNAstore reagent and homogenized as composite samples (five identical tissues per sample).
Effects of CaIFNi overexpression and knockdown on bacterial resistance in vivo
To overexpress CaIFNi in vivo, the coding sequence of CaIFNi was cloned and inserted into the EcoR V site of the pCN3 vector, yielding the recombinant plasmid pCaIFNi. Extraction of the endotoxin-free plasmid pCaIFNi was performed with an EndoFree Plasmid Kit (Tiangen, China). C. altivelis with an average weight of 14.6 ± 1.8 g were separated into three groups, and each group (n = 20) received intramuscular injections of 100 μL of PBS (set as the control group), pCN3 (200 μg/mL) or pCaIFNi (200 μg/mL). The expression levels of CaIFNi in the head kidney and spleen were measured at 5 d post-injection (dpi) via qRT‒PCR. Moreover, all the groups were injected i.p. with 100 μL V. harveyi suspension (5 × 105 CFU/mL), followed by head kidney and spleen collection at 6, 9 and 12 hpi for the quantification of bacterial loads.
To achieve in vivo knockdown of CaIFNi, the T7 RiboMAX Express RNAi System (Promega, USA) was adopted to synthesize the targeting siRNA, designated SiCaIFNi, with the specific primers listed in S1 Table. Healthy C. altivelis (20 individuals per group) were intramuscularly administered 100 μL of PBS (control) or siCaIFNi/siCaIFNi-C (200 μg/mL). Afterward, five fish were euthanized to assess the knockdown efficiency via qRT‒PCR at 12 hpi, and concurrently, the remaining fish were challenged with 100 μL of a V. harveyi suspension (5 × 105 CFU/mL). The head kidney and spleen were dissected to determine the bacterial burden at 6, 9 and 12 hpi.
Western blot analysis
The tissue samples were lysed in RIPA buffer (Beyotime, China) supplemented with protease inhibitors (Beyotime, China). The lysates were centrifuged at 14,000 rpm for 15 min at 4 °C, and the supernatants were mixed with loading buffer and heated. The proteins were separated by 12% SDS‒PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) using an eBlot semidry transfer system (GenScript, USA). The membrane was blocked with 5% skim milk for 1 h, washed with PBST, and incubated with primary antibodies for 1 h. The primary antibodies used were mouse anti-His (bsm-33004 M; Bioss) and mouse anti-β-actin (HC201-01; TransGen). Following three PBST washes, the membrane was incubated with the secondary antibody, HRP-goat anti-mouse IgG (bs-0296G-HRP, Bioss), for 1 h. Finally, protein levels were detected using Seven Super ECL Prime substrate (Seven, China).
Expression and preparation of the recombinant protein CaIFNi (rCaIFNi) and its truncation mutant rCaIFNi△148-165
The sequence encoding the mature peptide of CaIFNi (excluding the signal peptide) was ligated into the prokaryotic expression plasmid pET-32a at the EcoR V restriction site, resulting in the expression of a recombinant protein with a 21 kDa fusion partner (rTrx) that mainly consists of a Trx-His dual-tag [59]. Afterward, the recombinant plasmid was transformed into E. coli BL21 (DE3) cells, and protein expression was induced using 0.5 mM IPTG at 20 °C for 20 h, after which the truncation mutant rCaIFNi△148-165 was induced using 0.5 mM IPTG at 16 °C for 36 h. The induced proteins were detected by 12% SDS‒PAGE, while purification and concentration quantification were in accordance with our previously established methods [60].
Binding ability of rCaIFNi and its mutant to bacteria
The binding ability of rCaIFNi and its mutant to bacteria was assessed on the basis of a modified ELISA method from a previous study [60]. Briefly, 96-well plates were coated with 100 μL of bacterial suspension (1 × 108 CFU/mL) at 4 °C for 12 h. Then, the plates were rinsed three times with TBST and blocked with 5% BSA solution for 1 h. After being washed with TBST, 100 μL of rCaIFNi, rCaIFNi△148-165 or rTrx (tag-matched control) at various concentrations was added to the plates and incubated for 3 h at room temperature, with PBS serving as a blank control. The plates were subsequently washed with TBST three times and then incubated with anti-His tag antibody dilutions (1:2000, Bioss) for 1 h. After being washed with TBST, the plates were incubated with HRP-conjugated goat anti-mouse IgG antibody (1:5000, Bioss) for 1 h at room temperature. After the final washes, TMB substrate was added to each well, and the reactions were terminated with 2 M H₂SO₄. The absorbance of each well at 450 nm was detected via a microplate reader. This assay was performed in three independent experiments.
Antimicrobial activity of rCaIFNi and its mutant in vitro
In brief, V. harveyi, V. parahaemolyticus, S. agalactiae and S. iniae were allowed to grow to mid-exponential phase, after which they were harvested and diluted to a concentration of 1 × 104 CFU/mL in PBS. Subsequently, 100 μL of 50 μg/mL rTrx (tag-matched control), rCaIFNi, rCaIFNi△148-165 or PBS (control) was cocultured with 100 µL of diluted bacterial suspension at 30 °C for 3 h. Next, the mixture was spread onto LB or BHI solid plates, followed by incubation at 30 °C for 16 h for colony counting. This assay was performed in three independent experiments.
Peptide prediction, synthesis and structure modeling
The active antimicrobial region of CaIFNi was predicted using AMPA (https://tcoffee.crg.eu/apps/ampa/do) and CAMPR4 (https://camp.bicnirrh.res.in/) servers, while its potential for natural proteolytic release was evaluated with the ExPASy PeptideCutter tool. On the basis of these predictions, CaIFNi-18 (KFALQKHYHTCFTWRHHA) with an amidated C-terminus was synthesized by the solid-phase method in GL Biochem (Shanghai, China), with its purity exceeding 95% as guaranteed by high-performance liquid chromatography, and its quality was confirmed by mass spectrometry. The 3D structure of CaIFNi-18 was predicted using I-TASSER and visualized via PyMOL.
CD spectroscopy
CaIFNi-18 was dissolved in 50% (v/v) TFE, 30 mM SDS or 10 mM PBS to obtain a final concentration of 100 μM. Three types of peptide solutions were individually transferred to a quartz cuvette (10 mm path length). CD spectral analyses were performed on a J-1500 spectropolarimeter (Jasco, Japan) to monitor the peptide solutions in the range of 190–250 nm. The mean residual ellipticity (θ) was calculated from the acquired CD data.
Molecular dynamics simulations
The CHARMM-GUI server was employed to generate lipid bilayer systems, where CaIFNi-18, full-length CaIFNi, or the deletion mutant CaIFNi△148-165 was initially positioned 2 nm above the membrane interface [61]. All-atom systems have been constructed to represent peptides or proteins that interact with G- bacterial outer membranes or G+ bacterial membranes [62,63]. To achieve charge neutralization, 150 mM NaCl was added to the systems, after which the calcium ions stabilized the LPS within the outer leaflet of the G- bacterial membranes. The system parameters are documented in S3 Table. Adopting the CHARMM36 force field and TIP3P water model in GROMACS 2021.2, all-atom MD simulations were executed for 100 ns with a 2 fs time step [64,65]. The CHARMM-GUI standard NVT/NPT protocol with a temperature of 310 K was utilized for the preceding equilibration. Molecular visualization was performed using Visual Molecular Dynamics (VMD) v1.9.4. To evaluate the conformational dynamics of CaIFNi-18, CaIFNi and CaIFNi△148-165 in membrane systems, the root mean square deviation (RMSD), radius of gyration (Rg), center-of-mass (COM) distance and number of hydrogen bonds were analyzed from the resulting trajectories.
Isothermal titration calorimetry (ITC)
ITC assays were performed using a MicroCal PEAQ-ITC instrument (Malvern, USA) at 25 °C. LPS (Solarbio, China), LTA (Sigma, USA) and CaIFNi-18 were dissolved in 10 mM HEPES buffer (pH 7.0). In a typical titration, 13 injections of 500 μM CaIFNi-18 were added to the reaction cell containing either 25 μM LPS or 50 μM LTA at 180 s intervals, with a stirring speed of 750 rpm. The heat of CaIFNi-18 dilution in buffer was subtracted from the raw data prior to integration in MicroCal Origin 5.0 software, after which the corrected data were analyzed using one-site fitting models.
Disc diffusion assay
To preliminarily determine the antibacterial activity of CaIFNi-18, a disc diffusion assay was conducted with reference to a previous method [48]. In brief, the bacterial cells were harvested, diluted in PBS to a concentration of 1 × 107 CFU/mL and subsequently spread onto agar plates. After that, blank paper disks were placed onto the plates, after which 20 μL of PBS (negative control), 1 mg/mL ampicillin/kanamycin (positive control) or 1 mg/mL CaIFNi-18 was added to the discs dropwise, after which the plates were incubated for 16 h at either 37 °C or 30 °C. Finally, zones of inhibition were documented utilizing a GenoSens 2100 gel imaging system (Clinx, China).
MIC and MBC assays
The MIC and MBC of CaIFNi-18 were evaluated in accordance with a twofold dilution method [66]. Briefly, bacterial suspensions were prepared in the respective liquid culture media (LB broth for gram-negative strains and BHI broth for gram-positive strains) at a concentration of 2 × 106 CFU/mL. One hundred μL of bacterial suspension was mixed with equal volumes of CaIFNi-18 serial dilutions in 96-well plates, resulting in final concentrations ranging from 1 to 128 μM, whereas PBS and ampicillin/kanamycin served as the negative and positive controls, respectively. After incubation for 16 h, the absorbance of each well at 600 nm was measured using a microplate reader. The MIC was defined as the minimum concentration of CaIFNi-18 at which bacterial growth was completely suppressed. To determine the MBC of CaIFNi-18, 100 μL of the mixture was spread onto agar plates, followed by incubation for 16 h. The MBC was defined as the lowest concentration at which no colonies formed on the plates. This assay was performed in three independent experiments.
Inhibition curve assay
Various concentrations (100 μL) of the peptide solutions at the MIC were cultured with equal volumes of bacterial suspension (2 × 106 CFU/mL) in 96-well plates. Afterward, the absorbance of each well at 600 nm was detected every hour by a microplate reader for 9 consecutive hours. This assay was performed in three independent experiments.
Cytotoxicity and hemolytic activity assays
The cytotoxicity of CaIFNi-18 was evaluated using cell lines (RAW 264.7 and CAK) with reference to a previous method with some modifications [67]. The cells were inoculated into 96-well plates (1 × 104 cells/well) and cultivated overnight. Subsequently, 10 μL of serial dilutions of CaIFNi-18 were added to the cells to achieve a final concentration of 1–128 μM, followed by coculture for 24 h. Then, 10 μL of CCK-8 solution (Biosharp, China) was added to each well. After incubation for 2 h, the absorbance at 450 nm was measured by a microplate reader. The medium alone and containing cells functioned as the blank and positive control, respectively. The cell viability was calculated as (ODtreatment − ODblank)/(ODcontrol − ODblank) × 100%. This assay was performed in three independent experiments.
Mouse red blood cells (MRBCs) were utilized to assess the hemolytic activity of CaIFNi-18 as previously described [68]. Briefly, MRBC were isolated, harvested by centrifugation at 800 × g for 10 min, washed three times with 0.9% NaCl solution, and subsequently prepared as a 4% (v/v) suspension in 0.9% NaCl solution. Afterward, 100 μL MRBC suspension was mixed with 100 μL serial dilutions of CaIFNi-18 (the final concentration ranged from 1 to 128 μM) and incubated at 37 °C for 1 h. After centrifugation, the supernatant was collected, and the absorbance was measured at 540 nm. 0.9% NaCl and 1% Triton X-100 with MRBC served as the negative and positive controls, respectively. The hemolysis rate = (ODtreatment – ODnegative)/(ODpositive − ODnegative) × 100%. This assay was performed in three independent experiments.
Membrane depolarization assay
3,3’-Dipropylthiadicarbocyanine iodide (DiSC3-5), a cationic dye, was used to detect bacterial membrane depolarization levels, and the assay was conducted as previously described [48]. Briefly, bacteria were washed three times with HEPES buffer (5 mM HEPES, 20 mM glucose), and a bacterial suspension (OD600 = 0.05) was prepared either in buffer A for G- bacteria (5 mM HEPES, 20 mM glucose, 100 mM KCl and 2 mM EDTA) or in buffer B (5 mM HEPES, 20 mM glucose and 100 mM KCl) for G+ bacteria. Afterward, a 100 μL aliquot of the bacterial suspension was transferred to a black 96-well plate, and 1 μM DiSC3-5 was added. After incubation for 90 min, the fluorescence intensity (excitation λ = 622 nm; emission λ = 670 nm) of each well was measured by a microplate reader for 10 min. Subsequently, 50 μL of the MIC-fold CaIFNi-18 solution was added to the mixture, and the fluorescence intensity was monitored at one-minute intervals for 30 min, while deionized water functioned as a negative control. This assay was performed in three independent experiments.
Propidium iodide (PI) uptake assay
The effects of CaIFNi-18 on bacterial membrane permeability were estimated via a PI uptake analysis performed with flow cytometry. In brief, the bacterial suspension was adjusted to a concentration of 1 × 108 CFU/mL in PBS, followed by incubation with MIC-fold CaIFNi-18 for 1 h. Afterward, PI was added to the mixture at a final concentration of 10 μg/mL and incubated at room temperature for 30 min. Finally, the samples were analyzed by flow cytometry (Beckman, USA), and the data were processed with FlowJo software (v.10.9).
Scanning electron microscopy (SEM)
To investigate the morphological effects of CaIFNi-18 on bacterial cells, a modified SEM protocol was adopted [69]. A bacterial suspension (1 × 10⁸ CFU/mL) in 0.9% NaCl solution was treated with 5 × MIC CaIFNi-18 or 0.9% NaCl (control) for 1 h. Following incubation, the cells were rinsed three times with 0.9% NaCl and then fixed in 2.5% glutaraldehyde at 4 °C overnight. After fixation, the cells were pelleted, washed and subsequently dehydrated in a graded ethanol series (30%, 50%, 70%, 90%, and 100% absolute ethanol) for 15 min each. The dehydrated samples were lyophilized in a vacuum freeze dryer overnight, coated with gold and then observed by SEM (Verios G4 UC, Thermo Scientific, USA).
DNA binding assay
This assay was performed to evaluate the DNA binding ability of CaIFNi-18 to bacterial genomic DNA according to a previous method [70]. Briefly, bacterial genomic DNA was extracted using a TIANamp Bacteria DNA Kit (Tiangen, China). Afterward, the purified bacterial DNA (100 ng) was treated with CaIFNi-18 at concentrations ranging from 0.5 to 32 μM, while 32 μM BSA-treated DNA was used as a control. After a 30-minute incubation at 37 °C, the samples were analyzed by 1.2% agarose gel electrophoresis and visualized using a gel documentation system.
Therapeutic efficacy of CaIFNi-18 against V. harveyi infection in vivo
To determine whether CaIFNi-18 had therapeutic effects on V. harveyi, a grouper model of V. harveyi infection was established by administering 100 μL of bacterial suspension at a concentration of 5 × 105 CFU/mL. At 1 hpi, C. altivelis (12.7 ± 3.0 g) were randomly assigned to one of two groups (n = 50). One group was injected i.p. with 100 μL of PBS, whereas the other group received an injection of 100 μL of CaIFNi-18 (100 μg/mL). Afterward, the liver, spleen and head kidney were collected at 6, 9, and 12 hpi to quantify the bacterial load, and the mortality rate of the fish was monitored daily for one week.
Antibacterial activity of recombinant CaIFNi and its mutant against V. harveyi in vivo
To assess the antibacterial activity of the recombinant protein rCaIFNi and its truncation mutant in vivo, groupers (16.9 ± 2.7 g) were challenged via i.p. injection with 5 × 104 CFU of V. harveyi. At 1 hpi, the fish were randomly assigned to four groups (n = 20) and administered a 100 μL injection of PBS (control) or the respective recombinant proteins (rTrx as a tag-matched control, rCaIFNi, or rCaIFNi△148-165) at a concentration of 100 μg/mL. Tissue samples from the spleen and head kidney were collected at 6, 9, and 12 hpi to measure bacterial loads.
Statistical analysis
Statistical analyses were performed using GraphPad v.8.0 software. The data are presented as the means ± SDs. The significance of differences between two independent groups was determined by an unpaired Student’s t test. For multiple comparisons, one-way analysis of variance (ANOVA) was used for the plate counting assays, whereas two-way ANOVA followed by Tukey’s multiple comparisons test was used to evaluate the ELISA, inhibition curve, and membrane depolarization assay results. Survival rates were evaluated by the log-rank (Mante-Cox) test. Quantification of bacterial loads was performed via a nonparametric Mann‒Whitney U test. The significant differences are presented as *p < 0.05 and **p < 0.01.
Supporting information
S2 Table. Amino acid sequence of type I IFNs in teleosts involved in analysis of phylogenetic tree.
https://doi.org/10.1371/journal.ppat.1014419.s002
(DOCX)
S3 Table. Parameters for constructing membrane-peptide and membrane-protein systems in molecular dynamics.
https://doi.org/10.1371/journal.ppat.1014419.s003
(DOCX)
S1 Text. Rationale for the selection of bacterial surface components.
https://doi.org/10.1371/journal.ppat.1014419.s004
(DOCX)
S1 Raw Images. Raw images of Figs 3D, 4A, 8D and 10A.
https://doi.org/10.1371/journal.ppat.1014419.s005
(PDF)
Acknowledgments
We are grateful to all the study participants and to the staff whose dedicated efforts were essential to the collection and processing of the samples.
References
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