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Granulin peptide GRN-41 of Mozambique tilapia is a novel antimicrobial peptide against Vibrio species
Biochemical and Biophysical Research Communications 515 (2019) 706e711
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Granulin peptide GRN-41 of Mozambique tilapia is a novel antimicrobial peptide against Vibrio species Sheng-Han Wu a, Hsin-Yiu Chou a, b, Ping-Chung Liu a, Jen-Leih Wu c, Hong-Yi Gong a, b, * a
Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung, 20224, Taiwan Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan c Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, 11529, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 May 2019 Accepted 4 June 2019 Available online 8 June 2019
In our previous study, the novel GRN-41 peptide generated from alternative splicing of the Mozambique tilapia PGRN1 gene was identified to be a potent peptide that protected against V. vulnificus in the transgenic zebrafish model by modulating innate immune-related genes. In this study, the anti-bacterial activities of synthetic Mozambique tilapia GRN-41 peptide (OmGRN-41) against various bacterial pathogens were investigated. The results showed that OmGRN-41 had bactericidal activity against Vibrio species, including V. vulnificus, V. alginolyticus, and V. harveyi, but exhibited bacteriostatic activity against V. parahaemolyticus. OmGRN-41 maintained bactericidal activity (64 mM) against V. vulnificus at pH 2 to pH 10 or after heat treatment for 1 h at high temperatures between 40 C and 100 C. TEM observations revealed that the outer membrane of V. vulnificus was disrupted by OmGRN-41, leading to morphological rupture and loss of cytoplasmic contents. Additionally, little hemolytic activity against tilapia and sheep erythrocytes was detected after treatment with 128 mM OmGRN-41. OmGRN-41 can effectively enhance the survival of Nile tilapia infected by V. vulnificus. Our results suggest that the OmGRN-41 is a novel antimicrobial peptide possessing bactericidal activity, especially against Vibrio species. These results indicate that OmGRN-41 can be applied in human Vibriosis treatment and has the potential to defend against Vibrio spp. infection in critical aquaculture organisms. © 2019 Elsevier Inc. All rights reserved.
Keywords: Tilapia Granulin Bactericidal Bacteriostatic Vibrio Antimicrobial peptide
1. Introduction Aquaculture is a rapidly developing and crucial industry because of the insufficient food supply and deficiency of global fishery resources [1]. However, intensive farming has led to disease outbreaks and excessive use of antibiotics, which causes environmental pollution and the emergence of antibiotic-resistant strains [2]. Hence, the development of alternative antimicrobial agents is becoming very important [3]. In recent years, many natural products and antibacterial peptides (AMPs) from marine organisms have been identified and applied in the development of new drugs [4,5]. AMPs, which are composed of less than 100 amino acids and are positively charged, have broad-spectrum antimicrobial activity and regulate innate immunity [6].
* Corresponding author. Division of Life Science, Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, No.2, Beining Rd., Jhongjheng District, Keelung City, 20224, Taiwan. E-mail address: [email protected] (H.-Y. Gong). https://doi.org/10.1016/j.bbrc.2019.06.022 0006-291X/© 2019 Elsevier Inc. All rights reserved.
In 1990, Bateman et al. unexpectedly identified another family of proteins called granulins (GRNs) when purifying cysteine-rich defensin family AMPs from human leukocytes [7]. GRN is a 6-kDa peptide that contains 12 conserved cysteine residues that form 6 disulfide bonds [7]. The GRN unit is derived from progranulin (PGRN) by proteolytic cleavage [8]. PGRN is a multifunctional growth factor that regulates cell division, survival, and migration and is involved in tumorigenesis, tissue repair, embryogenesis, and inflammation [9]. There is only one PGRN gene that encodes PGRN, which is composed of 7.5 GRN units in the mammalian genome [9]. PGRN and GRN had been found to play the opposite roles in the regulation of inflammation [8,10,11]. Furthermore, GRN can serve as a cofactor for toll-like receptor 9 signaling and is involved in innate immunity to produce TNF-a in response to microbial DNA [12]. However, unlike in mammals, there are multiple PGRN genes in the teleost genome. They can be divided into long-form PGRN composed of at least 6 GRN units and short-form PGRNs composed of 1.5e3 GRN units [13,14]. In our previous study, we found that the short-form progranulin PGRN1 gene of Mozambique tilapia expressed two transcripts, PGRN1 encoding a 206-a.a. PGRN with
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two GRN units, GRN-A and GRN-B, and the alternatively spliced transcript GRN-41 encoding a secreted GRN peptide composed of a 20-a.a. ER signal peptide and 41-a.a. mature GRN peptide named GRN-41 [15]. PGRN1 and GRN-41 not only are abundantly expressed in immune-related organs but also are further induced in the spleen of tilapia challenged with Vibrio vulnificus. Mozambique tilapia GRN-41 can significantly activate and modulate the expression of innate immune-related genes in transgenic zebrafish and increase the survival rate after challenge with V. vulnificus [15]. According to previous studies, PGRN or GRN is involved in the modulation of host immunity to defend against pathogens. Moreover, the cysteine-rich eNAP-1 protein, a 7.2 kDa GRN peptide, was purified from horse neutrophils [16]. Interestingly, the horse eNAP1 has been the only GRN peptide demonstrated to possess antimicrobial peptide activity since 1992 [16]. In the present study, we investigated the antibacterial activities of different chemically synthetic tilapia GRN peptides, including 41-a.a. GRN-41 peptides from Mozambique and Nile tilapia (OmGRN-41 and OnGRN-41), the 56-a.a. intact GRN-A derived from the PGRN1 gene of Mozambique tilapia (OmGRN-A), and the common 30-a.a. GRN-30 (OmGRN-30) shared by GRN-41 and GRN-A [15], against critical bacterial pathogens in aquaculture. Our results demonstrate that only the Mozambique tilapia OmGRN-41 peptide had antimicrobial properties, indicating that it is a novel antimicrobial peptide, especially against Vibrio species. 2. Materials and methods 2.1. Peptide synthesis
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no visible growth after overnight incubation. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of peptide that killed more than 99% of the initial inoculum after overnight incubation. 2.3. Time-kill curve of Vibrio species treated with OmGRN-41 The preparation of bacterial cultures followed the same procedure described in section 2.2. Then, the bacterial culture was adjusted to achieve 5 105 CFU/ml and combined with synthetic peptides at an MIC of 0.5 , 1 , or 2 in 96-well microplates. The plates were incubated at 30 C for 0, 0.5, 1, 2, 4, 8, 12, 16, and 24 h, and viable bacteria were counted via the plate count method. 2.4. Effect of different treatments on the antimicrobial activity of OmGRN-41 To determine the effects of temperature and pH on the antibacterial activity of OmGRN-41, the peptide was incubated under different conditions for 1 h. In the temperature analysis, OmGRN-41 was incubated for 1 h at 25, 40, 60, 80, or 100 C. In the pH analysis, OmGRN-41 was incubated at 25 C for 1 h at pH 2, 4, 6, 8, 10, or 12. Subsequently, the antibacterial activity tests and analyses followed the procedures described in Materials and methods 2.2. 2.5. Examination of the morphology of V. vulnificus treated with OmGRN-41 by transmission electron microscopy (TEM)
The tilapia GRN peptides used in this study were synthesized using 9-fluorenylmethoxycarbonyl solid-phase synthesis methods (Kelowna International Scientific Inc., Taiwan). The MW and purity of synthetic peptides were measured by MS and HPLC, respectively. Furthermore, the characteristics of synthetic peptides, such as the isoelectric point and net charge, were predicted using Genscript's Peptide Property Calculator (https://www.genscript.com/tools/ peptide-property-calculator). The results of the characterization of synthetic tilapia GRN peptides are summarized in Table S1. Synthetic peptides were dissolved in 0.9% NaCl solution (saline) at concentrations of 2e4 mg/ml and stored at 80 C.
V. vulnificus was prepared as described in Materials and methods 2.2. Then, V. vulnificus was centrifuged at 2000 g for 5 min, and the pellet was washed with saline three times. The concentration of V. vulnificus was adjusted to 5 107 CFU/ml, after which the culture was incubated with 0, 16, 32, and 64 mM OmGRN-41 at 30 C for 10 min. Then, 10 ml of each sample was dropped onto a CF300-CU copper-mesh grid (Electron Microscopy Sciences, USA) for 10 min. The excess solution was absorbed with filter paper, and the grids were allowed to air-dry at room temperature for 2 h. Finally, the copper-mesh grids were examined to assess the morphology of bacteria using a HT7700 transmission electron microscope (Hitachi, Japan). Moreover, the viability of V. vulnificus was calculated using the plate count method after treatment with OmGRN-41 for 10 min.
2.2. Antibacterial activity assay of synthetic peptides
2.6. Hemolytic assay
A total of eight aquaculture pathogenic bacteria strains were used in this study, including six gram-negative bacteria, Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus [17], and two grampositive bacteria, Streptococcus agalactiae and Streptococcus iniae [18]. The antibacterial activity of synthetic tilapia GRN peptides was determined via the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. Briefly, the bacterial stock was removed from a 80 C freezer. Thirty microliter of bacterial stock was added to 3 ml MuellerHinton (MH) broth and incubated at 30 C for 16 h with shaking (200 rpm). Then, 30 ml of the overnight bacterial culture was added to MH broth and incubated at 30 C for 6 h with shaking. Next, the bacterial culture was diluted with MH broth to achieve 1 106 CFU/ ml. The synthetic peptides were diluted with saline to produce solutions of 0, 16, 32, 64, 128, 256, 512, and 1024 mg/ml. One hundred microliter of bacteria culture (1 106 CFU/ml) and 100 ml of synthetic peptides were mixed in 96-well microplates and incubated at 30 C for 24 h. The viable bacteria were counted via the plate count method. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of peptide that resulted in
Tilapia and sheep erythrocytes were collected from Mozambique tilapia and commercial defibrinated sheep blood (Creative LifeSciences, Taiwan), respectively, following a previously described procedure [20]. Then, the tilapia and sheep erythrocytes were diluted with saline to produce a 4% (v/v) erythrocyte solution. Next, one hundred microliter of erythrocyte solution was mixed with an equal volume of different concentrations of OmGRN-41 in a 96-well microplate. The plate was incubated at 37 C for 1 h and then centrifuged at 500 g for 5 min at 25 C. One hundred microliter of supernatant was transferred to a new 96-well microplate, and the OD540 values were measured with a microplate reader (Bio-Tek Instruments, Inc., USA). Erythrocytes treated with 1% Triton X-100 (TX) or saline alone were used as positive and negative controls, respectively. The percentage of hemolysis was calculated following a previously described formula [21]. 2.7. In vivo antibacterial activity of OmGRN-41 The challenge test was described in our previous study [15]. Briefly, Nile tilapia (body weight: 1.84 ± 0.25 g) were intraperitoneally injected with V. vulnificus (4 105 CFU/g body weight). After
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30 min, infected fish were injected with 0, 5, 10, or 20 mg OmGRN41 per gram body weight (BW) (10 individuals per group). The fish in each group were monitored daily for 7 days, and the test was repeated three times. The relative percent of survival (RPS) were calculated following a previously described formula [15]. The statistical analysis was performed by ANOVA and Duncan's multiple range test. Different letters indicate significant differences among the groups (P < 0.05). 3. Results 3.1. Antibacterial activity of synthetic tilapia GRN peptides First, the antibacterial activity of synthetic tilapia GRN peptides was determined via the broth microdilution method according to CLSI guidelines. The MIC and MBC values of OmGRN-41 against V. vulnificus were both 256 mg/ml (64 mM). The MIC and MBC values of OmGRN-41 against V. alginolyticus and V. harveyi were both 512 mg/ml (128 mM). However, OmGRN-41 had no MIC activity against V. parahaemolyticus, A. hydrophila, E. tarda, S. agalactiae, or S. iniae (Table S2). Moreover, OmGRN-A and OmGRN-30 had no antimicrobial activity against any of the tested strains (Table S2). In addition, the GRN-41 transcript has also been identified in Nile tilapia (OnGRN-41) [15]. Three amino acids are different between the mature peptides of OmGRN-41 and OnGRN-41 (Fig. S1). Thus, we also determined the antibacterial activity of OnGRN-41 to compare it with that of OmGRN-41. Unlike OmGRN-41, OnGRN-41 had no antibacterial activity against any of the tested strains (Table S3). These results indicate that the genetic variations in the PGRN1 gene encoding GRN-41 of Mozambique and Nile tilapia lead
to differences in their antimicrobial activities against Vibrio species. 3.2. Time-kill curves of OmGRN-41 against Vibrio species and other aquaculture bacterial pathogens Time-kill curve assays of OmGRN-41 against various bacterial pathogens were performed. More than 99% of V. vulnificus were destroyed after treatment with 64 mM or 128 mM OmGRN-41 for 1 h. All of the V. vulnificus were killed after treatment with 64 mM or 128 mM OmGRN-41 for 8 or 2 h, respectively (Fig. 1A). More than 97% and 99% of the V. alginolyticus were destroyed after treatment with 64 mM or 128 mM OmGRN-41, respectively, for 1 h. All of the V. alginolyticus were killed after treatment with 128 mM OmGRN-41 for 2 h. However, V. alginolyticus re-growth (1.75 106 CFU/ml) above the initial dose (5.02 105 CFU/ml) was observed during the treatment with 64 mM OmGRN-41 after 12 h (Fig. 1B). More than 92 and 99% of the V. harveyi were destroyed after treatment with 64 mM and 128 mM OmGRN-41 after around 2 h. All of the V. harveyi were killed after treatment with 128 mM OmGRN-41 for 4 h. Nevertheless, the significant re-growth of V. harveyi was observed (1.56 108 CFU/ml) above the initial dose (5.22 105 CFU/ml) during the treatment with 64 mM OmGRN-41 after 16 h (Fig. 1C). Although no MIC and MBC activities against V. parahaemolyticus, A. hydrophila, E. tarda, S. agalactiae, and S. iniae were detected after 24 h of incubation with OmGRN-41 at 30 C, we also analyzed the time-kill curve of these five bacterial pathogens during treatment with OmGRN-41. More than 99% of the V. parahaemolyticus were destroyed after treatment with 64 mM and 128 mM OmGRN-41 for 2 h (Fig. 1D). OmGRN-41 exhibited bacteriostatic activity at 64 mM and 128 mM after 8 h and 12 h, respectively, against
Fig. 1. Time-kill curve analysis of Vibrio species treated with OmGRN-41. (A) V. vulnificus, (B) V. alginolyticus, (C) V. harveyi, (D) and V. parahaemolyticus were co-cultured with OmGRN-41, and the viable counts were calculated by the plate count method at 0, 0.5, 1, 2, 4, 8, 12, 16, and 24 h. The saline alone and 32, 64, and 128 mM of OmGRN-41 treatment groups are indicated by a solid circle (z.cirf), solid box ( ), solid triangle (:), and cross (╳), respectively. Each point represents the mean ± SD from three experiments.
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V. parahaemolyticus. However, the significant bacterial re-growth of V. parahaemolyticus was observed (2.50 108 CFU/ml) above the initial dose (3.26 105 CFU/ml) during the treatment with 64 mM OmGRN-41 after 12 h (Fig. 1D). The bacterial re-growth of V. parahaemolyticus was observed above the initial dose (3.26 105 CFU/ml) during the treatment with 128 mM OmGRN-41 from 16 h (1.26 106 CFU/ml) to 24 h (9.33 108 CFU/ml) comparable to that in the control (no OmGRN-41 treatment) at 24 h (9.56 108 CFU/ml). However, OmGRN-41, even at the dose of 128 mM, exhibited no antimicrobial activity against A. hydrophila, E. tarda, S. agalactiae, and S. iniae (Fig. S2). 3.3. Effect of temperature and pH on the antimicrobial activity of OmGRN-41 against V. vulnificus Next, to determine the effects of temperature and pH on OmGRN-41, its antibacterial activity was analyzed after incubation at different temperatures and pH values for 1 h. The results showed that OmGRN-41 still possessed MIC and MBC activities against V. vulnificus after heating at temperatures between 40 and 100 C (Table S4 and Fig. S3). In addition, OmGRN-41 had MIC and MBC activities against V. vulnificus after treatment with pH 2 to pH 10 buffers; however, it lost MIC and MBC activities against V. vulnificus after treatment with pH 12 buffer (Table S5 and Fig. S4). 3.4. TEM analysis of V. vulnificus treated with OmGRN-41 Further, we observed the integrity of V. vulnificus by TEM after treatment with OmGRN-41 for 10 min. The results indicated that the bacterial appearance of the control group was that of typical rod-shaped cells with an intact cell outer membrane and flagella, and the cytoplasmic region showed a homogeneous electron density (Fig. 2A). However, the outer membrane of V. vulnificus was clearly destroyed by 16 mM OmGRN-41 (Fig. 2A). Next, after exposure to 32 mM OmGRN-41, the outer membrane of V. vulnificus was clearly destroyed with loss of intracellular material (Fig. 2A). Finally, V. vulnificus displayed a non-homogeneous electron density in the cytoplasmic region and morphological changes after treatment with 64 mM OmGRN-41 (Fig. 2A). In addition, the visible
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counts of V. vulnificus after treatment with OmGRN-41 for 10 min before TEM observation were calculated using the plate count method (Fig. 2B). The results showed that V. vulnificus bacteria decreased from 5.40 107 CFU/ml to 5.38 106 CFU/ml, 2.89 104 CFU/ml, and 4.91 102 CFU/ml after treatment with 16, 32, and 64 mM OmGRN-41, respectively (Fig. 2B). 3.5. Hemolytic activity of OmGRN-41 Hemolytic activity was evaluated to determine the cytotoxic effect of OmGRN-41 on eukaryotic cells by measuring the percentage of hemolysis in tilapia and sheep erythrocytes after exposure to OmGRN-41 at concentrations from 2 mM to 128 mM. Little hemolytic activity of OmGRN-41 was detected at all concentrations in tilapia and sheep erythrocytes (Fig. 3). Even at the highest dose of OmGRN-41 (128 mM), there was only 2.4 ± 1.0% hemolysis in tilapia erythrocytes compared to 100% hemolysis with Triton X-100 (Fig. 3A). 3.6. The survival of tilapia challenged with V. vulnificus was rescued by OmGRN-41 Finally, we evaluated the antibacterial activity of OmGRN-41 in vivo. The survival rate of control group Nile tilapia injected with saline alone was 100%. The survival rates of Nile tilapia challenged with V. vulnificus, followed by treatment with 0, 5, 10 and 20 mg OmGRN-41 per gram body weight (BW) were 6.7%, 3.3%, 53.3% and 80.0%, respectively. The Relative percent of survival (RPS) in V. vulnificus infected Nile tilapia was significantly increased after treatment with OmGRN-41 in dose of 10 mg/g BW (56.4%) and 20 mg/g BW (78.6%) compared with the V. vulnificus injection alone group (Fig. 4). 4. Discussion In recent years, many AMP genes have been discovered in fish genomes, and many lines of evidence indicate the critical roles of AMPs in fish innate immunity [22]. AMPs not only have bactericidal activity but also act as immune modulators to regulate host
Fig. 2. TEM analysis of V. vulnificus treated with OmGRN-41. (A) Morphology of V. vulnificus co-cultured without or with 16, 32, or 64 mM OmGRN-41 for 10 min examined by TEM. The scale bar is 1 mm. (B) The visible counts of V. vulnificus treated with OmGRN-41 for TEM observation. The dashed line indicates the bacterial inoculum concentration (CFU/ml). For each group, the mean ± SD from three experiments are presented.
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Fig. 3. Hemolytic assay of OmGRN-41. The percentage of hemolysis in tilapia (A) and sheep (B) erythrocytes after exposure to OmGRN-41 from 2 mM to 128 mM. Erythrocytes treated with 1% Triton X-100 (TX) and saline only were used as the positive and negative controls, for which the percentage of hemolysis was 100 and 0%, respectively. For each group, the mean ± SD from three experiments are presented.
Fig. 4. Survival curves of Nile tilapia challenged with V. vulnificus, followed by treatment with OmGRN-41. Tilapia were infected with V. vulnificus (4 105 CFU/g BW) by i.p. injection. After 30 min, the infected fish were treated with OmGRN-41 (0, 5, 10, 20 mg/g BW), and the survival rates were monitored for 7 days. Each point represents the mean ± SD (n ¼ 10) from three experiments. Different letters indicate significant differences among the groups (P < 0.05). BW: body weight.
immune responses [23e25]. In our previous studies, we demonstrated that tilapia GRN-41 can significantly activate and modulate the expression of innate immune-related genes in transgenic zebrafish and increase the survival rate after challenge with V. vulnificus [15]. In this study, we further showed that OmGRN-41 exhibited bactericidal activity against pathogenic Vibrio species, including V. vulnificus, V. alginolyticus, and V. harveyi. (Fig. 1AeC and Table S2). Moreover, we also found that OmGRN-41 inhibited the growth of V. parahaemolyticus at a high concentration within 16 h (Fig. 1D). The Mozambique tilapia GRN-41 is the second GRN peptide proven to possess antibacterial peptide activity, after the granulin peptide eNAP-1 purified from equine neutrophils, since 1992 [16]. At present, AMPs are known to function via two main bactericidal mechanisms. First, AMPs bind to bacterial cell membranes and form pores through amphiphilic structures, directly leading to bacterial death. Second, AMPs can directly enter bacterial cells and interact with target molecules to affect bacterial growth or metabolism [26]. According to the time-kill curve and TEM data, OmGRN-41 at 64 mM killed a high proportion of V. vulnificus within a short time (Figs. 1A and 2). We propose that OmGRN-41 may bind to certain molecules on the surface of Vibrio species and severely
damage the outer membrane of V. vulnificus, leading to the outflow of intracellular substances. Previous studies have shown that many antibacterial peptides are capable of dissolving erythrocytes, so the hemolytic assay is generally used to evaluate the safety of antibacterial peptides in clinical applications [27]. Because OmGRN-41 has the potential to be used for the treatment of Vibriosis in fish and mammals, tilapia and sheep erythrocytes were used for the hemolytic analyses in this study. The data showed that OmGRN-41 had no hemolytic activity against tilapia and sheep erythrocytes (Fig. 3); thus, it may be used in clinical applications in fish or mammals. Although the antibacterial activity of OmGRN-41 was not as strong as antibiotics, it was more stable in acidic solution (pH 2), such as gastric juice, and much safer with little hemolytic activity (Fig. 3 and Table S5). These results indicate that OmGRN-41 is more suitable for oral administration as a functional additive in aquafeed. In vivo results showed that OmGRN-41 in dose of 10 mg/g and 20 mg/g BW by i.p. injection can effectively increase the survival of Nile tilapia infected by V. vulnificus (Fig. 4). Mass production of recombinant OmGRN-41 by yeast or other expression system [28] is critical for application in aquafeed to defend against Vibrio species in cultured fish, shrimp and shellfish.
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In our previous report, we found that GRN-41 was also expressed in Nile tilapia [15], but there are three amino acid differences in the mature peptide of GRN-41 between Mozambique and Nile tilapia (Fig. S1). The results indicated that OmGRN-41 had bactericidal activity against V. vulnificus, V. alginolyticus, and V. harveyi and bacteriostatic activity against V. parahaemolyticus, whereas OnGRN-41 had no antibacterial activity against the tested strains (Tables S2e3). These results suggest that the genetic variations leading to the amino acid differences in the mature peptides of OmGRN-41 and OnGRN-41 might play an essential role in their antibacterial activity. Factors such as the complexity of PGRN gene numbers, different transcripts formed from alternative splicing, and sequence variations in GRN units that occur during evolution provide opportunities for GRN peptides from immune modulators to gain the function of antimicrobial peptides, such as OmGRN-41, to defend against pathogens. In conclusion, our results suggest that the OmGRN-41 is a novel, potent antimicrobial peptide and can be used to defend against Vibrio species infection in humans and aquaculture animals. Funding This work was supported by the Ministry of Science and Technology of Taiwan (MOST), Taiwan grant numbers MOST 102-2313B-019-004-MY3 and 104-2321-B-019-003-MY3, to Hong-Yi Gong. Acknowledgments We would like to thank Dr. Chun-Yao Chen (Department of Life Science, Tzu Chi University, Hualien, Taiwan) for kindly providing V. vulnificus 93U204 [17] and Dr. Jyh-Yih Chen (Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan) for providing S. agalactiae used in this study. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.022. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.022. References [1] R. Goldburg, R. Naylor, Future seascapes, fishing, and fish farming, Front. Ecol. Environ. 3 (2005) 21e28. [2] F.C. Cabello, Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment, Environ. Microbiol. 8 (2006) 1137e1144. [3] T. Defoirdt, P. Sorgeloos, P. Bossier, Alternatives to antibiotics for the control of bacterial disease in aquaculture, Curr. Opin. Microbiol. 14 (2011) 251e258.
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Granulin peptide GRN-41 of Mozambique tilapia is a novel antimicrobial peptide against Vibrio species Sheng-Han Wu a, Hsin-Yiu Chou a, b, Ping-Chung Liu a, Jen-Leih Wu c, Hong-Yi Gong a, b, * a
Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung, 20224, Taiwan Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan c Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, 11529, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 May 2019 Accepted 4 June 2019 Available online 8 June 2019
In our previous study, the novel GRN-41 peptide generated from alternative splicing of the Mozambique tilapia PGRN1 gene was identified to be a potent peptide that protected against V. vulnificus in the transgenic zebrafish model by modulating innate immune-related genes. In this study, the anti-bacterial activities of synthetic Mozambique tilapia GRN-41 peptide (OmGRN-41) against various bacterial pathogens were investigated. The results showed that OmGRN-41 had bactericidal activity against Vibrio species, including V. vulnificus, V. alginolyticus, and V. harveyi, but exhibited bacteriostatic activity against V. parahaemolyticus. OmGRN-41 maintained bactericidal activity (64 mM) against V. vulnificus at pH 2 to pH 10 or after heat treatment for 1 h at high temperatures between 40 C and 100 C. TEM observations revealed that the outer membrane of V. vulnificus was disrupted by OmGRN-41, leading to morphological rupture and loss of cytoplasmic contents. Additionally, little hemolytic activity against tilapia and sheep erythrocytes was detected after treatment with 128 mM OmGRN-41. OmGRN-41 can effectively enhance the survival of Nile tilapia infected by V. vulnificus. Our results suggest that the OmGRN-41 is a novel antimicrobial peptide possessing bactericidal activity, especially against Vibrio species. These results indicate that OmGRN-41 can be applied in human Vibriosis treatment and has the potential to defend against Vibrio spp. infection in critical aquaculture organisms. © 2019 Elsevier Inc. All rights reserved.
Keywords: Tilapia Granulin Bactericidal Bacteriostatic Vibrio Antimicrobial peptide
1. Introduction Aquaculture is a rapidly developing and crucial industry because of the insufficient food supply and deficiency of global fishery resources [1]. However, intensive farming has led to disease outbreaks and excessive use of antibiotics, which causes environmental pollution and the emergence of antibiotic-resistant strains [2]. Hence, the development of alternative antimicrobial agents is becoming very important [3]. In recent years, many natural products and antibacterial peptides (AMPs) from marine organisms have been identified and applied in the development of new drugs [4,5]. AMPs, which are composed of less than 100 amino acids and are positively charged, have broad-spectrum antimicrobial activity and regulate innate immunity [6].
* Corresponding author. Division of Life Science, Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, No.2, Beining Rd., Jhongjheng District, Keelung City, 20224, Taiwan. E-mail address: [email protected] (H.-Y. Gong). https://doi.org/10.1016/j.bbrc.2019.06.022 0006-291X/© 2019 Elsevier Inc. All rights reserved.
In 1990, Bateman et al. unexpectedly identified another family of proteins called granulins (GRNs) when purifying cysteine-rich defensin family AMPs from human leukocytes [7]. GRN is a 6-kDa peptide that contains 12 conserved cysteine residues that form 6 disulfide bonds [7]. The GRN unit is derived from progranulin (PGRN) by proteolytic cleavage [8]. PGRN is a multifunctional growth factor that regulates cell division, survival, and migration and is involved in tumorigenesis, tissue repair, embryogenesis, and inflammation [9]. There is only one PGRN gene that encodes PGRN, which is composed of 7.5 GRN units in the mammalian genome [9]. PGRN and GRN had been found to play the opposite roles in the regulation of inflammation [8,10,11]. Furthermore, GRN can serve as a cofactor for toll-like receptor 9 signaling and is involved in innate immunity to produce TNF-a in response to microbial DNA [12]. However, unlike in mammals, there are multiple PGRN genes in the teleost genome. They can be divided into long-form PGRN composed of at least 6 GRN units and short-form PGRNs composed of 1.5e3 GRN units [13,14]. In our previous study, we found that the short-form progranulin PGRN1 gene of Mozambique tilapia expressed two transcripts, PGRN1 encoding a 206-a.a. PGRN with
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two GRN units, GRN-A and GRN-B, and the alternatively spliced transcript GRN-41 encoding a secreted GRN peptide composed of a 20-a.a. ER signal peptide and 41-a.a. mature GRN peptide named GRN-41 [15]. PGRN1 and GRN-41 not only are abundantly expressed in immune-related organs but also are further induced in the spleen of tilapia challenged with Vibrio vulnificus. Mozambique tilapia GRN-41 can significantly activate and modulate the expression of innate immune-related genes in transgenic zebrafish and increase the survival rate after challenge with V. vulnificus [15]. According to previous studies, PGRN or GRN is involved in the modulation of host immunity to defend against pathogens. Moreover, the cysteine-rich eNAP-1 protein, a 7.2 kDa GRN peptide, was purified from horse neutrophils [16]. Interestingly, the horse eNAP1 has been the only GRN peptide demonstrated to possess antimicrobial peptide activity since 1992 [16]. In the present study, we investigated the antibacterial activities of different chemically synthetic tilapia GRN peptides, including 41-a.a. GRN-41 peptides from Mozambique and Nile tilapia (OmGRN-41 and OnGRN-41), the 56-a.a. intact GRN-A derived from the PGRN1 gene of Mozambique tilapia (OmGRN-A), and the common 30-a.a. GRN-30 (OmGRN-30) shared by GRN-41 and GRN-A [15], against critical bacterial pathogens in aquaculture. Our results demonstrate that only the Mozambique tilapia OmGRN-41 peptide had antimicrobial properties, indicating that it is a novel antimicrobial peptide, especially against Vibrio species. 2. Materials and methods 2.1. Peptide synthesis
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no visible growth after overnight incubation. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of peptide that killed more than 99% of the initial inoculum after overnight incubation. 2.3. Time-kill curve of Vibrio species treated with OmGRN-41 The preparation of bacterial cultures followed the same procedure described in section 2.2. Then, the bacterial culture was adjusted to achieve 5 105 CFU/ml and combined with synthetic peptides at an MIC of 0.5 , 1 , or 2 in 96-well microplates. The plates were incubated at 30 C for 0, 0.5, 1, 2, 4, 8, 12, 16, and 24 h, and viable bacteria were counted via the plate count method. 2.4. Effect of different treatments on the antimicrobial activity of OmGRN-41 To determine the effects of temperature and pH on the antibacterial activity of OmGRN-41, the peptide was incubated under different conditions for 1 h. In the temperature analysis, OmGRN-41 was incubated for 1 h at 25, 40, 60, 80, or 100 C. In the pH analysis, OmGRN-41 was incubated at 25 C for 1 h at pH 2, 4, 6, 8, 10, or 12. Subsequently, the antibacterial activity tests and analyses followed the procedures described in Materials and methods 2.2. 2.5. Examination of the morphology of V. vulnificus treated with OmGRN-41 by transmission electron microscopy (TEM)
The tilapia GRN peptides used in this study were synthesized using 9-fluorenylmethoxycarbonyl solid-phase synthesis methods (Kelowna International Scientific Inc., Taiwan). The MW and purity of synthetic peptides were measured by MS and HPLC, respectively. Furthermore, the characteristics of synthetic peptides, such as the isoelectric point and net charge, were predicted using Genscript's Peptide Property Calculator (https://www.genscript.com/tools/ peptide-property-calculator). The results of the characterization of synthetic tilapia GRN peptides are summarized in Table S1. Synthetic peptides were dissolved in 0.9% NaCl solution (saline) at concentrations of 2e4 mg/ml and stored at 80 C.
V. vulnificus was prepared as described in Materials and methods 2.2. Then, V. vulnificus was centrifuged at 2000 g for 5 min, and the pellet was washed with saline three times. The concentration of V. vulnificus was adjusted to 5 107 CFU/ml, after which the culture was incubated with 0, 16, 32, and 64 mM OmGRN-41 at 30 C for 10 min. Then, 10 ml of each sample was dropped onto a CF300-CU copper-mesh grid (Electron Microscopy Sciences, USA) for 10 min. The excess solution was absorbed with filter paper, and the grids were allowed to air-dry at room temperature for 2 h. Finally, the copper-mesh grids were examined to assess the morphology of bacteria using a HT7700 transmission electron microscope (Hitachi, Japan). Moreover, the viability of V. vulnificus was calculated using the plate count method after treatment with OmGRN-41 for 10 min.
2.2. Antibacterial activity assay of synthetic peptides
2.6. Hemolytic assay
A total of eight aquaculture pathogenic bacteria strains were used in this study, including six gram-negative bacteria, Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus [17], and two grampositive bacteria, Streptococcus agalactiae and Streptococcus iniae [18]. The antibacterial activity of synthetic tilapia GRN peptides was determined via the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. Briefly, the bacterial stock was removed from a 80 C freezer. Thirty microliter of bacterial stock was added to 3 ml MuellerHinton (MH) broth and incubated at 30 C for 16 h with shaking (200 rpm). Then, 30 ml of the overnight bacterial culture was added to MH broth and incubated at 30 C for 6 h with shaking. Next, the bacterial culture was diluted with MH broth to achieve 1 106 CFU/ ml. The synthetic peptides were diluted with saline to produce solutions of 0, 16, 32, 64, 128, 256, 512, and 1024 mg/ml. One hundred microliter of bacteria culture (1 106 CFU/ml) and 100 ml of synthetic peptides were mixed in 96-well microplates and incubated at 30 C for 24 h. The viable bacteria were counted via the plate count method. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of peptide that resulted in
Tilapia and sheep erythrocytes were collected from Mozambique tilapia and commercial defibrinated sheep blood (Creative LifeSciences, Taiwan), respectively, following a previously described procedure [20]. Then, the tilapia and sheep erythrocytes were diluted with saline to produce a 4% (v/v) erythrocyte solution. Next, one hundred microliter of erythrocyte solution was mixed with an equal volume of different concentrations of OmGRN-41 in a 96-well microplate. The plate was incubated at 37 C for 1 h and then centrifuged at 500 g for 5 min at 25 C. One hundred microliter of supernatant was transferred to a new 96-well microplate, and the OD540 values were measured with a microplate reader (Bio-Tek Instruments, Inc., USA). Erythrocytes treated with 1% Triton X-100 (TX) or saline alone were used as positive and negative controls, respectively. The percentage of hemolysis was calculated following a previously described formula [21]. 2.7. In vivo antibacterial activity of OmGRN-41 The challenge test was described in our previous study [15]. Briefly, Nile tilapia (body weight: 1.84 ± 0.25 g) were intraperitoneally injected with V. vulnificus (4 105 CFU/g body weight). After
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30 min, infected fish were injected with 0, 5, 10, or 20 mg OmGRN41 per gram body weight (BW) (10 individuals per group). The fish in each group were monitored daily for 7 days, and the test was repeated three times. The relative percent of survival (RPS) were calculated following a previously described formula [15]. The statistical analysis was performed by ANOVA and Duncan's multiple range test. Different letters indicate significant differences among the groups (P < 0.05). 3. Results 3.1. Antibacterial activity of synthetic tilapia GRN peptides First, the antibacterial activity of synthetic tilapia GRN peptides was determined via the broth microdilution method according to CLSI guidelines. The MIC and MBC values of OmGRN-41 against V. vulnificus were both 256 mg/ml (64 mM). The MIC and MBC values of OmGRN-41 against V. alginolyticus and V. harveyi were both 512 mg/ml (128 mM). However, OmGRN-41 had no MIC activity against V. parahaemolyticus, A. hydrophila, E. tarda, S. agalactiae, or S. iniae (Table S2). Moreover, OmGRN-A and OmGRN-30 had no antimicrobial activity against any of the tested strains (Table S2). In addition, the GRN-41 transcript has also been identified in Nile tilapia (OnGRN-41) [15]. Three amino acids are different between the mature peptides of OmGRN-41 and OnGRN-41 (Fig. S1). Thus, we also determined the antibacterial activity of OnGRN-41 to compare it with that of OmGRN-41. Unlike OmGRN-41, OnGRN-41 had no antibacterial activity against any of the tested strains (Table S3). These results indicate that the genetic variations in the PGRN1 gene encoding GRN-41 of Mozambique and Nile tilapia lead
to differences in their antimicrobial activities against Vibrio species. 3.2. Time-kill curves of OmGRN-41 against Vibrio species and other aquaculture bacterial pathogens Time-kill curve assays of OmGRN-41 against various bacterial pathogens were performed. More than 99% of V. vulnificus were destroyed after treatment with 64 mM or 128 mM OmGRN-41 for 1 h. All of the V. vulnificus were killed after treatment with 64 mM or 128 mM OmGRN-41 for 8 or 2 h, respectively (Fig. 1A). More than 97% and 99% of the V. alginolyticus were destroyed after treatment with 64 mM or 128 mM OmGRN-41, respectively, for 1 h. All of the V. alginolyticus were killed after treatment with 128 mM OmGRN-41 for 2 h. However, V. alginolyticus re-growth (1.75 106 CFU/ml) above the initial dose (5.02 105 CFU/ml) was observed during the treatment with 64 mM OmGRN-41 after 12 h (Fig. 1B). More than 92 and 99% of the V. harveyi were destroyed after treatment with 64 mM and 128 mM OmGRN-41 after around 2 h. All of the V. harveyi were killed after treatment with 128 mM OmGRN-41 for 4 h. Nevertheless, the significant re-growth of V. harveyi was observed (1.56 108 CFU/ml) above the initial dose (5.22 105 CFU/ml) during the treatment with 64 mM OmGRN-41 after 16 h (Fig. 1C). Although no MIC and MBC activities against V. parahaemolyticus, A. hydrophila, E. tarda, S. agalactiae, and S. iniae were detected after 24 h of incubation with OmGRN-41 at 30 C, we also analyzed the time-kill curve of these five bacterial pathogens during treatment with OmGRN-41. More than 99% of the V. parahaemolyticus were destroyed after treatment with 64 mM and 128 mM OmGRN-41 for 2 h (Fig. 1D). OmGRN-41 exhibited bacteriostatic activity at 64 mM and 128 mM after 8 h and 12 h, respectively, against
Fig. 1. Time-kill curve analysis of Vibrio species treated with OmGRN-41. (A) V. vulnificus, (B) V. alginolyticus, (C) V. harveyi, (D) and V. parahaemolyticus were co-cultured with OmGRN-41, and the viable counts were calculated by the plate count method at 0, 0.5, 1, 2, 4, 8, 12, 16, and 24 h. The saline alone and 32, 64, and 128 mM of OmGRN-41 treatment groups are indicated by a solid circle (z.cirf), solid box ( ), solid triangle (:), and cross (╳), respectively. Each point represents the mean ± SD from three experiments.
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V. parahaemolyticus. However, the significant bacterial re-growth of V. parahaemolyticus was observed (2.50 108 CFU/ml) above the initial dose (3.26 105 CFU/ml) during the treatment with 64 mM OmGRN-41 after 12 h (Fig. 1D). The bacterial re-growth of V. parahaemolyticus was observed above the initial dose (3.26 105 CFU/ml) during the treatment with 128 mM OmGRN-41 from 16 h (1.26 106 CFU/ml) to 24 h (9.33 108 CFU/ml) comparable to that in the control (no OmGRN-41 treatment) at 24 h (9.56 108 CFU/ml). However, OmGRN-41, even at the dose of 128 mM, exhibited no antimicrobial activity against A. hydrophila, E. tarda, S. agalactiae, and S. iniae (Fig. S2). 3.3. Effect of temperature and pH on the antimicrobial activity of OmGRN-41 against V. vulnificus Next, to determine the effects of temperature and pH on OmGRN-41, its antibacterial activity was analyzed after incubation at different temperatures and pH values for 1 h. The results showed that OmGRN-41 still possessed MIC and MBC activities against V. vulnificus after heating at temperatures between 40 and 100 C (Table S4 and Fig. S3). In addition, OmGRN-41 had MIC and MBC activities against V. vulnificus after treatment with pH 2 to pH 10 buffers; however, it lost MIC and MBC activities against V. vulnificus after treatment with pH 12 buffer (Table S5 and Fig. S4). 3.4. TEM analysis of V. vulnificus treated with OmGRN-41 Further, we observed the integrity of V. vulnificus by TEM after treatment with OmGRN-41 for 10 min. The results indicated that the bacterial appearance of the control group was that of typical rod-shaped cells with an intact cell outer membrane and flagella, and the cytoplasmic region showed a homogeneous electron density (Fig. 2A). However, the outer membrane of V. vulnificus was clearly destroyed by 16 mM OmGRN-41 (Fig. 2A). Next, after exposure to 32 mM OmGRN-41, the outer membrane of V. vulnificus was clearly destroyed with loss of intracellular material (Fig. 2A). Finally, V. vulnificus displayed a non-homogeneous electron density in the cytoplasmic region and morphological changes after treatment with 64 mM OmGRN-41 (Fig. 2A). In addition, the visible
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counts of V. vulnificus after treatment with OmGRN-41 for 10 min before TEM observation were calculated using the plate count method (Fig. 2B). The results showed that V. vulnificus bacteria decreased from 5.40 107 CFU/ml to 5.38 106 CFU/ml, 2.89 104 CFU/ml, and 4.91 102 CFU/ml after treatment with 16, 32, and 64 mM OmGRN-41, respectively (Fig. 2B). 3.5. Hemolytic activity of OmGRN-41 Hemolytic activity was evaluated to determine the cytotoxic effect of OmGRN-41 on eukaryotic cells by measuring the percentage of hemolysis in tilapia and sheep erythrocytes after exposure to OmGRN-41 at concentrations from 2 mM to 128 mM. Little hemolytic activity of OmGRN-41 was detected at all concentrations in tilapia and sheep erythrocytes (Fig. 3). Even at the highest dose of OmGRN-41 (128 mM), there was only 2.4 ± 1.0% hemolysis in tilapia erythrocytes compared to 100% hemolysis with Triton X-100 (Fig. 3A). 3.6. The survival of tilapia challenged with V. vulnificus was rescued by OmGRN-41 Finally, we evaluated the antibacterial activity of OmGRN-41 in vivo. The survival rate of control group Nile tilapia injected with saline alone was 100%. The survival rates of Nile tilapia challenged with V. vulnificus, followed by treatment with 0, 5, 10 and 20 mg OmGRN-41 per gram body weight (BW) were 6.7%, 3.3%, 53.3% and 80.0%, respectively. The Relative percent of survival (RPS) in V. vulnificus infected Nile tilapia was significantly increased after treatment with OmGRN-41 in dose of 10 mg/g BW (56.4%) and 20 mg/g BW (78.6%) compared with the V. vulnificus injection alone group (Fig. 4). 4. Discussion In recent years, many AMP genes have been discovered in fish genomes, and many lines of evidence indicate the critical roles of AMPs in fish innate immunity [22]. AMPs not only have bactericidal activity but also act as immune modulators to regulate host
Fig. 2. TEM analysis of V. vulnificus treated with OmGRN-41. (A) Morphology of V. vulnificus co-cultured without or with 16, 32, or 64 mM OmGRN-41 for 10 min examined by TEM. The scale bar is 1 mm. (B) The visible counts of V. vulnificus treated with OmGRN-41 for TEM observation. The dashed line indicates the bacterial inoculum concentration (CFU/ml). For each group, the mean ± SD from three experiments are presented.
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Fig. 3. Hemolytic assay of OmGRN-41. The percentage of hemolysis in tilapia (A) and sheep (B) erythrocytes after exposure to OmGRN-41 from 2 mM to 128 mM. Erythrocytes treated with 1% Triton X-100 (TX) and saline only were used as the positive and negative controls, for which the percentage of hemolysis was 100 and 0%, respectively. For each group, the mean ± SD from three experiments are presented.
Fig. 4. Survival curves of Nile tilapia challenged with V. vulnificus, followed by treatment with OmGRN-41. Tilapia were infected with V. vulnificus (4 105 CFU/g BW) by i.p. injection. After 30 min, the infected fish were treated with OmGRN-41 (0, 5, 10, 20 mg/g BW), and the survival rates were monitored for 7 days. Each point represents the mean ± SD (n ¼ 10) from three experiments. Different letters indicate significant differences among the groups (P < 0.05). BW: body weight.
immune responses [23e25]. In our previous studies, we demonstrated that tilapia GRN-41 can significantly activate and modulate the expression of innate immune-related genes in transgenic zebrafish and increase the survival rate after challenge with V. vulnificus [15]. In this study, we further showed that OmGRN-41 exhibited bactericidal activity against pathogenic Vibrio species, including V. vulnificus, V. alginolyticus, and V. harveyi. (Fig. 1AeC and Table S2). Moreover, we also found that OmGRN-41 inhibited the growth of V. parahaemolyticus at a high concentration within 16 h (Fig. 1D). The Mozambique tilapia GRN-41 is the second GRN peptide proven to possess antibacterial peptide activity, after the granulin peptide eNAP-1 purified from equine neutrophils, since 1992 [16]. At present, AMPs are known to function via two main bactericidal mechanisms. First, AMPs bind to bacterial cell membranes and form pores through amphiphilic structures, directly leading to bacterial death. Second, AMPs can directly enter bacterial cells and interact with target molecules to affect bacterial growth or metabolism [26]. According to the time-kill curve and TEM data, OmGRN-41 at 64 mM killed a high proportion of V. vulnificus within a short time (Figs. 1A and 2). We propose that OmGRN-41 may bind to certain molecules on the surface of Vibrio species and severely
damage the outer membrane of V. vulnificus, leading to the outflow of intracellular substances. Previous studies have shown that many antibacterial peptides are capable of dissolving erythrocytes, so the hemolytic assay is generally used to evaluate the safety of antibacterial peptides in clinical applications [27]. Because OmGRN-41 has the potential to be used for the treatment of Vibriosis in fish and mammals, tilapia and sheep erythrocytes were used for the hemolytic analyses in this study. The data showed that OmGRN-41 had no hemolytic activity against tilapia and sheep erythrocytes (Fig. 3); thus, it may be used in clinical applications in fish or mammals. Although the antibacterial activity of OmGRN-41 was not as strong as antibiotics, it was more stable in acidic solution (pH 2), such as gastric juice, and much safer with little hemolytic activity (Fig. 3 and Table S5). These results indicate that OmGRN-41 is more suitable for oral administration as a functional additive in aquafeed. In vivo results showed that OmGRN-41 in dose of 10 mg/g and 20 mg/g BW by i.p. injection can effectively increase the survival of Nile tilapia infected by V. vulnificus (Fig. 4). Mass production of recombinant OmGRN-41 by yeast or other expression system [28] is critical for application in aquafeed to defend against Vibrio species in cultured fish, shrimp and shellfish.
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In our previous report, we found that GRN-41 was also expressed in Nile tilapia [15], but there are three amino acid differences in the mature peptide of GRN-41 between Mozambique and Nile tilapia (Fig. S1). The results indicated that OmGRN-41 had bactericidal activity against V. vulnificus, V. alginolyticus, and V. harveyi and bacteriostatic activity against V. parahaemolyticus, whereas OnGRN-41 had no antibacterial activity against the tested strains (Tables S2e3). These results suggest that the genetic variations leading to the amino acid differences in the mature peptides of OmGRN-41 and OnGRN-41 might play an essential role in their antibacterial activity. Factors such as the complexity of PGRN gene numbers, different transcripts formed from alternative splicing, and sequence variations in GRN units that occur during evolution provide opportunities for GRN peptides from immune modulators to gain the function of antimicrobial peptides, such as OmGRN-41, to defend against pathogens. In conclusion, our results suggest that the OmGRN-41 is a novel, potent antimicrobial peptide and can be used to defend against Vibrio species infection in humans and aquaculture animals. Funding This work was supported by the Ministry of Science and Technology of Taiwan (MOST), Taiwan grant numbers MOST 102-2313B-019-004-MY3 and 104-2321-B-019-003-MY3, to Hong-Yi Gong. Acknowledgments We would like to thank Dr. Chun-Yao Chen (Department of Life Science, Tzu Chi University, Hualien, Taiwan) for kindly providing V. vulnificus 93U204 [17] and Dr. Jyh-Yih Chen (Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan) for providing S. agalactiae used in this study. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.022. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.022. References [1] R. Goldburg, R. Naylor, Future seascapes, fishing, and fish farming, Front. Ecol. Environ. 3 (2005) 21e28. [2] F.C. Cabello, Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment, Environ. Microbiol. 8 (2006) 1137e1144. [3] T. Defoirdt, P. Sorgeloos, P. Bossier, Alternatives to antibiotics for the control of bacterial disease in aquaculture, Curr. Opin. Microbiol. 14 (2011) 251e258.
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