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An active domain HF-18 derived from hagfish intestinal peptide effectively in-hibited drug-resistant bacteria in vitro/vivo

Meiling Jiang, Xiaoqian Yang, Haomin Wu, Ya Huang, Jie Dou, Changlin Zhou,Lingman Ma

PII: S0006-2952(19)30445-9DOI: https://doi.org/10.1016/j.bcp.2019.113746Reference: BCP 113746

To appear in: Biochemical Pharmacology

Received Date: 17 September 2019Accepted Date: 3 December 2019

Please cite this article as: M. Jiang, X. Yang, H. Wu, Y. Huang, J. Dou, C. Zhou, L. Ma, An active domain HF-18derived from hagfish intestinal peptide effectively inhibited drug-resistant bacteria in vitro/vivo, BiochemicalPharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113746

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© 2019 Published by Elsevier Inc.

1

1 An active domain HF-18 derived from hagfish intestinal peptide

2 effectively inhibited drug-resistant bacteria in vitro/vivo

3

4

5 Meiling Jiang1, Xiaoqian Yang1, Haomin Wu, Ya Huang, Jie Dou, Changlin Zhou* and

6 Lingman Ma*

7

8 School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu

9 211198, PR China

10 1 These authors were contributed equally to this work.

11 Correspondence: School of Life Science and Technology, China Pharmaceutical

12 University, 639 Longmian Road, Nanjing, Jiangsu 211198, PR China. Telephone: +86

13 2586185921, Fax number: +86 2586185921, Email address: cl_zhou@cpu.edu.cn (Changlin

14 Zhou), 1620174416@cpu.edu.cn (Lingman Ma).

15

16 Abbreviations: His, Histidine; Gly, Glycine; Lys, Lysine; Phe, Phenylalanine; Trp,

17 Tryptophan; Arg, Argnine; CFU, Colony forming unit; SDS, Sodium dodecyl sulfate; DMSO,

18 Dimethyl sulfoxide; CMC, Sodium carboxymethylcellulose; TEM, Transmission electron

19 microscopy; NPN, N-Phenyl-1-naphthylamine; PI, Propidium iodide; FITC, Fluorescein

20 isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; Histological examination, H&E.

2

21 Abstract

22 Antibiotic resistance is spreading faster than the development of new antibiotics into

23 clinical practice. Currently, the design of antimicrobial peptides (AMPs), potential new

24 antibacterial agents with rare antimicrobial resistance, is the available strategy to enhance the

25 antimicrobial activity and lower the toxicity of AMPs. In this study, a peptide derived from

26 hagfish intestinal peptide was designed and termed as HF-18 (GFFKKAWRKVKKAFRRVL).

27 After antimicrobial/bactericidal test in vitro, we found that HF-18 exhibited a potent

28 antimicrobial activity with MIC of only 4 μg/ml against drug-resistant Staphylococcus aureus

29 (S. aureus). Meanwhile, it eliminated the test bacteria within 1 h, suggesting its rapid

30 bactericidal effect. Importantly, this peptide had no obvious hemolytic activity and

31 cytotoxicity to mammalian cells. Furthermore, its notable antimicrobial effects in vivo was

32 confirmed again in S. aureus induced mouse bacteremia and skin wound infection, reflecting

33 as the decrease in bacterial counts in mouse lung or skin (up to 1.9 or 3.5 log CFU

34 respectively), and including the inhibitory activity on inflammatory cytokines secretion. The

35 possible mechanisms underlying HF-18 against drug-resistant S. aureus may attribute that

36 HF-18 neutralized the negative charge in S. aureus surface and then disrupted the integrity of

37 cell membranes to enhance the permeation of bacterial membrane, showing as the increased

38 uptake of NPN and PI and the obvious morphology changes of S. aureus. In addition, this

39 peptide bound to bacterial genomic DNA to suppress the expression of Panton-Valentine

40 leukocidin (pvl) and nuclease (nuc) genes, which play major roles in S. aureus virulence. The

41 properties of HF-18 suggest a path towards developing antibacterial agents that has stronger

42 antibacterial activity and greater security for clinical treatment of infection induced by S.

3

43 aureus, especially drug-resistant S. aureus.

44 Keywords: Antimicrobial peptide; Antimicrobial activity; Drug-resistant bacteria;

45 Bacteremia; Skin infection

46

47

48 1. Introduction

49

50

51 The frequent emergence of multidrug-resistant bacteria has posed significant threats to

52 public health [1]. S. aureus is a commensal Gram-positive (G+) bacterium with a high

53 population of drug-resistant types that can cause potentially life-threatening infections, such

54 as soft tissue infection, sepsis and pneumonia, leading to 500,000 hospital visits per year [2-4].

55 Over the last two decades, much interest has been focused on natural compounds known as

56 antimicrobial peptides (AMPs). This wide group of molecules is expressed by unicellular

57 organisms, animals and plants. They offer little opportunity for the development of resistance

58 [5], and they have broad-spectrum antimicrobial activity [6, 7]. However, in previous study,

59 we extensively examined the cathelicidin-like antimicrobial peptides, such as Cbf-K16,

60 Cbf-14 and Cbf-14-2, which are all the mutants of BF-30, and found that they have prominent

61 activity against gram negative (G-) bacteria [8-10]. Thus, it is meaningful to design new

62 peptides that have superior antimicrobial activity against drug-resistant S. aureus. Despite

63 intense research on AMPs, only a handful of peptides have been clinically approved. There

4

64 are some obstacles that limit the direct development of AMPs into applicable antibiotics.

65 These obstacles include the limitations of using natural AMPs, high cytotoxicity and low

66 activity [11]. Given the inherent limitations of using AMPs, various modifications have been

67 employed to overcome these obstacles, such as peptides interception, unnatural amino acid

68 incorporation [12, 13], amino acid mutations and terminal modifications [14, 15].

69

70 Hagfish are the oldest living craniates, lacking the main components of adaptive

71 immunity. As scavengers, hagfish must protect themselves against microbes ingested with

72 rotten marine organisms that breach the physical barrier afforded by their intestinal epithelium.

73 In search of novel ingredients for innate immunity, hagfish intestinal antimicrobial peptides

74 from the tissues of Myxine glutinosa (Atlantic hagfish) were isolated and characterized as a

75 cationic peptide family [16]. In the present study, in order to obtain a peptide with excellent

76 antimicrobial activity, low cytotoxicity and a short amino acid sequence, five peptides

77 (HF-18-0, HF-18, HF-18-1, HF-18-2 and HF-14) were respectively obtained by intercepting

78 and mutating of weak charged and high hydrophobic amino acids to enhance the positive

79 charges and reduce the hydrophobicity.

80

81 In this study, in order to obtain a desirable peptide that improved activity with

82 modulation of toxicity, we evaluated the antibacterial activities and the selectivity of action

83 against drug-resistant bacteria of the modified analog peptides via antimicrobial/bactericidal

84 test and cytotoxicity test in vitro. Subsequently, we further investigated its protective effects

85 on acute systemic and/or local drug-resistant S. aureus-infected mice and elucidated the

5

86 possible mechanisms underlying its mode of action.

87

88

89 2. Materials and methods

90

91

92 2.1 Antimicrobial peptides and reagents

93 The acetate of peptides HF-18-0, HF-18, HF-18-1, HF-18-2, HFIAP-1 and FITC-HF-18

94 (>98.0%) were synthesized using the solid-phase method (GL Biochem Co., Ltd, Shanghai,

95 China). The following kits and reagents were utilized: mouse TNF-α, mouse IL-6 and mouse

96 IL-1β ELISA Kits (Multisciences Biotech Co., Ltd, Hangzhou, China); HiScript Q RT

97 SuperMix for qPCR kit and ChamQ SYBR qPCR Master Mix kit (Vazyme Biotech Co., Ltd,

98 Nanjing, Jiangsu, China); bacterial genomic DNA extraction kit, bacterial RNA extraction Kit,

99 polymyxin, ampicillin, ciprofloxacin, norfloxacin, mupirocin, SDS, DMSO, CMC, NPN,

100 DAPI, PI, paraformaldehyde and glutaraldehyde (Sangon Biotech Co., Ltd, Shanghai, China);

101 DMEM, fetal bovine serum (FBS), penicillin and streptomycin (Gibco, Vienna, NY, USA);

102 LPS, MTT, pentobarbital sodium and FITC (Sigma-Aldrich, St. Louis, USA).

103

104 2.2 Bacterial strains, cells and mice

105 S. aureus ATCC 25923, Streptococcus pneumonia (S. pneumonia) ATCC 49619,

6

106 Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853, Escherichia coli (E. coli) ATCC

107 25922, Propionibacterium acnes (P. acnes) ATCC 11827, and Klebsiella pneumonia (K.

108 pneumonia) ATCC 13883 were purchased from the American Type Culture Collection

109 (ATCC, VA, USA). The corresponding clinical strains of above and other strains, including

110 Staphylococcus epidermis (S. epidermis), Streptococcus pyogenes (S. pyogenes),

111 Enterococcus, and Dysentery bacillus (D. bacillus) were isolated from human clinical

112 specimens and identified by the Medical Laboratory Center of Zhongda Hospital Southeast

113 University (Nanjing, Jiangsu, China). All of the strains were kept in glycerin and stored at

114 -80°C until required.

115

116 Immortalized human skin keratinocyte HaCaT and human gastric epithelial GES-1 cells

117 were purchased from ATCC. The cells were cultured in DMEM supplemented with 10% FBS

118 and 1% penicillin/streptomycin. Sheep red blood cells (sRBCs) were purchased from

119 Bio-Channel Biotechnology Co., Ltd (Nanjing, Jiangsu, China). Cell culture was maintained

120 at 37°C humidified incubator supplemented with 5% CO2.

121

122 ICR 6-week-old male and female mice were obtained from Yangzhou University

123 (Yangzhou, Jiangsu, China). The animals were provided with continuous access to standard

124 rodent chowand water and housed in a rodent facility at 22 ± 1°C with a 12 h light–dark cycle.

125 Manipulations of animals were performed in accordance with the guidelines of the National

126 Institutes of Health Guide for the Care and Use of Laboratory Animals (Eighth Edition,

7

127 revised 2011), and was approved by the Science and Technology Department of Jiangsu

128 Province (SYXK 2016-0011) and the Ethics Committee of China Pharmaceutical University.

129

130 2.3 Antimicrobial activity assay in vitro

131 Minimum inhibitory concentration (MIC) of AMPs was evaluated using a standard

132 micro-broth serial dilution assay as previously described [17]. The MICs were interpreted as

133 the lowest concentration of peptide or antibiotic that completely inhibited the visible growth

134 of bacteria after 24 h of incubation at 37 °C.

135

136 The potential bactericidal effect of HF-18 was evaluated using time-killing assay.

137 Bacteria cells were diluted to 5 × 105 CFU/ml and incubated with HF-18 or polymyxin (4 ×

138 MIC). At 0, 1, 2, 4, 8 and 12 h, samples were obtained for bacterial counts. Time-killing

139 curves were constructed by plotting mean colony counts (log10 CFU/ml) vs time.

140

141 Zone of inhibition: S. aureus，E. coli, S. epidermis and P. aeruginosa were poured evenly

142 onto the plate. After incubation, filter paper disk, which impregnated with HF-18 or

143 polymyxin (16 μg), was placed on the surface of agar. Plates were incubated at 37°C for 12 h.

144 Normal saline was employed as negative control.

145

146 2.4 The hemolytic activity and the toxicity against HaCaT and GES-1 cells

147 The hemolytic activity of HF-18 was assessed according to previous methods [18]. The

8

148 sRBCs were washed with PBS and centrifuged at 900 g, 4°C for 10 min. The washed cells

149 were re-suspended in PBS to attain a dilution of 3%. The cell suspension was incubated with

150 serially diluted HF-18 (8, 32, 128, and 512 μg/ml) for 1 h at 37°C. After centrifugation, the

151 supernatant was detected at 450 nm for hemolytic activity assay.

152

153 The cytotoxic activity of HF-18 toward mouse spleen cells, human HaCaT and GES-1

154 cells were detected using an MTT assay [19]. The mice were anesthetized with 10% chloral

155 hydrate. Spleen cells were suspended in RPMI-1640 supplemented with 10% FBS and seeded

156 in 96-well plates. Serially diluted HF-18 (50, 100, 200, 400, and 800 μg/ml) were added.

157 HaCaT and GES-1 cells were resuspended and seeded in 96-well plates. The wells were

158 added with serially diluted HF-18 (16 and 128 μg/ml). Cells were incubated for 48 h at 37°C

159 in 5% CO2. MTT assay was performed at 490 nm.

160

161 2.5 Antimicrobial activity assay in S. aureus induced mouse bacteremia and skin wound

162 infection

163 ICR male mice were used for a mouse bacteremia model and it was established via an

164 intraperitoneal injection of 0.5 ml bacterial suspension (5 × 109 CFU/ml) or vehicle. After 0.5

165 and 2 h post-infection, HF-18 (2.5, 5, and 10 mg/kg, n=16/group) was intraperitoneally

166 injected. Polymyxin (5 mg/kg) was intravenously injected after inoculation for 2 h. Eight

167 hours later, six mice in each group were euthanized by exsanguination to count bacterial

168 colonies in blood, lung, liver, spleen and kidney. Serum TNF-α and IL-6 were detected by

169 ELISA kits. Mouse lung was fixed with 4% paraformaldehyde for H&E staining or directly

9

170 homogenated for Gram staining. The survival and weight of other mice were recorded for

171 7-day continuous observation.

172

173 For bacterial skin infection, ICR female mice were anesthetized with pentobarbital

174 sodium (50 mg/kg, i. p.) and shaved on the dorsal surfaces. One full-thickness skin wound

175 was created on the dorsal skin using a biopsy puncher with a diameter of 1 cm, then the

176 wounds were infected with 20 μl of bacterial suspension (5 × 107 CFU/ml of drug-resistant S.

177 aureus) and a gentle stream of air was aimed over the inoculation site until skin appeared wet

178 but absent of any standing volume from the inoculum suspension. Two hours later, wounds

179 (15 mice per group) were topically treated with vehicle (3% CMC), HF-18 (2.5, 5, and 10

180 mg/kg) or polymyxin (5 mg/kg) at once-daily application volume of 1 ml/kg. Hereinto, HF-18

181 and polymyxin were suspended in 3% CMC in PBS at concentration of 2.5, 5, and 10 mg/ml

182 (HF-18) or 5 mg/ml (polymyxin). Each mouse was housed separately to avoid cross

183 contamination. The wounds were photographed at the indicated time points and wound sizes

184 were determined using Image J. On days 3 and 7, bacterial counts of wound specimens were

185 recorded, serum cytokines were detected by ELISA kit and wound specimens were collected

186 for H&E staining.

187

188 2.6 Membrane permeability assay

189 The morphological changes of bacteria treated with peptide were observed under TEM

190 [20]. S. aureus (1 × 107 CFU/ml) was mixed with HF-18 (16 μg/ml) for 40 min incubation,

191 fixed with 2.5% glutaraldehyde and subjected to routine TEM assay. Untreated bacteria were

10

192 used as negative control.

193

194 Zeta-potential values of S. aureus (1 × 107 CFU/ml) treated with HF-18 (2, 4, 8 and 16

195 μg/ml) were obtained by a Zeta Potential & Partical Size Analyser (Brookhaven Instruments

196 Corporation, Austin, Texas, USA) [21].

197

198 Peptide-induced outer membrane permeability was measured as previously described

199 [22]. Briefly, S. aureus (1 × 107 CFU/ml) suspended in 5 mM HEPES buffer (pH7.4) was

200 mixed with NPN reagent (10 μM). After the addition of HF-18 (2, 4, 8 and 16 μg/ml) or 0.1%

201 Triton X-100, the time-dependent effect of HF-18 on NPN fluorescence was recorded at the

202 excitation/emission wavelengths of 350/420 nm, respectively.

203

204 The ability of HF-18 to adhere to the cell membrane of S. aureus was assessed by BD

205 AccuriC6 flow cytometer (Bd Biosciences, New York, USA) assay. S. aureus (1 × 107

206 CFU/ml) were incubated with FITC or FITC-HF-18 (4 μg/ml) for 40 min and analyzed by

207 flow cytometry.

208

209 The ability of HF-18 or HF-14 to permeate the inner membrane of S. aureus was

210 assessed by flow cytometer assay. That is, S. aureus (1 × 107 CFU/ml) were incubated with

211 HF-18 (2, 4, 8 and 16 μg/ml) or HF-14 (32, 64, 128 and 256 μg/ml) for 40 min at 37°C.

212 Following the addition of PI (20 μg/ml) mixture for 5 min in the dark, cells were analyzed by

213 flow cytometry.

11

214

215 2.7 DNA binding assay

216 Bacteria suspensions (1 × 105 CFU/ml) were cultured with sub-MICs HF-18 (0.5, 1, and

217 2 μg/ml) or HF-14 (8, 16, and 32 μg/ml) for 0, 16 and 24 h for bacterial concentrations

218 determination by spectrophotometer at 600 nm.

219

220 To test the ability of HF-18 or HF-14 to block DNA, DNA samples were extracted using

221 bacterial genomic DNA extraction kit and incubated with HF-18 (1, 2, 4, 8, and 16 μg/ml) or

222 HF-14 (2, 4, 8, 16, and 32 μg/ml) at 25°C for 10 min. Then, DNA binding assay was carried

223 out on the basis of Habash’s method [23].

224

225 The localization of sub-MIC HF-18 on S. aureus was detected by confocal laser scanning

226 microscopy. Bacteria were incubated with FITC-HF-18 (2 μg/ml) for 40 and 60 min and then

227 re-suspended in DAPI (0.1 μg/ml) for 10 min. The samples were collected for fluorescence

228 detection.

229

230 2.8 RT-qPCR analysis

231 Bacteria suspensions were cultured with HF-18 (0.5, 1, and 2 μg/ml) for 40 min. Total

232 bacterial RNA was extracted using bacteria RNA extraction Kit. The cDNA were obtained

233 using a HiScript Q RT SuperMix for qPCR kit. The qPCR was subsequently performed

234 according to a ChamQ SYBR qPCR Master Mix kit. The sequences of the forward and

12

235 reverse primers were as follows: pvl forward, 5′-ATG GTC AAA AAA AGA CTA TT-3′ and

236 reverse, 5′-TCA ATT ATG TCC TTT CAC TTT AAT TTC-3′; sea forward, 5′-TTG GAA

237 ACG GTT AAA ACG AA-3′ and reverse, 5′-GAA CCT TCC CAT CAA AAA CA-3′; nuc

238 forward, 5′-GCG ATT GAT GGT GAT ACG GTT-3′ and reverse, 5′-AGC CAA GCC TTG

239 ACG AAC TAA AGC-3′; seb forward, 5′-TGT TAG GGT ATT TGA AGA TGG-3′ and

240 reverse, 5′-CGT TTC ATA AGG TGA GTT GTT-3′; 16S rRNA forward, 5′-AGA GTT TGA

241 TCM TGG CTC AG-3′ and reverse, 5′-TAC GGY TAC CTT GTT ACG ACT T-3′. The

242 relative mRNA expression was normalized to internal control and determined using the

243 comparative Cq method (2-ΔΔCq).

244

245 2.9 Statistical analysis

246 All data are presented as the mean ± standard deviation of at least three independent

247 experiments. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software,

248 Inc., La Jolla, CA, USA). Data were analyzed using one-way analysis of variance. Differences

249 were considered to be statistically significant at P < 0.05.

250

251

252 3. Results

253

254

255 3.1 The design and characterizations of designed peptides

13

256 In this design, 18 amino acids were intercepted from hagfish intestinal peptide

257 (HFIAP-1), generating HF-18-0. His12 and Gly14 were mutated to Lys12 and Phe14,

258 respectively, to increase the positively charged amino acids, resulting in HF-18. To reduce

259 cytotoxicity, Trp7 was mutated to Arg7 or Lys7 to obtain HF-18-1 and HF-18-2, respectively.

260 To reduce synthetic cost, 14 amino acids were intercepted from HF-18-2, generating HF-14.

261 The peptides design process is shown in Fig. 1A. The optimal peptide was selected based on

262 its antimicrobial activity and cytotoxicity.

263

264 The values of key physicochemical properties in HF-18, including hydrophobic moment,

265 hydrophobicity and instability index, were 0.7, 0.244 and 26.6, respectively (Fig. 1B). In 25

266 mM SDS, the CD spectrum of HF-18 showed a positive peak at approximately 195 nm and

267 two negative peaks near 208 and 222 nm (Fig. 1C), which are characteristic of an α-helix

268 structure. HF-18 had 60.4% α-helicity in SDS, while the α-helical content was 0.0% in water,

269 indicating that HF-18 possessed high hydrophobicity and presented mostly as an α-helix in a

270 membrane-mimetic environment.

271

272 3.2 The functional screening of the peptides based on their antimicrobial activity and

273 cytotoxicity

274 Hemolytic activity is an important parameter to evaluate the eukaryotic cytotoxicity of

275 peptides. Five peptides exhibited low hemolytic activity against sRBCs (Fig. 2A). The spleen

276 cell viability of all four mutated peptides was enhanced relative to that of HFIAP-1. Both

277 HF-18 and HF-18-1 showed lower cell viability than HF-18-0 and HF-18-2 at doses of more

14

278 than 100 μg/ml (Fig. 2B). For HaCaT and GES-1 cells, these peptides nearly had no

279 cytotoxicity at a concentration of 32 μg/ml. HF-18 (128 μg/ml) had higher cell viability than

280 HFIAP-1 (73.9%) and lower cell viability than other three peptides (Fig. 2C), suggesting that

281 this peptide had low cytotoxicity.

282

283 As summarized in Table 1, HF-18-0 displayed a reduced antimicrobial activity against

284 both G- and G+ bacteria with an MIC range of 16-128 μg/ml when compared to the parent

285 peptide with an MIC range of 8-64 μg/ml. However, there was no significant change in the

286 antimicrobial activity of HF-18-1 and HF-18-2 compared to HF-18-0. Meanwhile, HF-14,

287 which possessed the shortened amino acid sequence, displayed no activity. Excitingly, HF-18

288 showed a superior activity with an MIC range of 4-32 μg/ml and exhibited more than twice

289 the activity than HF-18-0, especially against clinical norfloxacin or mupirocin resistant S.

290 aureus (MIC 4 μg/ml), which was similar to polymyxin.

291

292 HF-18 also had a remarkable bacteriolytic ring against drug-resistant S. aureus compared

293 with that of polymyxin (Fig. 2D). The results illustrated that HF-18 had powerful

294 antimicrobial activity against drug-resistant S. aureus.

295

296 The time-kill curves showed that HF-18 (4 × MIC) exerted killing effects on S. aureus, S.

297 epidermisa, E. coli and P. aeruginosa. This peptide was able to eliminate bacteria by 12 h and

298 exhibited impressive bactericidal activity against S. aureus than that of polymyxin (1h vs 8 h)

299 (Figs. 2E-H), indicating its efficacious bactericidal effect.

15

300

301 3.3 HF-18 effectively protected mouse from drug-resistant S. aureus infection

302 Since lethal bacteremia is a common outcome of drug-resistant S. aureus infections, we

303 investigated the ability of HF-18 to work systemically and to rescue mice from this type of

304 infection. As shown in Figs. 3A and B, the infected mice with HF-18 (10 mg/kg) treatment

305 achieved 90% survival rate, the same as that of polymyxin. Mice lung injury was also

306 alleviated by HF-18 in a dose-dependent manner (Fig. 3C). HF-18 also displayed remarkable

307 weight maintenance (Fig. 3D). Gram staining results exhibited the same tendency, showing a

308 barely visible round of S. aureus in the HF-18-treated (10 mg/kg) group (Fig. 3E). Treatment

309 with HF-18 (10 mg/kg) significantly reduced bacterial titers in blood, lung, liver, spleen and

310 kidney tissues with 1.6, 1.9, 2.5, 2.8, and 2.1 log CFU compared with the model group (Fig.

311 3F). Alveolar interstitial congestion, edema, pulmonary blood vessel dilatation and substantial

312 inflammatory cell infiltration were obvious in infected mice. The administration of HF-18

313 notably improved these lung injuries (Fig. 3G) and showed a maximum reduction in the

314 secretion of TNF-α and IL-1β from 451.2 to 316.9 pg/ml and 302.4 to 202.9 pg/ml,

315 respectively (Figs. 3H and I). The data obtained above demonstrated that HF-18 exerted

316 potent antimicrobial effects in a mouse bacteremia model.

317

318 3.4 HF-18 significantly ameliorated skin wound infection induced by drug-resistant S.

319 aureus

320 S. aureus is the leading cause of skin and soft tissue infections. Here, we established a

16

321 mouse model of skin infection induced by inoculation of S. aureus on skin wounds to test the

322 potential effectiveness of HF-18. The sizes of the wounds were measured to evaluate the

323 severity of infection. It was further demonstrated that, after an 11-day therapeutic course,

324 treatment with HF-18 (5 and 10 mg/kg) obviously alleviated the drug-resistant S. aureus

325 infection in mice with skin injuries, showing wound healing and no obvious ulceration (Figs.

326 4A and B). In the 13-day inoculation period, the wound healing rates of the HF-18-treated

327 groups were higher than polymyxin-treated group (81.7%), especially for that of HF-18 high

328 group (Fig. 4C). However, in the absence of drug treatment, mouse wound healing was slow,

329 showing nonreduced unclosed wound areas and purulent wounds. These results demonstrated

330 that HF-18 could significantly alleviate skin infection induced with drug-resistant S. aureus.

331

332 The ability of HF-18 to reduce drug-resistant S. aureus colonization was determined

333 within the 13-day treatment course. The bacterial titers of HF-18 (10 mg/kg) on day 7

334 compared with those on day 3 were decreased up to 5.4 log CFU, while there were 5.0 log

335 CFU reductions in the polymyxin group. After a 7-day therapeutic course, there were

336 remarkable 2.7 and 3.5 log CFU reductions in the HF-18 (5 and 10 mg/kg) groups compared

337 with those in the model group (Fig. 4D), indicating that HF-18 could effectively suppress the

338 skin bacterial infection.

339

340 H&E staining showed that HF-18 exhibited a better wound-healing effect on day 11. The

341 skin ulcer had healed completely, and the epithelium was mainly covered of HF-18 (5 and 10

342 mg/kg) groups, and the therapeutic effects resembled those of the polymyxin group, while the

17

343 model group was still covered with a layer of inflamed hemorrhagic tissue showing

344 hyperemia and edema. On day 3, the surface of each group was covered with a thick layer of

345 inflamed hemorrhagic and necrotic tissue, showing deep tissue hyperemia and edema (Fig.

346 4E).

347

348 Furthermore, severe inflammatory infiltration was developed as characterized by the

349 presence of a large number of inflammatory cells in wounds after infection. We also detected

350 the contents of serum TNF-α and IL-6 and found that the secretion of these two

351 pro-inflammatory cytokines was reduced from 328.9 to 211.4 pg/ml and 134.6 to 87.9 pg/ml

352 (p < 0.05), respectively, after 11 days post HF-18 (10 mg/kg) treatment (Figs. 4F and G),

353 indicating that HF-18 may decrease the systemic inflammatory response induced by local

354 infection by inhibiting the bacterium from spreading systemically.

355

356 3.5 HF-18 disrupted the cell membrane of drug-resistant S. aureus

357 To explore whether HF-18 could interfere with bacterial membrane integrity, a TEM

358 assay was used to observe the ultrastructure of cells. Untreated cells showed smooth, clearly

359 visible structures, with distinct outer membranes and intact inner membranes. HF-18-treated

360 bacteria exhibited a complete loss of cytoplasmic content and significant morphological

361 changes, resulting in a substantial disruption of the cell membrane (Fig. 5A).

362

363 The affinity of this peptide for bacterial membrane was evaluated by FITC-HF-18. After

364 40 min of incubation, HF-18 had a high affinity for the bacterial membrane with 98.0%

18

365 adherence rates (Fig. 5B). To confirm whether the accumulation of HF-18 on the bacterial

366 surface is due to charge attraction, a zeta-potential assay was performed. After adding peptide,

367 there was a concentration-dependent zeta-potential increase from -30.2 ± 3.3 mV to -1.2 ± 0.2

368 mV (Fig. 5C), demonstrating that HF-18 could effectively neutralize the negative charge on

369 the bacterial surface.

370

371 In NPN uptake assay, as expected, the addition of HF-18 resulted in a dramatic increase

372 in fluorescence intensity, because this hydrophobic fluorescent probe was taken up by the

373 bacterial outer membrane. The fluorescent uptake rate gradually increased to more than 40%

374 after 5 min and achieved 98.9% after 30 min at higher concentrations of HF-18, which was in

375 accordance with the results obtained from cells treated with 0.1% Triton X-100 (Fig. 5D).

376

377 PI uptake is used as an indicator to analyze the ability of HF-18 and HF-14 to rupture the

378 membrane of intact S. aureus cells. HF-18 (2, 4, 8, and 16 μg/ml) caused a 29.6%, 73.9%,

379 86.5% and 91.9% fluorescence uptake of PI, respectively, suggesting that the permeability of

380 bacterial inner membrane was increased by HF-18 in a dosage-dependent manner (Fig. 5E).

381 These results were consistent with the findings from zeta-potential neutralization and NPN

382 uptake. However, HF-14 had no effect on PI fluorescence uptake of cell membrane (Fig. 5F).

383 Data above revealed the potential binding ability of HF-18 to the bacterial surface, which may

384 cause a membrane disruption cascade.

385

386 3.6 HF-18 bound to genomic DNA and inhibited the expression of pvl and nuc genes

19

387 To confirm the suppressive effect of the peptide on intracellular DNA at sub-MIC

388 concentrations, an electrophoretic gel mobility shift assay was conducted in vitro. The

389 results showed that HF-18 (2 μg/ml) was able to bind to the genomic DNA of drug-resistant

390 S. aureus. The electrophoretic movement of DNA was completely inhibited by HF-18 when

391 the peptide concentration was higher than 4 μg/ml compared with the movement of

392 unprocessed DNA (Figs. 6A and B). Interestingly, HF-14 (8, 16 and 32 μg/ml) could

393 completely inhibit the electrophoretic movement of genomic DNA from drug-resistant S.

394 aureus but did not result in evident growth retardation (Figs. 6C and D).

395

396 In addition, the location of sub-MIC HF-18 within bacteria cells was observed

397 simultaneously. HF-18 (2 μg/ml) was able to penetrate the bacterial membrane and then bind

398 to DNA after 60 min of coincubation (Fig. 6E). This DNA binding effect indicated that

399 HF-18 achieved antimicrobial activity against drug-resistant S. aureus at sub-MIC

400 concentrations mainly by binding the genomic DNA instead of disrupting the bacterial

401 membrane.

402

403 The wide range of pathogenicity of S. aureus can be attributed to its ability to produce

404 various secreted virulence factors. In this study, the results showed that pvl and nuc genes

405 were reduced 2.1- and 1.5-fold, in HF-18 (2 μg/ml)-treated groups, while there were no

406 obvious inhibitory effects on staphylococcal enterotoxin A (sea) and staphylococcal

407 enterotoxin B (seb) genes (Fig. 6F), suggesting that HF-18 could suppress the expression of

408 virulence genes under nonmembrane-disrupting conditions.

20

409

410

411 4. Discussion

412

413

414 Currently, bacterial resistance to conventional antibiotics has reached hazardous levels

415 and may represent an impending return to the pre-antibiotic era [24]. AMPs are

416 broad-spectrum antimicrobials that are not easy to acquire resistance and have been candidate

417 alternatives to conventional antibiotics [25]. In our design, HF-18 stood out in the analog

418 peptides, for its powerful antimicrobial activity (4-32 μg/ml) (Table 1), featuring high net

419 charges, hydrophobicity and α-helicity while low cytotoxicity. As reported, the increasing

420 number of positively charged amino acids in peptides augmented their ability to combine with

421 the negatively charged bacterial membrane [26-28], and the increase of hydrophobicity

422 enhanced their affinity for the lipid components of the bacterial membrane, which contributed

423 to the increased antimicrobial activity. Trp is a hydrophobic amino acid. The antimicrobial

424 activity of HF-18 was significantly reduced attribute to the mutation of Trp into Arg and Lys.

425 This may partly account for the cytotoxicity of peptide, showing as the decreased cytotoxicity

426 of HF-18-1 and HF-18-2 towards mouse spleen cells, human HaCaT and GES-1 cells

427 compared with that of HF-18. Importantly, no cytotoxicity of HF-18 was observed at HF-18

428 dosage of 32 μg/ml, which was higher than the concentration at which the peptide exerted

429 antimicrobial activity (4 μg/ml). In contract, as Mario et al have reported that peptide Bac5

21

430 (1-31) exhibited good antimicrobial activity against erythromycin-resistant E. coli, but

431 showed high cytotoxicity [29]. In summary, these data suggest that positive charges and

432 hydrophobicity play important roles in enhancing peptide antimicrobial activity and reducing

433 peptide cytotoxicity.

434

435 Here, we investigated whether this design is conducive to the antimicrobial activity of

436 HF-18 against drug-resistant G+ bacteria. S. aureus, as a representative G+ bacterium, are

437 multidrug-resistant bacteria responsible for several cases of hospital-acquired infections,

438 which constitute a global public health problem. RP557 obtained by Woodbum et al could not

439 kill S. aureus thoroughly at 8 × MIC [30]. 2IH3 and 2IH4 obtained by Lai et al exhibited a

440 superior antimicrobial activity against G- bacteria; while for G+ bacteria, no activity was

441 detected [31]. Therefore, the discovery of new molecules capable of inhibiting the growth or

442 killing S. aureus would have a large impact on the treatment of S. aureus-mediated diseases.

443 As expected, HF-18 exhibited a stronger and faster antimicrobial effects against G+ bacteria

444 compared with that of other four peptides, especially for norfloxacin and mupirocin-resistant

445 S. aureus. For acute systemic infection and chronic local infection of skin wound induced by

446 S. aureus, it could eliminate bacterial counts in tissues or skin wounds to inhibit the systemic

447 dissemination of bacteria, accompanied by the improved mouse survival rate, alleviated lung

448 injury and enhanced wound healing rates. S. aureus is the leading cause of human infections

449 capable of invading most tissues of the human body, giving rise to skin inflammation,

450 pneumonia and septicemia [32, 33]. Inflammatory cytokines, including TNF-α, IL-1β and

451 IL-6 along with superoxide anions and other pro-inflammatory cytokines, may increase tissue

22

452 injury [29, 34, 35]. Here, we found that HF-18 effectively decreased the excessive secretion

453 of TNF-α, IL-1β and IL-6 and then inhibited the bacteria-induced inflammatory response to

454 alleviate lung injury or reduce purulence production. These data suggest that HF-18 can

455 improve infectious diseases by effectively eliminating drug-resistant bacteria.

456

457 The binding of cationic AMPs to the cell membrane is crucial to its biological activity.

458 Alpha-helical peptides, such as membrane lysis AMPs, have obvious hydrophobic and

459 hydrophilic surfaces that favor the insertion of AMPs into bacterial cell membranes and

460 subsequent membrane permeability enhancement and membrane damage [36]. HF-18

461 exhibited a typical α-helix configuration in SDS (a membrane-mimetic environment) (Fig. 1D)

462 with α-helical contents of 60.4%. It had a destructive effect on the bacterial cell membrane in

463 a dose-dependent manner, reflecting as the increased uptake of NPN and PI by 91.9% and

464 98.9%, respectively. In addition, the ultimate morphological changes in the membrane

465 structure and pore formation on the membrane surface caused the leakage of cytoplasmic

466 important contents of S. aureus after HF-18 treatment.

467

468 At sub-MIC levels, HF-18 could not interfere with the basic viability of S. aureus but

469 bound to S. aureus genomic DNA, as shown by the complete inhibition of the electrophoretic

470 movement of the DNA in gel retardation assay. Because the favorable electrostatic potential

471 and amphipathic hydrophobicity of cationic AMPs make they cause aggregation of ribosomes

472 which are polyanionic, and mediate this interaction in bacteria. Magainin had little effect on

473 DNA binding even at concentrations up to 128 μg/ml [37]. Excitedly, HF-18 could effectively

23

474 bind to the DNA of drug-resistant S. aureus at a concentration of 2 μg/ml. As previously

475 described, S. aureus can produce a myriad of virulence factors that allow the bacteria to attach

476 to host cells, invade the tissues, evade the immune system and release an array of exoproteins

477 and toxins, such as pvl, nuc, sea and seb virulence genes [38, 39]. These are well-known

478 virulence factors of S. aureus causing severe disease. Wang Y et al. reported that peptide

479 GH12 could significantly downregulate virulence genes, thus inhibiting the synthesis of

480 related virulence factors and inducing the activity of related enzymes [40]. Similarly, we also

481 confirmed that HF-18 notably inhibited the expression of pvl and nuc virulence genes of

482 drug-resistant S. aureus to relieve severe infections. However, from the data obtained, it is

483 still unclear whether HF-18 could bind to virulence genes specifically, such as pvl and nuc,

484 which needs to be further clarified.

485

486 HF-18 was shown to have activities against the cell membrane and genomic DNA. Its

487 mutant HF-14 also had the DNA binding ability in vitro; however, it could not disrupt the

488 bacterial cell membrane to exert the antibacterial activity, as shown in PI fluorescence uptake

489 and MIC experiment, indicating that the peptide could bind to genomic DNA, but it doesn't

490 necessarily kill the bacteria. As one of the candidates of antibacterial agents, the bacterial cell

491 membrane disruption of peptides plays a more important role in antimicrobial activity.

492

493 In summary, the antimicrobial activity of the active domain HF-18 has been investigated,

494 with the interception and mutation of amino acids within the hagfish intestinal peptide. This

495 peptide has no obvious hemolytic activity and cytotoxicity towards mammalian cells.

24

496 Moreover, the peptide design endows HF-18 a much lower cost and enhanced

497 antibacterial/bactericidal activities against various G+ bacteria, even drug-resistant bacteria. In

498 vivo, HF-18 exhibits a potent protective effect against norfloxacin and mupirocin-resistant S.

499 aureus-induced mice with acute bacteremia or local skin infection after wound. The possible

500 mechanisms underlying its mode of action involve direct antimicrobial activity via significant

501 membrane rupture properties at high MIC concentrations and the dysfunction of bacterial

502 biological function by binding to bacterial DNA and the remarkable suppression on the

503 expression of pvl and nuc virulence genes at the sub-MIC concentrations. The data above

504 suggest that this peptide has potential clinical application prospects.

505

506

507 Acknowledgements

508 This work was supported by National Natural Science Foundation of China (No.

509 81673483, 81803591), Young Scientists of Natural Science Foundation of Jiangsu Province

510 of China (No. BK20180563), National Mega-project for Innovative Drugs

511 (2019ZX09721001-004-005), the “111 Project” from the Ministry of Education of China and

512 the State Administration of Foreign Expert Affairs of China (No. 111-2-07) and National Key

513 Research and Development Program of China (2018YFA0902000).

514

515

516 Conflict of interest

25

517 The authors declare that no conflict of interest exists in the present study.

518

519

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33

689 Figure legends

690 Fig. 1: Design and characterizations of the peptides. (A) The peptide domain design process

691 from hagfish intestinal antimicrobial peptide to different peptides. (B) The helical wheel

692 projection peptide sequence and key physicochemical properties of HF-18. (C) The spectra of

693 HF-18 in water, 50 mM LPS, and 25 mM SDS. The final spectra were the average of 6 scans

694 after subtracting the spectrum obtained under the same conditions of a sample without

695 peptide.

696

697

698 Fig. 2: The functional screening of the peptides based on the antimicrobial activity and

699 cytotoxicity in vitro. (A) Hemolysis of five peptides against sRBCs at 8, 32, 128, and 512

700 μg/ml. Positive control: 1% Triton X-100. (B) Cytotoxicity of five peptides against mouse

701 spleen cells at 50, 100, 200, 400, and 800 μg/ml. (C) Cytotoxicity of five peptides against

702 epithelial cell lines (human HaCaT and GES-1) at 16 and 128 μg/ml. (D) Images of the

703 inhibition zones of HF-18 against S. aureus, S. epidermisa, E. coli and P. aeruginosa. Effect

704 of peptides on the viability of different bacterial strains. The time-kill curves of HF-18 against

705 (E) S. aureus, (F) S. epidermisa, (G) E. coli and (H) P. aeruginosa.

706

707

708 Fig. 3: Antimicrobial activity of HF-18 in S. aureus-infected mice. (A) Survival plot of mice

34

709 inoculated via an intraperitoneal injection with S. aureus (5 × 109 CFU/ml) and treated with

710 HF-18 (2.5, 5, and 10 mg/kg) or polymyxin (5 mg/kg). (B) The infection, induction and

711 treatment approach in vivo. (C) The protective effect of HF-18 on acute lung damage in

712 infected mice, and the gross appearance of lung lesions after infection. (D) Body weight plot

713 of mice inoculated via an intraperitoneal injection with S. aureus and treated with HF-18 or

714 polymyxin. (E) Gram staining of homogenized lung tissue. (F) Colonization of S. aureus

715 inoculum in the blood, lungs, liver, spleen, and kidneys of mice at 8 h after infection. (G)

716 H&E images (× 200) of lung lesions after infection. (H) Serum levels of TNF-α and (I) IL-1β

717 in mice at 8 h after infection. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the model.

718

719

720 Fig. 4: HF-18 protected mice from S. aureus infection. (A) Schematic illustration of the

721 antimicrobial activity of HF-18. (B) Images of the infected wounds on days 0, 3, 7, and 11

722 after treatment with HF-18. (C) Percent changes in the healing rate in wound size over time.

723 (D) Colonization of S. aureus inoculum in wounds on days 3 and 7 after injury. ###P < 0.001

724 compared with the model on day 3, **P < 0.01, ***P < 0.001 compared with the model on day

725 7. (E) H&E stained sections (× 100) of infected wounds and (F-G) serum levels of TNF-α and

726 IL-6 on days 3 and 11. #P < 0.05, ###P < 0.001 compared with control, *P < 0.05, **P < 0.01

727 compared with model.

728

729

35

730 Fig. 5: HF-18-induced membrane-destabilizing effects. (A) TEM of HF-18-induced bacterial

731 membrane disruption. (B) Flow cytometry assay of HF-18 adhering to the cell membrane of S.

732 aureus strains. (C) The zeta-potential of S. aureus treated with HF-18 (2, 4, 8, and 16 μg/ml).

733 (D) Outer membrane permeability induced by HF-18 (2, 4, 8, and 16 μg/ml) was detected by

734 NPN uptake in S. aureus. The enhanced uptake of NPN was measured by the increased

735 fluorescence of NPN. Positive control: 0.1% Triton X-100. (E) The inner membrane rupture

736 induced by HF-18 was detected by PI in S. aureus with 2, 4, 8, and 16 μg/ml peptide

737 concentrations. (F) The effect of HF-14 on inner membrane was detected by PI in S. aureus

738 with 32, 64, 128, and 256 peptide concentrations.

739

740

741 Fig. 6: Sub-MIC HF-18 bound to genomic DNA and downregulated the virulence genes of S.

742 aureus. (A) Effect of HF-18 at sub-MIC concentrations on the growth curve of S. aureus. (B)

743 DNA binding activity of HF-18 to S. aureus genomic DNA. (C) Effect of HF-14 at different

744 concentrations on the growth curve of S. aureus. (D) DNA binding activity of HF-14 to S.

745 aureus genomic DNA. (E) The localization of sub-MIC FITC-HF-18 within S. aureus was

746 determined by confocal laser scanning microscopy. The permeability of the cytoplasmic

747 membrane of HF-18 was indicated by FITC fluorescence (green), and the intracellular DNA

748 of bacteria was stained by DAPI (blue). (F) Expression of various virulence genes of S.

749 aureus in response to treatment with HF-18 (sub-MIC). 16S rRNA servers as an internal

750 control. *P < 0.05, **P < 0.01 compared with control.

36

751 Author Contributions Section

752 Meiling Jiang: Conceptualization, Methodology, Software. Meiling Jiang, Xiaoqian Yang:

753 Data curation, Writing - Original draft preparation. Haomin Wu, Ya Huang: Visualization,

754 Investigation. Jie Dou: Supervision, Validation. Changlin Zhou and Lingman Ma: Writing

755 - Reviewing and Editing.

756

757 Table 1 MIC of the Designed Peptides and Antibiotics against Microorganisms 758759

MIC（μg/ml）Microorganism

strains HF-18

HF-18-0

HF-18-1

HF-18-2

HF-14 HFIAP-1 Polymyxin Penicillin Ciprofloxacin Norfloxacin Mupirocin

S. aureus ATCC 25923 8 64 32 32 >128 16 16 2 2 8 1

S. aureus a 4 16 32 16 >128 32 4 32 16 >128 >128S. epidermis a 8 16 16 16 >128 64 8 2 2 8 64S. pneumoniae ATCC 49619 8 32 64 32 >128 16 4 >128 64 2 >128

S. pneumoniae a 16 128 128 128 >128 32 8 >128 >128 >128 >128S. pyogenes a 8 128 128 128 >128 16 64 >128 >128 >128 0.5P. acnes ATCC 11827 8 128 128 128 >128 8 >128 0.25 8 >128 >128

G+

Enterococcus a 8 16 8 8 >128 16 16 0.5 0.5 16 >128

E. coli ATCC 25922 8 64 8 8 >128 8 4 64 2 4 128

E. coli a 8 32 8 8 >128 8 4 >128 32 >128 >128P. aeruginosa ATCC 27853 16 32 32 32 >128 16 4 2 2 4 1

P. aeruginosa a 8 16 16 8 >128 32 4 >128 64 >128 >128K. pneumoniae ATCC 13883 16 128 128 128 >128 16 1 >128 2 1 >128

K. pneumoniae a 32 128 128 64 >128 16 8 >128 8 >128 >128

G-

D. bacillus a 8 16 8 8 >128 8 8 >128 8 16 64760 aThe strains are the clinical isolates.761762763

37

764765

766

767

38

768

39

769

40

770

41

771

42

772

43

773