海参抗菌肽的活性筛选与功效评价

doi:10.11978/2022208

热带海洋学报 ›› 2023, Vol. 42 ›› Issue (4): 184-194.doi: 10.11978/2022208CSTR: 32234.14.2022208

海参抗菌肽的活性筛选与功效评价

袁华标¹^,²^,⁴(), 黄靖彤¹^,²^,⁴, 万鹏¹^,³^,⁴, 蔡冰娜¹^,³^,⁴, 潘剑宇¹^,³^,⁴, 张煜航¹^,²^,⁴, 凌娟¹^,³^,⁴, 陈华¹^,³^,⁴()

1.中国科学院南海海洋研究所, 中国科学院热带海洋生物资源与生态重点实验室, 广东广州 510301
2.中国科学院大学, 北京 100049
3.南方海洋科学与工程广东省实验室(广州), 广东广州 511458
4.中国科学院南海生态环境工程创新研究院, 广东广州 510301

收稿日期:2022-09-30 修回日期:2022-11-09 出版日期:2023-07-10 发布日期:2022-12-05
作者简介:
袁华标(1997—), 男, 广东湛江人, 硕士研究生, 从事海洋生物活性肽的分离纯化与活性研究。email: yuanhuabiao20@mails.ucas.ac.cn
基金资助:
广东省海洋经济发展专项(粤自然资合[2022]036); 广东省海洋经济发展专项(粤自然资合[2020]036); 广东省海洋经济发展专项(粤自然资合[2020]041); 广东省自然科学基金项目(2022A1515010767); 广东省重点领域研发计划(2020B1111030004); 中国科学院南海生态环境工程创新研究院自主部署项目(ISEE2021PY05); 海南省重点研发计划(ZDYF2021SHFZ109)

Screening and efficacy evaluation of antibacterial peptides from Holothuroidea

YUAN Huabiao¹^,²^,⁴(), HUANG Jingtong¹^,²^,⁴, WAN Peng¹^,³^,⁴, CAI Bingna¹^,³^,⁴, PAN Jianyu¹^,³^,⁴, ZHANG Yuhang¹^,²^,⁴, LING Juan¹^,³^,⁴, CHEN Hua¹^,³^,⁴()

1. CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
3. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
4. Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou 510301, China

Received:2022-09-30 Revised:2022-11-09 Online:2023-07-10 Published:2022-12-05
Supported by:
Marine Economy Development Special Project of Guangdong Province(GDNRC[2022]036); Marine Economy Development Special Project of Guangdong Province(GDNRC[2020]036); Marine Economy Development Special Project of Guangdong Province(GDNRC[2020]041); National Natural Science Foundation of Guangdong, China(2022A1515010767); Key-Area Research and Development Program of Guangdong Province(2020B1111030004); Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences(ISEE2021PY05); Key Research and Development Program of Hainan Province(ZDYF2021SHFZ109)

摘要/Abstract

摘要：

本文将已有文献报道的海洋动物源抗菌肽(毒素除外)与海参蛋白组进行比对, 结合生物信息学软件, 设计、筛选海参抗菌肽, 并对其理化特性、抗菌活性与机制、生物相容性与细胞活性等进行研究。研究结果发现, 通过软件模拟与抗菌活性预测, 最终筛选并固相合成7条海参抗菌肽(H1—H7)。其中, H4(RVHRFLRR)可通过细菌膜电位去极化、膜透化等方式, 抑制金黄色葡萄球菌(Staphylococcus aureus)、铜绿假单胞菌(Pseudomonas aeruginosa)、创伤弧菌(Vibrio vulnificus)生长, 在较低浓度(31.25µg·mL^-1)时, 可使S. aureus细菌总数降至84.93%±4.21%(p<0.01), P. aeruginosa细菌总数降至95.92%±0.52%(p<0.01), V. vulnificus细菌总数降至77.14%±1.37%(p<0.01)。H4生物相容性好, 在<1000µg·mL^-1时均未表现出溶血性或细胞毒性, 其可能通过与细胞膜表面受体(EGFR、VEGFR2、FGFR1等)发生相互作用进而促进细胞迁移。在浓度为62.50µg·mL^-1和250µg·mL^-1时, H4均可显著促进L929细胞发生迁移(p<0.05)。实验结果表明, 海参抗菌肽H4生物相容性好, 兼有抗菌、促愈合功效, 可望作为一种潜在功能物质用于感染创面的修复与再生。

关键词: 海参, 抗菌肽, 生物信息学, 细胞迁移

Abstract:

In this paper, we compared the existing marine animal origin antibacterial peptides reported (Toxin except) with the holothurian proteome, combined with bioinformatics software to design, screen holothurian antibacterial peptides, and to investigate their physicochemical properties, antibacterial activity, mechanism, biocompatibility and cellular activity. It was found that through software simulation and antibacterial activity prediction, 7 holothurian bacteriostatic peptides (H1~H7) were finally screened and solid-phase synthesized. Among them, H4 (RVHRFLRR) could inhibit the growth of Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio vulnificus through bacterial membrane potential depolarisation or membrane permeabilisation. At a lower concentration (31.25 µg·mL^-1), it reduced the total bacterial count of S. aureus to 84.93%±4.21%(p<0.01), P. aeruginosa to 95.92%±0.52%(p<0.01) and V. vulnificus to 77.14%±1.37%(p<0.01). It was also found that H4 was biocompatible and did not exhibit haemolytic or cytotoxic properties at < 1000 µg·mL^-1. In addition, H4 may interact with cell membrane receptors (EGFR, VEGFR2, FGFR1, etc.) to promote cell migration. H4 significantly promoted the migration of L929 cells at both 62.50 µg·mL^-1 and 250 µg·mL^-1 (p<0.05). The above experimental results indicate that holothurian antibacterial peptide H4 is biocompatible, has both antibacterial and pro-healing effects, and can be used as a potential functional active substance for the repair and regeneration of infected wounds.

Key words: Holothuroidea, antibacterial peptide, bioinformatics, cell migration

袁华标, 黄靖彤, 万鹏, 蔡冰娜, 潘剑宇, 张煜航, 凌娟, 陈华. 海参抗菌肽的活性筛选与功效评价[J]. 热带海洋学报, 2023, 42(4): 184-194.

YUAN Huabiao, HUANG Jingtong, WAN Peng, CAI Bingna, PAN Jianyu, ZHANG Yuhang, LING Juan, CHEN Hua. Screening and efficacy evaluation of antibacterial peptides from Holothuroidea[J]. Journal of Tropical Oceanography, 2023, 42(4): 184-194.

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参考文献 33

[1]	侯晓艳, 2019. 基于生物信息学与多肽组学的花椒籽抗菌肽筛选及抑菌机理研究[D]. 成都: 四川农业大学.
	HOU XIAOYAN, 2019. Screening of antibacterial peptides and inhibition mechanism of pepper seeds based on bioinformatics and peptidomics[D]. Chengdu: Sichuan Agricultural University (in Chinese with English abstract).
[2]	李成华, 2007. 栉孔扇贝核心组蛋白的基因结构及H2A抗菌活性的研究[D]. 青岛: 中国科学院海洋研究所.
	LI CHENGHUA, 2007. Study on the gene structure and H2A antibacterial activity of the core histone of the ctenophore scallop (Pecten spp.)[D]. Qingdao: Graduate School of Chinese Academy of Sciences (Institute of Oceanography) (in Chinese with English abstract).
[3]	李冠楠, 夏雪娟, 隆耀航, 等, 2014. 抗菌肽的研究进展及其应用[J]. 动物营养学报, 26(1): 17-25. doi: 10.3969/j.issn.1006-267x.2014.01.003
	LI GUANNAN, XIA XUEJUAN, LONG YAOHANG, et al, 2014. Research progresses and applications of antimicrobial peptides[J]. Journal of Animal Nutrition, 26(1): 17-25 (in Chinese with English abstract).
[4]	马健, 2010. 海参再生研究进展[J]. 安徽农业科学, 38(11): 5694-5695, 5768.
	MA JIAN, 2010. Research Progress on the Regeneration of the Holothurians[J]. Anhui Agricultural Science, 38(11): 5694-5695, 5768 (in Chinese with English abstract).
[5]	聂竹兰, 李霞, 2006. 海参再生的研究[J]. 海洋科学, 30(5): 78-82.
	NIE ZULAN, LI XIA, 2006. Study on the regeneration of sea cucumber[J]. Marine Science, 30(5): 78-82 (in Chinese with English abstract).
[6]	张婷婷, 高珊, 矫建, 等, 2018. 香丹注射液溶血性实验及药物安全性检测问题探讨[J]. 中国药事, 32(4): 529-532.
	ZHANG TINGTING, GAO SHAN, JIAO JIAN, et al, 2018. Discussion on the hemolytic experiment and drug safety testing of Xiangdan injection[J]. China Pharmaceutical Affairs, 32(4): 529-532 (in Chinese with English abstract).
[7]	朱宁艺, 2021. 抗菌肽Mastoparan-C新型类似物的设计、合成、构效关系及其抑制和逆转抗生素耐药性作用研究[D]. 兰州: 兰州大学.
	ZHU NINGYI, 2021. Design, synthesis, conformational relationship and inhibition and reversal of antibiotic resistance of novel analogues of the antimicrobial peptide Mastoparan-C[D]. Lanzhou: Lanzhou University (in Chinese with English abstract).
[8]	AHMAD F, ASHRAF N, ELAHI M I, et al, 2022. Fabrication of biogenic-silver nanoparticles functionalized electrospun membranes counteracting bacteria and enhance wound healing[J]. Materials Today Communications, 31: 103493. doi: 10.1016/j.mtcomm.2022.103493
[9]	BUSTILLO M E, FISCHER A L, LABOUYER M A, et al, 2014. Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments[J]. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1838(9): 2228-2233. doi: 10.1016/j.bbamem.2014.04.010
[10]	CHEN YUXIN, MANT C T, HODGES R S, 2002. Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix[J]. Journal of Peptide Research, 59(1): 18-33. pmid: 11906604
[11]	CHO J H, SUNG B H, KIM S C, 2009. Buforins: Histone H2A-derived antimicrobial peptides from toad stomach[J]. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1788(8): 1564-1569. doi: 10.1016/j.bbamem.2008.10.025
[12]	CIUMAC D, GONG HAONING, HU XUZHI, et al, 2019. Membrane targeting cationic antimicrobial peptides[J]. Journal of Colloid and Interface Science, 537: 163-185. doi: S0021-9797(18)31300-6 pmid: 30439615
[13]	DE ZOYSA M, NIKAPITIYA C, WHANG I, et al, 2009. Abhisin: A potential antimicrobial peptide derived from histone H2A of disk abalone (Haliotis discus discus)[J]. Fish & Shellfish Immunology, 27(5): 639-646.
[14]	HAMMAMI R, FLISS I, 2010. Current trends in antimicrobial agent research: chemo- and bioinformatics approaches[J]. Drug Discovery Today, 15(13-14): 540-546. doi: 10.1016/j.drudis.2010.05.002 pmid: 20546918
[15]	HANCOCK R E W, GILL DIAMOND, 2000. The role of cationic antimicrobial peptides in innate host defences[J]. Trends in Microbiology, 8(9): 402-410. pmid: 10989307
[16]	KOO Y S, KIM J M, PARK I Y, et al, 2008. Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide[J]. Peptides, 29(7): 1102-1108. doi: 10.1016/j.peptides.2008.02.019 pmid: 18406495
[17]	LI CHUN, BLENCKE H -M, HAUG T, et al, 2015. Antimicrobial peptides in echinoderm host defense[J]. Developmental & Comparative Immunology, 49(1): 190-197.
[18]	LIMA P G, OLIVEIRA 0 J T A, AMARAL J L, et al, 2021. Synthetic antimicrobial peptides: Characteristics, design, and potential as alternative molecules to overcome microbial resistance[J]. Life Sciences, 278: 119647. doi: 10.1016/j.lfs.2021.119647
[19]	MATELUNA C, TORRWS P, RODRIGUEZ-PRÑA M, et al, 2022. Identification of VEGFR2 as the Histatin-1 receptor in endothelial cells[J]. Biochemical Pharmacology, 201: 115079. doi: 10.1016/j.bcp.2022.115079
[20]	MI BOBIN, LIU JING, LIU YI, et al, 2018. The designer antimicrobial peptide A-hBD-2 facilitates skin wound healing by stimulating keratinocyte migration and proliferation[J]. Cellular Physiology and Biochemistry, 51(2): 647-663. doi: 10.1159/000495320 pmid: 30463067
[21]	NIYONSABA F, USHIO H, NAKANO N, et al, 2007. Antimicrobial peptides human β-defensins stimulate epidermal keratinocyte migration, proliferation and production of proinflammatory cytokines and chemokines[J]. Journal of Investigative Dermatology, 127(3): 594-604. doi: 10.1038/sj.jid.5700599
[22]	ONG Z Y, WIRADHARMA N, YANG YIYAN, 2014. Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials[J]. Advanced Drug Delivery Reviews, 78: 28-45. doi: 10.1016/j.addr.2014.10.013
[23]	OSAKABE A, MOLARO A, 2022. Histone renegades: Unusual H2A histone variants in plants and animals[J]. Seminars in Cell & Developmental Biology, 135: 35-42.
[24]	RAMOS R, SILVA J P, RODRIGUES A C, et al, 2011. Wound healing activity of the human antimicrobial peptide LL37[J]. Peptides, 32(7): 1469-1476. doi: 10.1016/j.peptides.2011.06.005 pmid: 21693141
[25]	SAKTHIGURU N, MANIMOHAN M, JAGANATHAN G, et al, 2021. Biologically active Chitosan/ZnO/Acalypha indica leaf extract biocomposite: An investigation of antibacterial, cell proliferation and cell migration aptitude for wound healing application[J]. Sustainable Chemistry and Pharmacy, 19: 100357. doi: 10.1016/j.scp.2020.100357
[26]	SHAO CHANGXUAN, TIAN HAOTIAN, WANG TIANYU, et al, 2018. Central β-turn increases the cell selectivity of imperfectly amphipathic α-helical peptides[J]. Acta Biomaterialia, 69: 243-255. doi: S1742-7061(18)30020-5 pmid: 29355714
[27]	SHEN CUKUAN, LIN YUNZHI, MOHAMMADI T N, et al, 2022. Characterization of novel antimicrobial peptides designed on the basis of amino acid sequence of peptides from egg white hydrolysate[J]. International Journal of Food Microbiology, 378: 109802. doi: 10.1016/j.ijfoodmicro.2022.109802
[28]	SHI PUJIE, LIU MENG, FAN FENGJIAO, et al, 2018. Identification and mechanism of peptides with activity promoting osteoblast proliferation from bovine lactoferrin[J]. Food Bioscience, 22: 19-25. doi: 10.1016/j.fbio.2017.12.011
[29]	TAN PENG, FU HUIYANG, MA XI, 2021. Design, optimization, and nanotechnology of antimicrobial peptides: From exploration to applications[J]. Nano Today, 39: 101229. doi: 10.1016/j.nantod.2021.101229
[30]	TANIGUCHI M, SAITO K, AIDA R, et al, 2019. Wound healing activity and mechanism of action of antimicrobial and lipopolysaccharide-neutralizing peptides from enzymatic hydrolysates of rice bran proteins[J]. Journal of Bioscience and Bioengineering, 128(2): 142-148. doi: S1389-1723(18)31134-4 pmid: 30799089
[31]	THAPA R K, DIEO D B, TØNNESE H H, 2020. Topical antimicrobial peptide formulations for wound healing: Current developments and future prospects[J]. Acta Biomaterialia, 103: 52-67. doi: S1742-7061(19)30854-2 pmid: 31874224
[32]	ZHANG HAN, GAO ZHENJIE, DIAO QIANYING, et al, 2022. Antimicrobial activity and mechanisms of a derived antimicrobial peptide TroNKL-27 from golden pompano (Trachinotus ovatus) NK-lysin[J]. Fish & Shellfish Immunology, 126: 357-369.
[33]	ZHUANG XUE, LI HUI, WANG XIULI, et al, 2015. A review of the immune molecules in the sea cucumber[J]. Fish & Shellfish Immunology, 44(1): 1-11.

序号	序列	SVM	RF	ANN	DA	蛋白来源
1	RVHRFLRR	1.00	0.61	AMP	0.89	组蛋白H2A
2	KKPSKKPATKKAAKKKPAAKPKKAVKPKKPAAKKPAKKTSPKKKTAKKPAKKA	1.00	0.68	AMP	0.99	组蛋白H1β
3	PGKKTPQKKKPAAKKTKKPV	1.00	0.67	AMP	0.99	组蛋白H5
4	AKKPVRRLLQRRRRLRRRER	1.00	0.67	AMP	0.98	组蛋白H5
5	KPKKAVKPKKP	1.00	0.62	AMP	0.97	组蛋白H1β
6	KKAAKKKPAAKPKKAVKPKKP	1.00	0.69	AMP	1.00	组蛋白H1β
7	KKAAKKKPAAKPKKAVKPKKPAAKKPAKK	1.00	0.71	AMP	1.00	组蛋白H1β
8	KPAIRRLARR	1.00	0.62	AMP	0.77	组蛋白H4
9	RSARAGLQFPVGRVHRFLRR	0.99	0.81	AMP	0.98	组蛋白H2A
10	RGKGGKAWAKAKSRSARAGLQFPVGRVHRFLRR	0.99	0.92	AMP	1.00	组蛋白H2A
11	KKAAKKK	0.98	0.57	AMP	0.53	组蛋白H1β
12	FLGIKQTLKSLRQGKAKLII	0.98	1.00	AMP	1.00	核糖体L30
13	KAWAKAKSRSARAGLQFPVGRVHRFLRR	0.96	0.97	AMP	1.00	组蛋白H2A
14	KKKPVAAKKPVRRLLQRRRR	0.95	0.71	AMP	1.00	组蛋白H5
15	ILRLAVGLKGLPSVNPAWLV	0.93	0.97	AMP	0.99	组蛋白H3
16	ANFITHPYKRILRLAVGLKG	0.92	0.98	AMP	0.99	组蛋白H3
17	IKVELKRLAANNVLVHTKGT	0.90	0.86	AMP	0.84	组蛋白H5
18	IFVKTLTGKT	0.81	0.40	AMP	0.82	海参泛素
19	KYFLGIKQTLKSLRQGKAKLIIL	0.81	0.95	AMP	0.99	核糖体L30
20	RGKGGKAWAKAK	0.74	0.47	AMP	0.41	组蛋白H2A
21	MQIFVKTLTGKTITL	0.38	0.23	NAMP	0.25	海参泛素
22	MTGRGKGGKAWAKAKSRSARAGLQFPVGRVHRFLRRGNY	0.36	0.51	AMP	0.92	组蛋白H2A
23	FDKGKLKTVETQEKNTLPTKDTIDQEK	0.21	0.11	NAMP	0.03	胸腺素
24	MQIFVKTLTGKTITLEVEPSDTIENVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLR	0.21	0.26	NAMP	0.04	海参泛素
25	MSDKPDVSEVTK	0.06	0.07	NAMP	0.01	胸腺素
26	MSDKPDVSEVTKFDKGKLKTVETQEKNTLPTKDTIDQEKAAS	0.02	0.04	NAMP	0.00	胸腺素
27	STLHLVLRLR	0.00	0.33	NAMP	0.15	海参泛素

抗菌肽	肽序列	疏水性/%	净电荷/C	pI	分子量/Da
H1	RGKGGKAWAKAK	33.30	+5.00	11.83	1257.51
H2	RSARAGLQFPVGRVHRFLRR	45.00	+11.10	13.18	2379.81
H3	KAWAKAKSRSARAGLQFPVGRVHRFLRR	46.43	+9.10	13.19	3250.86
H4	RVHRFLRR	37.50	+4.10	12.96	1139.38
H5	KPKKAVKPKKP	45.45	+6.00	11.21	1248.63
H6	KKAAKKK	28.57	+5.00	11.11	801.05
H7	KKAAKKKPAAKPKKAVKPKKP	47.62	+11.00	11.51	2270.93

海参抗菌肽的活性筛选与功效评价

Screening and efficacy evaluation of antibacterial peptides from Holothuroidea

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