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  微生物与感染  2018, Vol. 13 Issue (1): 56-64      DOI: 10.3969/j.issn.1673-6184.2018.01.010
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新型抗菌肽——表面活性素、伊枯草菌素和丰原素
金清 , 肖明     
上海师范大学生命与环境科学学院,上海 200234
摘要:表面活性素(surfactin)、伊枯草菌素(iturin)和丰原素(fengycin)是一类主要由革兰阳性芽胞杆菌通过非核糖体合成途径产生的抗菌肽,一般是由1个β-羟基脂肪酸与7~10个氨基酸肽链以酰胺键连接而成的环肽,具有抗细菌、抗真菌、抗病毒、抗肿瘤等生物活性,具有良好的医疗应用前景。目前,人们对这3种新型抗菌肽在医药领域中的研究进展所知甚少,故本文对其发现历史、结构特点、作用机制、生物合成和应用价值进行阐述,为后续研究提供借鉴。
关键词抗菌肽    表面活性素    伊枯草菌素    丰原素    
Novel antimicrobial peptides: surfactin, iturin and fengycin
JIN Qing , XIAO Ming     
College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, China
Abstract: Surfactin, iturin and fengycin are novel antimicrobial peptides produced mainly by Gram-positive bacilli through non-ribosome synthesis. The structure is generally composed of β-hydroxy fatty acids and 7 to 10 amino acid peptide chains linked by the amide bond. With antibacterial, antifungal, antiviral, antitumoral and other biological activities, they have a good application prospect in medical field. This article describes the discovery history, structural characteristics, action mechanisms, biosynthesis and potential applications of the peptides.
Key words: Antimicrobial peptide    Surfactin    Iturin    Fengycin    

抗菌肽(antimicrobial peptide)具有抗菌谱广、作用迅速强大、不易产生耐药性等优点,在医药、化妆品、食品工业中具有广阔的应用前景[1-3]。表面活性素(surfactin)、伊枯草菌素(iturin)和丰原素(fengycin)是芽胞杆菌产生的主要活性物质,可抑制农作物病害[4-6]。近年来发现这3类物质在医药领域有重要应用前景,具有抗病毒[5, 7]、抗肿瘤[8-12]、抗细菌[13-15]、抗真菌[16-18]、抗支原体[19]、抗炎[20]作用,展示出极大的临床应用潜力[21],是一种新型抗菌肽[22-26],但人们对它们在医药领域中的研究进展所知甚少。因此,本文就表面活性素、伊枯草菌素和丰原素的发现历史、结构特点、作用机制、生物合成及应用价值进行综述。

1 发现历史

表面活性素于1968年首次在枯草芽胞杆菌(Bacillus subtilis)的培养液中发现,由4种异构体(枯草菌素A~D)组成,表现出各种生理活性,包括作为纤维蛋白凝固抑制剂和细胞裂解物[27]。伊枯草菌素于1957年首次在从土壤中分离到的枯草芽胞杆菌中发现[28],丰原素则首次在枯草芽胞杆菌F29-3中发现[29]

表面活性素、伊枯草菌素和丰原素具有广泛的工业应用价值,如制造洗涤剂[30-31]、抑制植物病害[32-34]、提高油采收率[31]等。近年来,这3种新型抗菌肽的医疗应用研究也取得重大突破,在医药领域中扮演着越来越重要的角色。

与其他菌肽的生产类似,这3种抗菌肽也是通过发酵生产。目前研究者从各方面努力,包括优良菌种选育[5]、发酵过程优化[35-36]、高效分离纯化方式的探索等来提高产率[37-38],取得了一系列成果。

2 结构特点及作用机制

表面活性素、伊枯草菌素和丰原素是一类主要由革兰阳性芽胞杆菌产生的抗菌肽,一般由1个疏水的脂肪烃链以羧基、羰基或氨基与亲水的由7~10个氨基酸构成的肽链以酰胺键或内酯的形式连接构成环肽,其结构上的差异主要在于脂肪链中碳原子的个数、氨基酸的种类及脂肪酸链与肽链连接键的不同(图 1)。

A: Surfactin is formed by β-hydroxy fatty acids (12-17 carbon atoms) with peptide chain through lactone bonds. The molecular peptide chain is composed of seven α-amino acids. B: Iturin is a ring formed by β-amino fatty acids (14-17 carbon atoms) with peptide chain through amide bond. The molecular peptide chain is composed of seven α-amino acids. C: Fengycin is composed of β-hydroxy fatty acids (14-17 carbon atoms) and peptide chain through lactone bond. Fengycin is different from surfactin and iturin. The macrocyclic ring is composed of peptide chain of ten α-amino acids. The Arabic numerals represent the position of the amino acid. 图 1 表面活性素、伊枯草菌素和丰原素的分子结构 Fig. 1 Molecular structures of surfactin, iturin and fengycin

表面活性素由β-羟基脂肪酸与肽链以内酯键结合而成[39-41]。多数细菌代谢产生的表面活性素的肽链为七元肽,即分子中的肽链由7个α-氨基酸组成,再与带有12~17个碳原子的β-羟基脂肪酸构成一个大内酯环,相对分子质量(Mr)为1 000左右。多肽中典型的氨基酸顺序为[27, 42-43]L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu(图 1A)。表面活性素家族常见的有surfactin A[44]、surfactin C[44]、lichenysin[44-46]、pumilacidin等[46]

伊枯草菌素由β-氨基脂肪酸与肽链以酰胺键成环[47]。多数细菌代谢产生的伊枯草菌素的肽链也为七元肽,与带有14~17个碳原子的β-氨基脂肪酸构成一个大环,Mr为1 000左右。多肽中典型的氨基酸顺序为[47-48]L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser(图 1B)。伊枯草菌素家族种类较多,如iturin A[49-54]、iturin C[49, 51]、iturin D[51]、iturin E[51-53]、iturin F[53]、杆菌霉素(bacillomycin)D[49-52]、bacillomycin F[49-51]、bacillomycin L[49-51]、mixirins[9, 51-52]、mojavensin[52]、抗霉枯草菌素(mycosubtilin)[49-51, 53-54]和subtulene[51]等。

丰原素由β-羟基脂肪酸与肽链以内酯键结合而成。与表面活性素不同的是,丰原素的大环由肽链自行构成,β-羟基脂肪酸并不参与[54-55]。多数细菌代谢产生的丰原素的肽环为十元肽,第三位的D-Tyr和最后一位的L-Ile以内酯键结合成环,再与带有14~17个碳原子的β-羟基脂肪酸以酯键结合,Mr为1 500左右[54-56]。多肽中典型的氨基酸顺序为[54-58]L-Glu-D-Orn-D-Tyr-D-allo-Thr-L-Glu-D-Ala-L-Pro-L-Gln-L-Tyr-L-Ile(图 1C)。丰原素家族常见的有fengycin A[54-58]、fengycin B[54-55, 57-58]、fengycin C[54-55, 58]、fengycin S[55]和制磷脂菌素(plipastatin)[54-55, 57-59]

表面活性素可通过溶解和破坏细胞膜发挥抗菌作用,其与膜中的极性头部和疏水酰基链均可相互作用,高浓度时引起磷脂双分子层高度不稳定,中等浓度时在细胞膜中形成离子传导孔与Ca2+结合,有助于膜渗透;其还可通过有机屏障驱动其他单价和二价阳离子,导致cAMP磷酸二酯酶活性被抑制[60-62]。伊枯草菌素能迅速引起细胞膜损伤,细胞通透性改变,细胞内物质外泄,从而达到抑制真菌孢子萌发、菌丝体生长的效果;其还可与细胞内靶点(如细胞DNA)相互作用,破坏细胞内钙稳态,导致细胞死亡[63-65]。丰原素对细胞膜有显著扰乱作用,通过基于丰原素浓度的两态跃迁过程使磷脂双分子层损伤,从而导致细胞死亡。高浓度时,丰原素作为洗涤剂促进细胞膜溶解,该过程主要由其对脂质双分子层的吸引力等物理化学性质所驱动;低浓度时,丰原素聚合形成孔洞,导致膜的渗透率发生变化[61, 66-68]

3 生物合成

芽胞杆菌能分泌多种肽类及由肽类衍生的抗菌活性物质,按合成途径分为核糖体肽和非核糖体肽[4]。非核糖体肽Mr较小,一般为3 000以下,通过非核糖体肽链合成酶(non-ribosomal peptide synthetase,NRPS)来合成,多发生于菌体生长停止之后;而核糖体肽Mr较大,大多于菌体快速生长时期合成[4, 69]

非核糖体途径合成的脂肽类抗菌活性物质合成于菌体生长停止之后,属于微生物次级代谢产物,能绕开核糖体,不以mRNA为模板,也不需tRNA作为运载工具,而是通过NRPS识别特定的氨基酸并连接成多肽链[4, 70]。表面活性素、伊枯草菌素和丰原素就是NRPS合成的次级代谢产物,由胞内游离氨基酸经活化后结合到合成酶系特定的结构域,从而实现肽链的延长和环化[71-72](图 2)。

A: amino acid activating domain; PCP: peptidyl carrier protein; C: condensation domain; E: epimerization domain; TE: thioesterase domain; MCT: monocarboxylate transporter. 图 2 表面活性素、伊枯草菌素和丰原素的代谢通路 Fig. 2 Metabolic pathways of surfactin, iturin and fengycin

NRPS是由多个功能模块组成的复合酶体系,按功能分为必不可少模块和可供选择模块,各模块负责活化不同的氨基酸使肽链延长。必不可少模块有氨基酸激活结构域(amino acid activating domain)、氨酰载体结构域(acyl carrier)、缩合结构域(condensation domain)和硫酯酶结构域(thioesterase domain)。氨基酸激活结构域由550个氨基酸残基构成,负责识别和腺苷酰化特定的氨基酸,又称为腺苷酰化结构域(adenylation domain);氨酰载体结构域负责运载氨基酸,又称为巯基化结构域T或肽酰载体蛋白(peptidyl carrier protein,PCP);缩合结构域负责肽键形成;硫酯酶结构域负责释放多肽和肽的环化。可供选择模块包括环化结构域(cyclization domain)、甲基转移酶结构域(methyltransferase domain)、差向异构酶结构域(epimerization domain)等。差向异构酶结构域负责将被激活的L-氨基酸转化为D-氨基酸。全酶由多个模块按特定的空间顺序排列而成,模块的数量、种类及排列次序决定了氨基酸种类、顺序和最终产物肽链的长短[73-74](图 2)。

NRPS合成多肽的一般过程为:首先,氨基酸激活结构域选择并结合特定的氨基酸,在ATP作用下激活氨基酸(腺苷化),形成氨酰腺苷酸。氨酰腺苷酸与氨酰载体结构域上的4-磷酸泛酰巯基辅基以共价键形式结合,形成氨酰载体复合体。然后,携带有活化氨基酸的氨酰载体与缩合结构域特定部位结合,在其合成酶的作用下,按相邻合成酶各组成模块的顺序依次向前形成肽键。肽键形成后,进入下一循环,即肽链延伸过程。最后,硫酯酶结构域终止肽链合成,将肽链从磷酸泛酰巯基辅基释放下来,并进行环化[75-77]

3.1 表面活性素

表面活性素合成酶包括3个亚单位:SrfA、ComA(SrfB)和SrfC[4, 26, 78-86]。编码表面活性素合成酶的基因srfA-AsrfA-BsrfA-C共同构成srfA操纵子(长约25 kb),分别负责编码Mr为401 000、402 000和144 000的3个合成单体酶E1A、E1B和E2。srfA-C编码的第一个硫酯酶结构域负责终止肽链延伸并释放多肽产物(图 2A)。

sfp基因(约4.5 kb)是参与表面活性素合成的第二调控元件,位于srfA操纵子下游30.5 kb处,与srfA-D末端相距约4 kb。sfp基因编码的SFP酶具有编码磷酸泛酰巯基乙胺基转移酶(phosphopantetheinyl transferase,PPTase)的功能,可催化非核糖体肽和载铁蛋白前体的形成,并借此将表面活性素合成酶激活,属PPTase超家族。有研究认为sfpsrfA-C-TE基因共同发挥主导作用,还有研究认为sfp基因在表面活性素合成中有更直接的调节作用[85]。在基因图谱中,srfAsfp相邻,而与srfB相隔,srfB功能等同于comA基因,即激活srfA启动子的转录。

SrfA-A负责组装前3位氨基酸;SrfA-B负责组装第4~6位氨基酸;SrfA-C负责组装第7位氨基酸,并将羟基脂肪酸转移到蛋白SrfA-A上。

3.2 伊枯草菌素

伊枯草菌素通过聚酮合酶(polyketide synthase,PKS)-NRPS杂合体系合成[78, 87-88]

编码伊枯草菌素合成酶的基因ituDituAituBituC共同构成itu操纵子(长约38 kb)。ituD负责编码丙二酰辅酶A转酰酶,对伊枯草菌素的形成起重要作用,被破坏可导致iturin A产生特异性缺陷;其还可调控伊枯草菌素产量,可能与脂肪酸合成有关。ituA编码Mr为449 000的蛋白,与脂肪酸合成酶、氨基酸转移酶和肽合成酶具有同源性,可能与β-氨基脂肪酸形成有关;部分基因与聚酮合酶相关,其编码的聚酮合酶参与脂肽类分子碳链合成的最终步骤,并为肽段部分氨基酸分子的组装做好准备。ituB编码Mr为609 000的具有4个氨基酸腺苷酸化结构域的肽合成酶。ituC编码Mr为297 000的另一种肽合成酶,具有2个腺苷酸化结构域、1个差向异构酶结构域和硫酯酶结构域,这可能有助于肽环化。ItuA负责组装第1位氨基酸,ItuB负责组装第2~5位氨基酸,ItuC负责组装第6和7位氨基酸(图 2B)。

3.3 丰原素

编码丰原素合成酶的基因fenCfenDfenEfenAfenB共同构成fen操纵子(长约37 kb),分别编码5个亚基,即5个单体酶——FenC、FenD、FenE、FenA和FenB。每个单体酶一般含1~3个氨基酸激活模块,且每个模块具有接受特定氨基酸及形成相应肽键的功能。丰原素合成从肽单体酶FenC开始,途经FenD、FenE和FenA,终止于肽单体酶FenB[60, 89-91]

FenC的Mr为287 000,负责活化并组装第1和2位氨基酸;FenD的Mr为290 000,负责活化第3和4位氨基酸;FenE的Mr为286 000,活化第5和6位氨基酸;FenA的Mr为406 000,负责活化第7~9位氨基酸;FenB的Mr为146 000,负责组装最后一位氨基酸。FenB含有能中断肽链合成的硫酯酶结构域,具有释放肽链的功能,在其下游也发现了与脂肪酸代谢有关的基因(图 2C)。

4 应用价值

研究表明,表面活性素对新城疫病毒(Newcastle disease virus,NDV)不仅具有直接灭活作用,还可阻断其对细胞的吸附;随着浓度升高还可抑制NDV的生物合成,具有明显的量-效关系。表面活性素的治疗指数为12.16,高于病毒唑的9.70,有望开发成为一种有效的抗病毒药物,这对养殖业生产及防治NDV感染所致人类疾病具有重要意义[7]。另有研究表明,表面活性素对人乳腺癌细胞Michigan Cancer Foundation-7(MCF-7)表现出较强的抑制作用。噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide,MTT)法显示,表面活性素能抑制MCF-7增殖,呈现浓度与时间依赖关系,细胞处理24 h后的半抑制浓度(half maximal inhibitory concentration,IC50)为10 μg/mL。随着发酵期间表面活性素含量升高(0.3~48.2 mg/kg),SEC(surfactin extractions of cheonggukjang)(100 μg/mL)的抗癌活性逐渐升高(20.3%~54.7%)[8]。表面活性素除具有抗病毒、抗肿瘤作用,还可抗细菌[13-15]、抗真菌[16-17]、抗支原体[19]、抗炎[20],抗菌谱较广,同时具有不易产生耐药性、可被动物消化酶降解、无残留等优点,均显示其应用于医药业的潜力。

伊枯草菌素具有强烈的抗真菌、抗肿瘤作用,还具有低毒、低残留、低过敏性和抗菌谱广的特点,是一种潜在的具有极大开发应用价值的医药产品。研究表明,伊枯草菌素对红色毛癣菌具有较强抑制作用[18]。Mixirins A、B和C具有抗肿瘤作用,可抑制人结肠癌细胞HCT-116的生长,其IC50分别为0.68、1.6和1.3 μg/mL[9]

丰原素具有显著的抗肿瘤效果,对肿瘤细胞及肿瘤组织具有较好的选择性,对肿瘤凋亡相关蛋白有明显影响,而对正常造血系统和白细胞无影响,为寻找新型抗肿瘤药物提供了方向。研究表明,与对照组相比,丰原素浓度达20 μg/mL时即可抑制人结肠癌细胞HT-29的生长,并呈浓度与时间依赖关系。蛋白免疫印迹分析发现,加入丰原素后,HT-29细胞中的Bax、Caspase-3和Caspase-6表达明显增加,而Bcl-2和CDK4/cyclin D1表达降低,表明丰原素可通过影响人HT-29细胞周期的G1期停滞和诱导细胞凋亡而对其产生抑制作用[10]。此外,大量研究也表明丰原素可抑制人结肠癌HCT-15细胞增殖[11],调节人肺癌95D细胞G0/G1期引起细胞周期停滞和促进细胞凋亡来抑制癌细胞生长[12],显示其具有极大的抗肿瘤潜力。

综上所述,抗菌肽是极具价值的新一代抗菌药物,能作为抗病原体药物,并可能发展成为抗肿瘤药物,在免疫调节、促进伤口愈合等方面有应用价值。多数文献表明,表面活性素、伊枯草菌素和丰原素在医疗领域有着巨大价值与广阔前景,具备低毒、抗菌谱广、不易产生耐药性等优势及工业化生产潜力,但临床应用还不广泛。随着对抗菌肽研究的不断深入及技术的不断改进,如何将其应用于临床、应用于人类医疗事业将成为研究的主要方向。

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文章信息

金清, 肖明
JIN Qing, XIAO Ming
新型抗菌肽——表面活性素、伊枯草菌素和丰原素
Novel antimicrobial peptides: surfactin, iturin and fengycin
微生物与感染, 2018, 13(1): 56-64.
Journal of Microbes and Infections, 2018, 13(1): 56-64.
通信作者
肖明
E-mail:xiaom88@shnu.edu.cn

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