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文章信息
- 杨航, 余军平, 危宏平
- YANG Hang, YU Jun-Ping, WEI Hong-Ping
- 裂解酶治疗的研究进展与应用前景
- Research progress in lysin therapy
- 微生物学通报, 2015, 42(1): 178-184
- Microbiology China, 2015, 42(1): 178-184
- 10.13344/j.microbiol.china.140623
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文章历史
- 收稿日期: 2014-08-16
- 接受日期: 2014-09-12
- 优先数字出版日期(www.cnki.net): 2015-09-28
近年来,多种超级耐药细菌的不断出现与传播给全球临床医学及细菌性传染病的治疗带来了严峻的挑战,加之新抗生素的研发面临着技术上以及商业上的障碍,近几年只有几种新抗生素上市。如果这种趋势继续,临床上有可能面临着无药可用的地步。人们现在越来越担心,是否我们将迈入因一个普通的细菌感染就会致命的后抗生素时代?如何有效应对细菌耐药性问题是当下亟待解决的难题。由于噬菌体对宿主细菌的裂解不受细菌耐药性的限制,噬菌体用于细菌感染的治疗受到了重新的考量与测试。但是,噬菌体治疗仍面临多种挑战,诸如噬菌体的细菌抗性、生物安全性、药代动力学等。噬菌体裂解酶是噬菌体感染宿主晚期表达的一类肽聚糖水解酶,具有与细菌共同进化的选择优势和高效的裂解活性。裂解酶体外应用时能快速水解革兰氏阳性细菌的细胞壁,其作用不受细菌耐药性的影响,而且不易诱导细菌产生新的抗性。近年来建立的多种动物感染模型,初步体现了裂解酶用于耐药细菌感染治疗的潜力与优势。本文将综述噬菌体裂解酶在耐药细菌控制以及抗感染治疗中的研究进展,同时也将介绍革兰氏阴性细菌噬菌体裂解酶研究的最近进展。
1 噬菌体裂解酶 1.1 噬菌体裂解酶的发现噬菌体裂解酶(Lysin)是双链DNA噬菌体感染宿主后晚期表达的一类肽聚糖水解酶。裂解酶在穴蛋白(Holin)的帮助下通过细胞膜孔洞抵达细胞壁上的肽聚糖靶点,对特定位点进行切割,直接造成细胞的破裂和瓦解,帮助子代噬菌体的释放。穴蛋白的作用是通过在细胞膜上形成低聚物对细胞膜进行穿孔,改变宿主细菌细胞膜的通透性,类似信号肽的功能。这种典型的穴蛋白-裂解酶裂解系统最早由Young等[1]提出,目前已得到普遍的认同。有些双链DNA噬菌体裂解酶不具备穴蛋白-裂解酶裂解系统。Loessner等[2, 3]研究发现,在蜡样芽胞杆菌(Bacillus cereus)的噬菌体裂解酶TP21和李斯特菌(Listeria monocytogenes)的噬菌体裂解酶A511中,裂解酶N端与信号肽序列同源性很高,具有与穴蛋白相似的功能,可以将自身引导至细胞壁靶点。
裂解酶的作用靶点是敏感细菌细胞壁上的肽聚糖链键,在体内借助穴蛋白的帮助而裂解靶细菌(自内裂解),这是噬菌体和细菌共进化的结果。当裂解酶体外应用时,可以直接作用于敏感革兰氏阳性菌的细胞壁而导致细菌的快速裂解(自外裂解)[4],而对革兰氏阴性菌没有作用。这种差异主要是由革兰氏阳性菌和革兰氏阴性菌细胞壁的构造不同决定的。革兰氏阴性菌细胞壁外的细胞膜阻碍了裂解酶与肽聚糖靶点的有效结合。裂解酶能自外裂解革兰氏阳性菌的发现开创了裂解酶用于耐药细菌控制与治疗的道路。
1.2 噬菌体裂解酶的结构革兰氏阳性菌DNA噬菌体编码的裂解酶大小通常为25−40 kD。一般来说,革兰氏阳性菌噬菌体裂解酶的结构相似[5, 6],具有一个决定该酶催化活性的N-端催化功能域(Catalytic domain,CD)和一个决定细胞壁结合特异性的C-端细胞壁结合功能域(Cell wall binding domain,CBD),二者之间由一个小片段连接(图 1)。革兰氏阴性细菌噬菌体裂解酶的催化功能域一般位于C端,而细胞壁结合功能域则位于N端[7]。最近的研究表明有些裂解酶结构中还存在独立的芽孢识别功能域(Spore binding domain,SBD),可以识别特定种属的细菌芽孢[8]。有些裂解酶拥有2个甚至3个不同的催化域[9]。裂解酶的细胞壁结合域可与宿主菌细胞壁上的特定基质(通常为糖类)高效、特异结合[10, 11],这是实现特异裂解靶细菌的前提[12]。基于序列比对发现,同一类裂解酶的N-端催化域具有较高的同源性,而C-端细胞壁结合域同源性低[12]。由于细胞裂解后溢出的裂解酶可能会杀死附近子代噬菌体的潜在宿主,阻碍噬菌体的进一步繁殖,这促使裂解酶不断进化其结合域,提高亲和力[12, 13],以限制游离裂解酶的释放。因此,有研究者认为裂解酶是一次性使用的酶类[14],即裂解作用完成后,裂解酶不能有效地从细胞壁上解离下来。
根据裂解酶催化域活性的不同,可将其分为多种类型。催化域活性可体现为N-乙酰-β-D-葡萄糖胺酶(N-acetyl-β-D-glucosaminidases)或N-乙酰-β-D-胞壁酸酶(N-acetyl-β-D-muramidases),或者内肽酶(Endopeptidases),或者N-乙酰胞壁酰-L-丙氨酸酰氨酶(N-acetylmuramoyl-L-alanine amidases)等(图 1)。N-乙酰-β-D-葡萄糖胺酶和N-乙酰-β-D-胞壁酸酶二者都能作用于细菌细胞壁上的半糖;内肽酶水解肽侧链和交联桥之间的氨基或肽键连接;N-乙酰胞壁酰-L-丙氨酸酰氨酶能够水解连接糖链和肽基的酰胺键[1, 15]。最近报道了一种具有γ-D-谷氨酰胺酰-L-赖氨酸肽链内切酶活性的酶[16],以及一个具有D-丙氨酰-L-丙氨酸内肽酶活性的酶[17]。
2 裂解酶治疗裂解酶最吸引人的地方在于裂解酶对病原细菌良好的杀灭能力,尤其是耐药细菌。裂解酶治疗正是在耐药细菌不断出现和传播的基础上提出来的。与传统的抗生素相比,裂解酶具有其独特的性质:(1) 裂解酶具有进化优势。裂解酶来自于噬菌体,噬菌体具有与宿主菌共同进化的特性。这是在长期的自然选择中保留下来的。(2) 裂解酶具有很强的特异性。裂解酶的细胞壁结合功能域只特异性的识别特定种属的细菌,这使得裂解酶具有很强的特异性。(3) 裂解酶具有高效的杀菌活力。裂解酶是释放子代噬菌体的“武器”,具有天然的高效性。(4) 裂解酶具有极低的产生耐药的可能性。迫于自然选择的压力,噬菌体只作用于宿主菌必不可少的保守部分,细菌很少能够产生抗性来逃避这种 识别。
为了探索裂解酶治疗之路,研究人员做了大量的体内外试验,充分展现了裂解酶用于细菌感染治疗的巨大潜力[18, 19, 20, 21]。截至目前,已经建立了大量的动物模型,包括皮肤感染模型[22]、粘膜去定殖模 型[4, 23, 24, 25]、阴道感染模型[26]、周身感染模型[27, 28, 29, 30, 31]、心内膜炎模型[32]、眼内炎模型[33]、脑膜炎模型[34]、肺炎感染模型[35, 36]等。在这些动物模型中,噬菌体裂解酶均能快速杀灭靶细菌,在短时间内将细菌数量降低几个数量级,有些甚至低至无法检出。
例如,在一个皮肤感染模型中,Pastagia等用嵌合裂解酶ClyS能有效地消灭小鼠烧伤皮肤感染的MRSA,使其降低了3个数量级[22]。作用效果比外用抗感染药莫匹罗星好(莫匹罗星作用后只能降低2个数量级),而且细菌对ClyS产生耐药性的可能性远低于莫匹罗星。
在一个粘膜去定殖模型中,Loeffler等[35]利用肺炎链球菌噬菌体裂解酶Pal清除小鼠鼻腔中的肺炎链球菌。Pal在体外能高效裂解15种常见类型的肺炎链球菌,包括青霉素抗性菌株。一次给药5 h后,能将小鼠鼻腔定殖的肺炎链球菌降到无法检测到的水平。Pal在杀灭靶细菌的同时对口腔中的其他微生物没有影响,而且也没有耐药细菌的产生。
在一个阴道感染模型中,Cheng等[26]利用裂解酶PlyGBS降低了小鼠阴道定殖的B族链球菌。研究者指出PlyGBS具有高效清除产前妇女阴道中的B族链球菌的潜力,可以降低新生儿感染的几率。而通常的做法是对产前妇女使用抗生素来预防和降低这种感染的风险。PlyGBS具有比抗生素更好的效果,而且不存在过敏现象和产生细菌耐药性等隐患。
在一个周身感染模型中,Gu等[28]在小鼠感染 1 h后一次性腹腔注射50 μg裂解酶LysGH15能有效提高2倍于致死剂量的MRSA感染的小鼠的存活率。在没有裂解酶保护的实验组中,在感染3.5 h后,小鼠血液中细菌浓度为107 CFU/mL。而在有裂解酶保护的实验组中,在感染3.5 h后小鼠血液中细菌浓度为104 CFU/mL,而在几天后,细菌数量低至无法检出。
与噬菌体治疗相比,裂解酶治疗有其独特的优势。譬如,裂解酶是蛋白质性质,可以建立明确的质控标准;裂解酶既可以从烈性噬菌体中分离得到,也可以从温和噬菌体中分离得到;可以方便地进行设计和改造等[37]。因此,裂解酶治疗得到了国内外许多学者的支持和倡导,各方面的研究均体现了裂解酶用于耐药细菌感染控制与治疗的潜力和发展空间。令人鼓舞的是,近年来有些裂解酶已经进入到了临床前的试验阶段(例如ClyS,P128等),而在美国已出现了开发裂解酶产品的高科技公司(如ContraFect)。ClyS是一个人工设计的嵌合裂解酶,由噬菌体Twort裂解酶plyTW的催化域和噬菌体phNM3裂解酶的细胞壁结合域拼接而成。这种设计增强了ClyS的水溶性和裂解活性,为临床试验提供了保障。但有关该裂解酶临床试验的结果目前还没有正式对外公布。值得一提的是,一个针对葡萄球菌的裂解酶P128已经进入到临床二期(http://www.gangagen.com/research.html)。从网站上了解到的情况来看,该裂解酶临床试验的效果很好。表 1简要列举了近年来裂解酶治疗研究的一些实践例子。
Lysin | Phage source | Bacteria | Murine sepsis challenge | Reference |
Cpl-1 | Cp1 | S. pneumoniae | Nasopharynx, mucosa, bloodstream, aortic valve | [31, 32, 34, 35, 38, 39, 40] |
Pal | Dp-1 | S. pneumoniae | Nasopharynx, oropharynx, intraperitoneal | [31, 35] |
C1 | C1 | S. pyogenes | Upper respiratory colonization | [4] |
PlyG | Gamma | B. anthracis | Intraperitoneal, bacteremia | [41] |
PlyPH | B. anthracis | Intraperitoneal, bacteremia | [42] | |
PlyV12 | Phi1 | E. faecalis and E. faecium | Intraperitoneal, bacteremia | [43] |
MV-L | MR11 | S. aureus | Intraperitoneal, bacteremia | [44] |
PlyGBS | NCTC 11261 | GBS | Upper respiratory colonization, vaginal colonization | [26] |
Lysin of phage ENB6 | ENB6 | Vancomycin-resistant E. faecium | Intraperitoneal, bacteremia | [45] |
Pal and Cpl-1 | Dp-1 and Cp-1 | S. pneumonia | Intraperitoneal, bacteremia | [31, 38] |
LytA | S. pneumoniae | Peritonitis-sepsis | [46] | |
PlyC | phiC1 | S. pyogenes | Oral mucosa, nasal mucosa | [4, 47] |
ClyS | MRSA | Nasal colonization, bacteremia | [29] | |
ClyS | MRSA and MSSA | Skin colonization | [22] | |
LysGH15 | GH15 | MRSA | Intraperitoneal, bacteremia | [28, 48] |
PlySs2 | S. suis phage | S. pyogenes and MRSA | Intraperitoneal, bacteremia | [49] |
SAL-1 | SAL | S. aureus | Intraperitoneal, bacteremia | [50] |
CF-301 | S. suis phage | S. aureus | Intraperitoneal, bacteremia | [51] |
ClyH | MRSA | Intraperitoneal, bacteremia | [52] | |
PlyPy | S. pyogenes | Streptococci | Intraperitoneal, bacteremia | [17] |
根据裂解酶的作用原理,裂解酶从外至内裂解细菌时只能作用于革兰氏阳性细菌。因革兰氏阴性细菌细胞壁外面的细胞膜能阻挡裂解酶与肽聚糖靶点的有效结合,裂解酶从外加入时不能裂解革兰氏阴性细菌。因此,不能在体外直接裂解革兰氏阴性细菌是裂解酶应用中的一个重大挑战。
但是最近一些研究表明,该挑战有可能通过一些设计而得到解决,显示了良好的前景。如Buchanan及其研究团队利用耶尔森氏菌细胞膜上的离子通道,将鼠疫菌素上能特异识别离子通道的序列与T4溶酶体融合,所得到的重组蛋白能借助离子通道通过耶尔森氏菌的细胞膜,导致耶尔森氏菌的死亡。而且对大肠杆菌也有明显的杀灭效果[53, 54, 55]。最近的其他研究表明由抗菌肽与裂解酶构成的融合蛋白可以在体外直接裂解铜绿假单胞菌[56]。该研究将抗菌肽SMAP-29融合在裂解酶KZ144的N端,通过大肠杆菌表达纯化后,SMAP-29-KZ144能够在其N端抗菌肽的帮助下通过细胞膜抵达细胞壁上的肽聚糖靶点,切割肽聚糖链键杀灭阴性菌铜绿假单胞菌。这种设计充分利用了抗菌肽的细胞膜破坏功能与裂解酶的肽聚糖水解功能,为多耐药阴性细菌的控制与治疗提供了新的思路。尤其值得指出的是,沿用这种思路设计的系列抗菌肽裂解酶对铜绿假单胞菌的体内杀灭效果已经在线虫感染模型中得到了证实[57]。
4 裂解酶存在的问题首先,有研究指出天然裂解酶在大肠杆菌中表达时有些对表达菌株具有明显的毒性,蛋白表达往往以包涵体的形式存在[29, 58, 59]。很难得到有高活力的裂解酶,这可能与裂解酶胞壁质水解酶的活力有关。该问题有可能通过构建合适的嵌合裂解酶(组合不同来源裂解酶的催化域和结合域)的方式进行解决。
其次,裂解酶本质上是蛋白质,进入机体后易受到蛋白酶的攻击,而具有较短的半衰期。这一问题有可能会制约裂解酶用于体内治疗的有效工作浓度和时间。和噬菌体不同,后者具有自我繁殖的能力,一次给药就可能产生持续的治疗效果。
此外,裂解酶还存在一个比较普遍的问题:可溶性差。裂解酶的C端是用来特异性结合宿主细菌的,该部分在结构上通常具有较强的疏水性和多个重复的跨膜区[60]。这使得裂解酶在水溶液中的溶解性较差。在体外应用时还需要摸索合适的缓冲体系。
5 展望噬菌体裂解酶具有抗生素与噬菌体所没有的优势:蛋白质性质、进化优势、高效的杀菌活性、清楚的结构、易于人工设计和改造、不易产生耐药性等。这些特点使得裂解酶具有用于临床耐药细菌控制与治疗的巨大潜力。目前的许多基础研究也支持这个论断。但是其对蛋白酶的敏感性、半衰期、可溶性、代谢途径、组织渗透性、免疫原性以及细胞毒性等因素仍然需要进一步的深入研究和寻找解决方法。譬如,裂解酶的大规模生产可能受到其可溶性差等因素的制约,而较短的半衰期对裂解酶的剂型和给药时间提出了新的要求。
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