噬菌体裂解酶的抗菌特性

摘要：噬菌体裂解酶是一类细胞壁水解酶,可水解肽聚糖,造成细菌的破裂。裂解酶一般具有两到三个结构域,参与对底物的催化和结合。作为一种新型的杀菌制剂,裂解酶已被越来越多地应用于化脓链球菌、肺炎链球菌、金黄色葡萄球菌等革兰氏阳性细菌病的治疗。与抗生素治疗相比,裂解酶不易使细菌产生抗性且作用相对专一,这可能是解决现在日趋严重的细菌耐药性的一种可行方法。另外,裂解酶还具有高效性,作用协同性,且自身抗体不削弱其作用等优势,使之成为未来预防、控制致病菌一种可能的新途径。     

鱼源副乳房链球菌前噬菌体裂解酶Sply828的抗菌活性

【背景】副乳房链球菌（Streptococcus parauberis）是重要的水产病原菌,该病原菌已逐渐出现新的血清型及多重耐药性状,因此亟须开发出一种新的抗菌药物用于该病害的防治。研究发现,前噬菌体编码的裂解酶能够有效地杀死其宿主,具有良好的抗菌应用前景。【目的】以副乳房链球菌前噬菌体裂解酶为对象,研究其杀菌宿主谱并优化其裂解活性的条件。【方法】利用PHASTER工具对副乳房链球菌菌株KRS02083全基因组序列分析发现,其前噬菌体包含一种裂解酶的基因Sply828;通过基因克隆、表达和纯化等技术得到裂解酶Sply828蛋白;通过浊度递减实验探究裂解酶Sply828对不同细菌的杀菌活性及其最适的裂解条件。【结果】裂解酶Sply828对鱼源副乳房链球菌具有最佳的杀菌活性,并发现该酶对处于指数生长期的细菌杀菌效果最好;其最适裂解温度为28°C,最适pH为6.2;Ca²⁺和Mg²⁺对该酶的杀菌活性有促进作用,但是Zn²⁺、Cu²⁺、Fe²⁺、Ni²⁺明显抑制...     

噬菌体裂解酶应用研究进展

近年来,随着抗生素的滥用,导致多重耐药性菌株出现的频率加快。因细菌感染导致死亡的人数逐年增多,人类健康面临巨大挑战,因此研制新型抗菌药物刻不容缓。噬菌体裂解酶因其高效的杀菌能力及高度的宿主专一性而成为新一代抗菌制剂的候选之一。其是一种细胞壁水解酶,在双链DNA噬菌体复制后期被合成,通过水解细胞壁肽聚糖上的化学键,从而裂解细菌细胞壁,释放出子代噬菌体。本文系统地介绍了噬菌体裂解酶的研究进展,为相关裂解酶抗菌药物的研发做出有益探索。     

噬菌体裂解酶——现状与未来

噬菌体裂解酶是一种由DNA噬菌体基因编码的高特异性酶, 可高效消化细菌细胞壁。革兰氏阳性菌噬菌体裂解酶的结构域相似, 裂解效率高, 与抗生素具协同抗菌作用, 且不易产生耐受性菌株, 抗体等体液因子对裂解酶的裂解活性影响小, 裂解酶作为一种潜在抗感染药物具有重要的研究价值。目前已建立了多种病原菌裂解酶应用的动物模型, 在防控耐药性病原菌感染上取得重要进展。本文就噬菌体裂解酶的抗菌作用进行综述。     

裂解酶治疗的研究进展与应用前景

多耐药病原细菌的不断出现和传播给公共医疗造成了严峻的威胁和挑战,开发新的抗菌分子迫在眉睫。噬菌体裂解酶来源于噬菌体,具有独特的进化和选择优势,不仅能高效快速的杀灭多耐药细菌,而且不易诱导细菌产生新的耐药性。本文对噬菌体裂解酶的结构和功能进行了简要的介绍,重点综述了裂解酶在抗细菌感染中近年的研究进展和应用前景。     

噬菌体裂解酶研究进展

噬菌体裂解酶是双链DNA噬菌体所特有的细胞壁水解酶。研究表明,所有噬菌体裂解酶在结构上具有相似性,即含有2个结构域：比较保守的N端催化区和差异较大的C端特异性结合区。裂解酶的高亲和性与种属特异的细胞壁糖基有关,而后常常是细菌存活的必要成分。所以,细菌难以产生对裂解酶的抗性。本简要综述噬茵体裂解酶的研究进展。     

链球菌噬菌体裂解酶在大肠杆菌中的表达、纯化及活性检测

噬菌体裂解酶是噬菌体产生的细胞壁水解酶,通过水解宿主菌细胞壁使子代噬菌体释放,在体外能高效且特异性地杀死细菌。本研究旨在克隆和表达链球菌噬菌体裂解酶PlyC,并测定其生物学活性。利用PCR方法扩增PlyC的2条肽链PlyCA和PlyCB,构建表达载体pET-32a(+)-PlyCA和pET-32a(+)-PlyCB,分别转化至大肠杆菌BL21(DE3)中,以0.7 mmol/L IPTG在30 oC诱导7 h实现了高效表达,SDS-PAGE分析表明PlyCA和PlyCB表达量均可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法纯化目的蛋白,其纯度大于95%。用透析复性方法得到目的产物重组链球菌噬菌体裂解酶PlyC,以浊度法和平板计数法检测其体外抗菌效果,扫描电子显微镜观察裂解酶作用前后细菌细胞形态变化。结果表明重组PlyC能特异性裂解化脓性链球菌(A组β-溶血性链球菌),以4μg/mL浓度作用于OD600为0.56的菌液60 min后杀菌率达99.6%,扫描电镜观察结果显示该酶作用于菌体后,链球菌细胞裂解,呈碎片状态。本研究为开发一种新型、高效的链球菌感染疾病治疗药物打下了基础。     

猪链球菌7型噬菌体裂解酶Ly7917催化域的裂菌活性

【目的】揭示链球菌噬菌体裂解酶Ly7917催化域CHAP的核心功能域,为进一步改造裂解酶提供理论依据。【方法】通过表达纯化分别从N端截短的Ly7917及从C端截短的Ly CHAP各蛋白,基于平板裂解试验和浊度递减试验,比较各截短蛋白之间的活性差异,以及添加Ca2+后各蛋白的活性变化。【结果】发现催化域蛋白Ly CHAP与Ly7917全酶的活性差异不显著,Ly CHAP的N端序列对其活性影响较大,不宜截短;而C端依次截短后,活性逐渐降低。C端截短20个氨基酸的Ly CHAP1-130,在添加1 mmol/L Ca2+后活性最强。【结论】Ly7917催化域CHAP的核心功能域为1-130 aa,推测其具有Ca2+结合区域,并发现Ly CHAP1-130在Ca2+参与下裂菌活性可媲美Ly7917全酶。预示着Ly CHAP1-130可以替代全酶应用于之后的临床试验。     

噬菌体及其裂解酶控制金黄色葡萄球菌的研究进展

近70年来,由于抗生素的广泛使用,耐药金黄色葡萄球菌不断出现。美国1999～2005年因感染耐甲氧西林金黄色葡萄球菌（MRSA）而入院的患者增加1倍多,其中诊断为败血症的患者增加81．2％。因此,寻找控制耐药菌的新对策十分迫切。目前有望替代抗生素的控菌手段有抗菌肽、噬菌体等。其中,噬菌体的发现早于抗生素,后因抗生素的普及而被忽视。如今,耐药菌株的流行使噬菌体治疗再次受到关注。本文就应用噬菌体及其裂解酶控制金黄色葡萄球菌的研究进展进行综述。     

高效广谱猪链球菌7型前噬菌体裂解酶的挖掘及活性研究

【目的】噬菌体具有防控耐药性病原菌的抗菌应用潜力,但是有些病原菌噬菌体的获得非常困难,研究发现大多数病原菌存在前噬菌体(prophage),且由前噬菌体编码的裂解酶(endolysin)具有良好的抗菌应用前景,本研究将挖掘猪链球菌前噬菌体及其编码的裂解酶。【方法】通过对GenBank中登录的数株猪链球菌前噬菌体裂解酶的基因信息分析,挖掘出一株猪链球菌7型菌株前噬菌体编码的裂解酶,研究其生物学活性。以猪链球菌7型菌株7917的基因组为模板,采用PCR技术扩增获得裂解酶基因ly7917,将其克隆至质粒pET28a(+)并转化大肠杆菌DH5α细胞,挑选基因序列正确的阳性克隆、抽提质粒、转化表达菌株大肠杆菌BL21,经IPTG诱导可高效表达裂解酶Ly7917。【结果】平板裂解试验结果显示Ly7917具有高效裂菌活性,能够裂解猪链球菌2型高致病力菌株HA9801;浊度递减试验结果显示该裂解酶能够高效裂解猪链球菌2型、7型、9型和马链球菌兽疫亚种参考株、金黄色葡萄球菌(包括耐甲氧西林金黄色葡萄球菌)等多种革兰氏阳性菌。【结论】基于前噬菌体挖掘的裂解酶Ly7917,具有高效广谱裂菌活性,为临床上利用裂解酶治疗耐药菌的混合感染提供了可能。     

负载噬菌体qdvp001的阳离子脂质体的制备及热稳定性和抗菌活性

[背景]副溶血弧菌噬菌体qdvp001在防治副溶血弧菌污染方面具有巨大的应用潜力,但其对温度的敏感性较高,在较高的温度环境下会失活.[目的]利用阳离子脂质体包埋保护噬菌体qdvp001以提高其热稳定性,同时探究其脂质体包被液的抗菌活性.[方法]制备和表征负载副溶血弧菌噬菌体qdvp001的阳离子脂质体,测定阳离子脂质体...     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

Selective growth promotion and growth inhibition of Gram-negative and Gram-positive bacteria by synthetic siderophore-β-lactam conjugates

Conjugates of a carbacephalosporin with hydroxamate, spermexatol, N,N-bis(2,3-dihydroxybenzoyl)-L-lysine, mixed catecholate/hydroxamate and cyanuric acid-based siderophores were investigated for their potential to promote growth of siderophore indicator strains of Gram-negative and Gram-positive bacteria under iron depleted conditions, for their antibacterial activity and for their ability to use iron transport path-ways to penetrate the Gram-negative bacterial outer membrane. The selective growth promotion of enter-obacterial and pseudomonas strains by hydroxamate, spermexatol and mixed catecholate-hydroxamate siderophore-based conjugates bearing a L- or D-amino acid spacer was correlated with TonB dependent uptake routes. The preferred outer membrane siderophore receptor used in Escherichia coli was found to be Fiu, followed by Cir. Antagonistic effects of siderophores administered with the conjugates to determine antibacterial activity confirmed the active transport of conjugates via siderophore receptors. All of the conjugates were still able to diffuse through the porin proteins OmpC and OmpF. Nevertheless, strong inhibition of E. coli and Pseudomones aeruginosa outer membrane mutants DC2 and K799/61 compared to the parent strains indicated inefficient penetrability of all types of conjugates tested. Mycobacterium smegmatis SG 987 was able to use all of the siderophore-cephalosporin conjugates as growth promotors. Consequently there was no growth inhibition of this strain. © Rapid Science 1998.     

Regulation of release of antibacterials from stressed scleractinian corals

Recently, we showed that mechanical stress on scleractinian (stony) corals caused a rapid release of antibacterial material (referred to as coral antibacterial activity, or CAA), which killed various bacterial species, including the coral pathogen Vibrio coralliilyticus . We now report on studies on the regulation of CAA release from stressed scleractinian corals. Corals can repeatedly release highly active CAA as a result of sequential stress inductions. Coral fragments were transferred 19 times from one beaker into another with a stress induction each time after 10 min. There was a decrease in the level of antibacterial activity released during the first four to five transfers. After the fifth transfer, the corals kept releasing CAA for the rest of the experiment with no significant decrease. Apparently, the release of CAA is downregulated by feedback inhibition, depending on the concentration of CAA in the surrounding water. By separating CAA-treated V. coralliilyticus from the surrounding water, it was shown that CAA was bound irreversibly to bacterial cells in a stoichiometric manner. Approximately 4 × 10² bacterial cells were sufficient to bind 1 U of CAA. Resident coral bacteria were more resistant to CAA than bacteria isolated from seawater, suggesting an ecological role for CAA. CAA release was obtained from corals after removal of the mucus layer, and the mucus itself contained antibacterial activity.