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Abstract：
UNASSIGNED: In the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.
UNASSIGNED: Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3\'-Dipropylthiacarbocyanine iodide (DiSC3(5)).
UNASSIGNED: The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.
UNASSIGNED: Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3\'-Dipropylthiacarbocyanine iodide (DiSC3(5)).
UNASSIGNED: The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.
摘要：
■在细菌对抗生素耐药性增加的时代，寻找新的杀菌物质，和来自极端生物的溶素具有在恶劣环境条件下稳定的无可置疑的优势。Phiko内溶素来源于感染革兰氏阴性嗜热热菌HB27的phiko噬菌体。该酶与先前研究的两种热稳定的2型酰胺酶相似，Ts2631和Ph2119来自于黑热菌噬菌体，这不仅显示出对嗜热细菌的高裂解活性，而且还显示出对革兰氏阴性嗜温细菌的高裂解活性。因此,在本文提出的研究中研究了PhiKo内溶素的抗菌潜力。
使用浊度降低测定(TRA)和抗细菌测试评估酶活性。采用差示扫描量热法评价蛋白质的稳定性。使用抗微生物肽(CAMP)和抗微生物肽计算器和预测因子(APD3)的集合来预测PhiKo一级序列中具有抗微生物潜力的区域。RAP-29合成肽的最小抑制浓度（MIC）是针对革兰氏阳性和革兰氏阴性选定的菌株确定的，并使用膜电位敏感的荧光染料3,3'-二丙硫氰青碘化物（DiSC3（5））研究了作用机理。
■PhiKo内溶素是高度热稳定的，熔融温度为91.70°C。然而,尽管它对极端微生物如：嗜热T.黄热菌,帕瓦蒂热虫，暗热,和耐辐射球菌,PhiKo对中温细菌显示出中等的抗菌活性。因此,它的蛋白质序列被搜索到具有潜在抗菌活性的区域。鉴定并合成了带高度正电荷的区域(PhiKo105-133)。新的RAP-29肽裂解葡萄球菌和革兰氏阴性菌的嗜温菌株，将细胞数量减少3.7-7.1log单位，并达到2-31μM范围内的最小抑制浓度值。该肽在水溶液中是未结构化的,但在洗涤剂存在下形成α-螺旋。此外，它结合脂磷壁酸和脂多糖，并导致细菌膜去极化。RAP-29肽是对抗细菌病原体的有希望的候选物。这种隐蔽肽的存在证明了比以前认为的更广泛的抗菌肽组。
使用浊度降低测定(TRA)和抗细菌测试评估酶活性。采用差示扫描量热法评价蛋白质的稳定性。使用抗微生物肽(CAMP)和抗微生物肽计算器和预测因子(APD3)的集合来预测PhiKo一级序列中具有抗微生物潜力的区域。RAP-29合成肽的最小抑制浓度（MIC）是针对革兰氏阳性和革兰氏阴性选定的菌株确定的，并使用膜电位敏感的荧光染料3,3'-二丙硫氰青碘化物（DiSC3（5））研究了作用机理。
■PhiKo内溶素是高度热稳定的，熔融温度为91.70°C。然而,尽管它对极端微生物如：嗜热T.黄热菌,帕瓦蒂热虫，暗热,和耐辐射球菌,PhiKo对中温细菌显示出中等的抗菌活性。因此,它的蛋白质序列被搜索到具有潜在抗菌活性的区域。鉴定并合成了带高度正电荷的区域(PhiKo105-133)。新的RAP-29肽裂解葡萄球菌和革兰氏阴性菌的嗜温菌株，将细胞数量减少3.7-7.1log单位，并达到2-31μM范围内的最小抑制浓度值。该肽在水溶液中是未结构化的,但在洗涤剂存在下形成α-螺旋。此外，它结合脂磷壁酸和脂多糖，并导致细菌膜去极化。RAP-29肽是对抗细菌病原体的有希望的候选物。这种隐蔽肽的存在证明了比以前认为的更广泛的抗菌肽组。
