金葡菌噬菌体裂解酶LysGH15关键位点的确定及其与黄芩苷联合治疗小鼠肺炎的研究
发布时间：2018-08-27 12:54
【摘要】：金黄色葡萄球菌是最常见的社区和医院感染相关病原菌。由于广谱耐药菌株的出现,预防和治疗由金葡菌所引起的疾病变得更加具有挑战性,寻求新的治疗策略已是势在必行。噬菌体裂解酶是双链DNA噬菌体在感染宿主周期期间产生的细菌细胞壁水解酶,其能够通过水解细胞壁肽聚糖从而破坏细胞壁的完整性,最终导致细菌崩解死亡。由于具有高效、特异的杀菌活性,噬菌体裂解酶被认为是控制细菌病原体的新型潜在药物。中药单体黄芩苷能靶向对抗金葡菌毒力因子α-溶血素,可以有效治疗金葡菌感染。前期,本实验室获得了金葡菌的噬菌体裂解酶Lys GH15,其对金葡菌表现出广谱、高效的杀菌活性。为进一步探索裂解酶作用的分子机制及其与中药联合应用的协同机理,本研究进一步对其催化区与底物互作的关键位点进行分析,并且将裂解酶Lys GH15与黄芩苷进行联合应用,对金葡菌的肺炎感染模型的治疗效果进行评价。裂解酶作用的分子机制应用Auto Dock 4.2软件对Lys GH15的CHAP片段与多肽DGln NH2-LLys-DAla-5Gly进行分子对接,预测Asp33、Ser35、Phe36、Tyr50、Arg71、Tyr72、Gln53和Asn75在CHAP与底物作用过程中应该会起作用。分别将这7个位点突变为丙氨酸后,通过诱导表达发现位点Asp33与Asn75突变为丙氨酸后蛋白无法成功表达,而Arg71、Tyr72、Ser35、Phe36和Tyr50这五个位点的突变体蛋白成功获得了表达。对CHAP片段和Arg71、Tyr72、Ser35、Phe36和Tyr50这五个位点的突变体进行了圆二色谱分析,CHAP片段的突变体在蛋白质二级结构组成上(α-螺旋和β-折叠的含量)没有显著的差异,表明这五个位点不影响CHAP的二级结构,也就不会影响CHAP片段的三级结构。酶谱实验结果表明,只有F36A不能形成透明条带,其余四个突变体均可以形成,这说明F36A突变体失去裂菌活性。天然的CHAP和S35A、Y50A、R71A、T72A这四个突变体均可以在60 min内使菌液的OD600值从0.6下降到几乎检测不到的状态,对照组加入了缓冲液作为空白对照,在加入同样的蛋白量的条件下(终浓度50μg/ml),只有F36A突变体的菌液OD600值变化趋势与对照组相似,菌液浓度没有下降,而同样,在菌落计数的试验中也发现只有突变体F36A活性完全丧失,而S35A、Y50A、R71A、T72A这四个突变体的抗菌活性与天然的CHAP没有显著的差异。由此表明,位点F36在CHAP蛋白与底物互作过程中起关键作用,而Asp33与Asn75对蛋白的折叠可能起关键的作用。裂解酶与黄芩苷联合应用对Lys GH15与黄芩苷在体外活性的相互影响进行了研究。单独的Lys GH15处理(剂量为50μg/ml,USA300菌悬液的OD600值在10 min内从0.785降至0.194)与Lys GH15(剂量50μg/ml)和黄芩苷(剂量16μg/ml)联合处理(USA300菌悬液的OD600值在10 min内从0.745降至0.124)相比,裂解活性没有显著差异(P0.05)。在溶血实验中,当兔红细胞与黄芩苷(16μg/ml)和Lys GH15(≥50μg/ml)联合同时孵育时,没有检测到溶血。相比之下,用黄芩苷单独处理时溶血率为13.1%,当用终浓度为25、50或100μg/ml的Lys GH15单独处理时,溶血率分别为34.5%、22.74%和20.7%。通过Western blot实验检测黄芩苷和Lys GH15对α-溶血素表达量的影响,表明同时加入黄芩苷(16μg/ml)和Lys GH15(100μg/ml)时几乎没有α-溶血素产生。进一步研究Lys GH15与黄芩苷联合使用治疗金葡菌肺炎的效果。结果表明,联合治疗组在提高小鼠的健康状况、降低肺脏W/T比值以及BALF中细胞数、蛋白含量及细胞因子水平上优于单独治疗组。在Lys GH15单独治疗和联合治疗的情况下,肺脏细菌菌落数降低至8.0×102CFU/mg和5.0×101CFU/mg,在黄芩苷单独治疗下,肺脏细菌菌落数达到1.0×104CFU/mg。作为对照,在没有治疗的情况下,24 h后的肺脏的细菌数量分别增加至3.3×1010CFU/mg,说明Lys GH15与黄芩苷联合使用对金葡菌肺炎能产生良好的治疗效果。
[Abstract]:Staphylococcus aureus is the most common pathogen associated with community and hospital infections. As a result of the emergence of broad-spectrum drug-resistant strains, prevention and treatment of diseases caused by Staphylococcus aureus have become more challenging. It is imperative to seek new treatment strategies. Phage lysozymes are produced by double-stranded DNA phages during the infection host cycle. Bacterial cell wall hydrolase, which can destroy the integrity of cell wall by hydrolyzing cell wall peptidoglycan, eventually leads to bacterial disintegration and death. Because of its high efficiency and specific bactericidal activity, bacteriophage lysozyme is considered as a new potential drug to control bacterial pathogens. Baicalin, a traditional Chinese medicine monomer, can target the virulence of Staphylococcus aureus. In the early stage, the phage lysozyme Lys GH15 of Staphylococcus aureus was obtained, which showed broad-spectrum and high-efficiency bactericidal activity against Staphylococcus aureus. The key sites of substrate interaction were analyzed and Lys GH15 and baicalin were combined to evaluate the therapeutic effect of Staphylococcus aureus pneumonia infection model. The molecular mechanism of Lys GH15 was docked with DGln NH2-LLys-DAla-5Gly by Auto Dock 4.2 software to predict Asp33. Ser35, Phe36, Tyr50, Arg71, Tyr72, Gln53 and Asn75 should play a role in the interaction between CHAP and substrates. After mutation of these seven sites into alanine, it was found that Asp33 and Asn75 could not be successfully expressed after mutation into alanine, but the mutants of Arg71, Tyr72, Ser35, Phe36 and Tyr50 were formed. The results showed that there was no significant difference in protein secondary structure composition (alpha-helix and beta-folding content) between the mutants of CHAP fragments and the mutants of Arg71, Tyr72, Ser35, Phe36 and Tyr50, indicating that these five sites did not affect the secondary structure of CHAP, nor did they affect the secondary structure of C. The results of Enzymogram showed that only F36A could not form transparent bands and the other four mutants could form, which indicated that F36A mutants lost fission activity. In the control group, buffers were added as the blank control. Under the same protein content (final concentration 50 ug/ml), the change trend of OD600 value of F36A mutant was similar to that of the control group, and the concentration of F36A did not decrease. Similarly, only F36A mutant completely lost its activity, but S35A, Y50A, R71A mutant completely lost its activity in the colony count test. These results suggest that site F36 plays a key role in the interaction between CHAP protein and substrate, and Asp33 and Asn75 may play a key role in protein folding. Lys GH15 treatment alone (dosage 50 ug/ml, USA 300 suspension OD600 value decreased from 0.785 to 0.194 in 10 minutes) and Lys GH15 (dosage 50 ug/ml) and Baicalin (dosage 16 ug/ml) combined treatment (USA 300 suspension OD600 value decreased from 0.745 to 0.124 in 10 minutes) showed no significant difference in lysis activity (P 0.05). In hemolysis test In contrast, the hemolysis rate was 13.1% when treated with baicalin alone, and 34.5%, 22.74% and 20.7% when treated with baicalin 25, 50 or 100 ug/ml respectively. The effect of baicalin and Lys GH15 on the expression of alpha-hemolysin showed that there was almost no production of alpha-hemolysin when baicalin (16 ug/ml) and Lys GH15 (100 ug/ml) were added at the same time. Bacterial colonies in lung were reduced to 8.0 *102 CFU/mg and 5.0 *101 CFU/mg in Lys GH15 alone and in combination. Bacterial colonies in lung were up to 1.0 *10 CFU/mg in baicalin alone. After 24 hours, the number of bacteria in the lungs increased to 3.3 *1010 CFU/mg, indicating that Lys GH15 combined with baicalin can produce a good therapeutic effect on staphylococcal pneumonia.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.1
本文编号：2207349
[Abstract]:Staphylococcus aureus is the most common pathogen associated with community and hospital infections. As a result of the emergence of broad-spectrum drug-resistant strains, prevention and treatment of diseases caused by Staphylococcus aureus have become more challenging. It is imperative to seek new treatment strategies. Phage lysozymes are produced by double-stranded DNA phages during the infection host cycle. Bacterial cell wall hydrolase, which can destroy the integrity of cell wall by hydrolyzing cell wall peptidoglycan, eventually leads to bacterial disintegration and death. Because of its high efficiency and specific bactericidal activity, bacteriophage lysozyme is considered as a new potential drug to control bacterial pathogens. Baicalin, a traditional Chinese medicine monomer, can target the virulence of Staphylococcus aureus. In the early stage, the phage lysozyme Lys GH15 of Staphylococcus aureus was obtained, which showed broad-spectrum and high-efficiency bactericidal activity against Staphylococcus aureus. The key sites of substrate interaction were analyzed and Lys GH15 and baicalin were combined to evaluate the therapeutic effect of Staphylococcus aureus pneumonia infection model. The molecular mechanism of Lys GH15 was docked with DGln NH2-LLys-DAla-5Gly by Auto Dock 4.2 software to predict Asp33. Ser35, Phe36, Tyr50, Arg71, Tyr72, Gln53 and Asn75 should play a role in the interaction between CHAP and substrates. After mutation of these seven sites into alanine, it was found that Asp33 and Asn75 could not be successfully expressed after mutation into alanine, but the mutants of Arg71, Tyr72, Ser35, Phe36 and Tyr50 were formed. The results showed that there was no significant difference in protein secondary structure composition (alpha-helix and beta-folding content) between the mutants of CHAP fragments and the mutants of Arg71, Tyr72, Ser35, Phe36 and Tyr50, indicating that these five sites did not affect the secondary structure of CHAP, nor did they affect the secondary structure of C. The results of Enzymogram showed that only F36A could not form transparent bands and the other four mutants could form, which indicated that F36A mutants lost fission activity. In the control group, buffers were added as the blank control. Under the same protein content (final concentration 50 ug/ml), the change trend of OD600 value of F36A mutant was similar to that of the control group, and the concentration of F36A did not decrease. Similarly, only F36A mutant completely lost its activity, but S35A, Y50A, R71A mutant completely lost its activity in the colony count test. These results suggest that site F36 plays a key role in the interaction between CHAP protein and substrate, and Asp33 and Asn75 may play a key role in protein folding. Lys GH15 treatment alone (dosage 50 ug/ml, USA 300 suspension OD600 value decreased from 0.785 to 0.194 in 10 minutes) and Lys GH15 (dosage 50 ug/ml) and Baicalin (dosage 16 ug/ml) combined treatment (USA 300 suspension OD600 value decreased from 0.745 to 0.124 in 10 minutes) showed no significant difference in lysis activity (P 0.05). In hemolysis test In contrast, the hemolysis rate was 13.1% when treated with baicalin alone, and 34.5%, 22.74% and 20.7% when treated with baicalin 25, 50 or 100 ug/ml respectively. The effect of baicalin and Lys GH15 on the expression of alpha-hemolysin showed that there was almost no production of alpha-hemolysin when baicalin (16 ug/ml) and Lys GH15 (100 ug/ml) were added at the same time. Bacterial colonies in lung were reduced to 8.0 *102 CFU/mg and 5.0 *101 CFU/mg in Lys GH15 alone and in combination. Bacterial colonies in lung were up to 1.0 *10 CFU/mg in baicalin alone. After 24 hours, the number of bacteria in the lungs increased to 3.3 *1010 CFU/mg, indicating that Lys GH15 combined with baicalin can produce a good therapeutic effect on staphylococcal pneumonia.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.1
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1 胡丽媛;金葡菌噬菌体裂解酶LysGH15关键位点的确定及其与黄芩苷联合治疗小鼠肺炎的研究[D];吉林大学;2017年
，本文编号：2207349
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