屎肠球菌PDF抑制剂的筛选及活性菌株的鉴定；I04-4776、I04-5195等生物活性菌株的多项分类研究
摘要
肽脱甲酰基酶(peptide deformylase,PDF)是微生物生长、繁殖的关键酶,肽脱甲酰基酶抑制剂具有广谱、不易产生耐药性、与现有的抗菌素不发生交叉耐药和选择性毒性好等优点,因此,Dan Ferbe等在发表于2002年2月Science上的一篇文章中,将PDF视为新一代广谱抗生素筛选最理想的分子靶点之一。
本室唐先兵博士已完成以屎肠球菌PDF为靶点的高通量筛选模型的构建,但尚未深入筛选。本研究对该模型进行了恢复,并应用屎肠球菌E.faecium(ATCC19434)为初筛模型,PDF酶为二筛模型,开展了PDF酶抑制剂的规模化筛选。从20261个微生物发酵样品中,挑选出6株活性较强且稳定的菌株,测定了它们的抗菌谱。菌株03—723和03—8772的抗菌谱十分引人注意,对所有耐药菌包括MRSA、MRSE和VREF都呈现抗菌活性,而且对PDF酶也有抑制作用。因此,本研究对这两株菌进行了鉴定,研究结果表明:这两株菌属于游动放线菌属的两个新种。其抗耐药菌的活性成分值得深入研究。
为扩大筛选范围,本研究还对13株红树林植物样品和2株药用植物样品进行了内生放线菌的分离,共分离出103株植物内生放线菌。经过表观形态比较、选取部分代表菌株进行16S rRNA基因的系统发育研究。研究结果表明,这103株分离株分布于放线菌目中的五个属:链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、诺卡氏菌属(Nocardia)、伦茨氏菌属(Lentzea)和糖丝菌属(Saccharothrix)。其中链霉菌占大多数,伦茨氏菌属(Lentzea)和糖丝菌属(Saccharothrix)菌株是本研究首次从植物中分离得到的。有8株放线菌与已知模式菌的相似性比较低,其中有3株低于98.5%,这些菌株可能代表了链霉菌属、伦茨氏菌属、糖丝菌属内未知的分类单元,有待进一步验证。这103株分离菌经过PDF酶筛选获得2株酶抑制阳性放线菌:I07-01838和I08-00453。经鉴别,菌株I07-01838为碳样小单孢菌(Micromonospora carbonacea),菌株I08-00453为一株稀有放线菌。本论文首次从植物内生菌中分离得到碳样小单孢菌,并首次发现碳样小单孢菌属菌株产生PDF酶抑制剂。
本研究还对近年来本所新药筛选重点实验室筛选得到的几株活性菌株进行了“种”水平的鉴定,其中,利用人高密度脂蛋白受体CLA-1表达上调剂筛选模型筛选得到的阳性菌株I04-4776被鉴定为链霉菌属的一个新种,命名为Streptomyces emeiensis,文章已发表在IJSEM上,活性成分4776B(4'-甲氧基-3',5,7-三羟基异黄酮)为首次从微生物代谢产物中获得;该模型筛得的另一个阳性菌株I04-5486与Streptomyces kunmingensis同种;以骨形态形成蛋白因子Ⅱ
Peptide Deformylase(PDF) is an essential enzyme for growth and multiplication of bacteria and bacteria can not survive if PDF has any mutation.The inhibitors of PDF have the characteristics of broad spectrum-of-activity,low frequency of antimicrobial resistance,good selective toxicity,so it is regarded as one of most promising target for the discovery of novel antibiotics.
The screening model targeted on E.faecium PDF for novel broad spectrum antibiotics has been establishted by Dr.Xianbing Tang in our laboratory.In this study,we renewed this model including overexpression,purification PDF of E.faecium according to the method by Dr.Tang.E.faecium ATCC 19434 was took as the cell screening model in our preliminary screening.The purified PDF of E.faecium was then used in a high throughout screening as the target for searching the inhibitor of it from the positive microbial fermentation broths samples obtained from the E.faecium screening model.6 trains with potent and stable activity were picked out from 20261 microbial fermentation broths samples.The antimicrobial spectrum against more than twenty strains including VRE, MRSA,MRSE was measured with MIC method.The result showed that the microbial fermentation broth of strain I07-01349 has the activity against MRSA,MRSE,MSSA, SE and MSSE but not against VREF,E.faecalis;the microbial fermentation broth of strain I06-01113 has the activity against Candida albicans,Staphylococcus aureus, Aspergillus niger、MSSA,VREF,E.faecalis but not MRSA,the microbial fermentation broth of strain I03-3506 has the activity against Candida albicans,Staphylococcus aureus,Aspergillus niger,E.faecalis but not all the other strains including VREF;the microbial fermentation broth of strain I03-01347 has the activity against MSSA15、MRSA 05-3、SE、MRSE 05-1、MSSE 05-20,but not all the other strains including VREF; the microbial fermentation broth of strain I03-723 and I03-8772 have the activity against many clinical antibiotic resistant strains including VREF,MRSA,MRSE with the potent inhibitory activity against E.faecium PDF at the same time.These two strains were classified as two novel species of the genus Actinoplanes based on a polyphasic approach and the DNA-DNA relatedness between them was 47.0%.
Based on the consideration to enrichment the microbial resource for screening,103 strains were islated from the plant samples of 15 kinds of plants including 13 kinds of Mangrove plants and 2 kinds of medicinal plants.After dereplication and grouping by their cultural characteristics on different media,representatives of the isolates were phylogeneticly analyzed based on 16S rRNA genes.These strains were determined to fall within 5 genera:Streptomyces,Micromonospora,Nocardia,Lentzea,Saccharothrix. Above 90%of them were Streptomycetes.Lentzea and Saccharothrix were isolted from plant for the first time.8 of the representatives hold low 16S rRNA gene similarities with validly published species.3 strains of them(the similarities with the closest type species are all below 98.5%) are probably novel strains of genus Streptomycetes,Lentzea and Saccharothrix.Endophytic rare actinomycete isolate I07-01838 and I I08-00453 were picked out with the good inhibitory activiry again PDF,and I07-01838 was classified to Micromonospora carbonacea.Micromonospora carbonacea was isolated from plant and was found to produce PDF inhibitor for the first time.
In this study,some bioactive strains obtained from some other screening models in the national key laboratory were classified with polyphasic approach.Actinomycete strain I04-4776 which produced up-regulators of CLA-1 was identified a novel strain of genus Streptomyces,for which the name Streptomyces emeienseis sp.nov.is proposed. Its bioactive compound 4776B is obtained from Actinomcete metabolites.An other strain I04-5486 from this model was determined to Streptomyces kunmingensis.In an other screening model targeting the BMP-2 promoter,strain 104-5195 which produces Amicoumacin B(a compound which can enhance the expression of the BMP-2) was classified as a novel strain of genus Nocardia;the name Nocardia jinanensis sp.nov.is proposed.Amicoumacin B was previously reported to be produced by Bacillus pumilus, and I04-5195 was the first actinomycete identified toproduce amicoumacin B.
本室唐先兵博士已完成以屎肠球菌PDF为靶点的高通量筛选模型的构建,但尚未深入筛选。本研究对该模型进行了恢复,并应用屎肠球菌E.faecium(ATCC19434)为初筛模型,PDF酶为二筛模型,开展了PDF酶抑制剂的规模化筛选。从20261个微生物发酵样品中,挑选出6株活性较强且稳定的菌株,测定了它们的抗菌谱。菌株03—723和03—8772的抗菌谱十分引人注意,对所有耐药菌包括MRSA、MRSE和VREF都呈现抗菌活性,而且对PDF酶也有抑制作用。因此,本研究对这两株菌进行了鉴定,研究结果表明:这两株菌属于游动放线菌属的两个新种。其抗耐药菌的活性成分值得深入研究。
为扩大筛选范围,本研究还对13株红树林植物样品和2株药用植物样品进行了内生放线菌的分离,共分离出103株植物内生放线菌。经过表观形态比较、选取部分代表菌株进行16S rRNA基因的系统发育研究。研究结果表明,这103株分离株分布于放线菌目中的五个属:链霉菌属(Streptomyces)、小单孢菌属(Micromonospora)、诺卡氏菌属(Nocardia)、伦茨氏菌属(Lentzea)和糖丝菌属(Saccharothrix)。其中链霉菌占大多数,伦茨氏菌属(Lentzea)和糖丝菌属(Saccharothrix)菌株是本研究首次从植物中分离得到的。有8株放线菌与已知模式菌的相似性比较低,其中有3株低于98.5%,这些菌株可能代表了链霉菌属、伦茨氏菌属、糖丝菌属内未知的分类单元,有待进一步验证。这103株分离菌经过PDF酶筛选获得2株酶抑制阳性放线菌:I07-01838和I08-00453。经鉴别,菌株I07-01838为碳样小单孢菌(Micromonospora carbonacea),菌株I08-00453为一株稀有放线菌。本论文首次从植物内生菌中分离得到碳样小单孢菌,并首次发现碳样小单孢菌属菌株产生PDF酶抑制剂。
本研究还对近年来本所新药筛选重点实验室筛选得到的几株活性菌株进行了“种”水平的鉴定,其中,利用人高密度脂蛋白受体CLA-1表达上调剂筛选模型筛选得到的阳性菌株I04-4776被鉴定为链霉菌属的一个新种,命名为Streptomyces emeiensis,文章已发表在IJSEM上,活性成分4776B(4'-甲氧基-3',5,7-三羟基异黄酮)为首次从微生物代谢产物中获得;该模型筛得的另一个阳性菌株I04-5486与Streptomyces kunmingensis同种;以骨形态形成蛋白因子Ⅱ
Peptide Deformylase(PDF) is an essential enzyme for growth and multiplication of bacteria and bacteria can not survive if PDF has any mutation.The inhibitors of PDF have the characteristics of broad spectrum-of-activity,low frequency of antimicrobial resistance,good selective toxicity,so it is regarded as one of most promising target for the discovery of novel antibiotics.
The screening model targeted on E.faecium PDF for novel broad spectrum antibiotics has been establishted by Dr.Xianbing Tang in our laboratory.In this study,we renewed this model including overexpression,purification PDF of E.faecium according to the method by Dr.Tang.E.faecium ATCC 19434 was took as the cell screening model in our preliminary screening.The purified PDF of E.faecium was then used in a high throughout screening as the target for searching the inhibitor of it from the positive microbial fermentation broths samples obtained from the E.faecium screening model.6 trains with potent and stable activity were picked out from 20261 microbial fermentation broths samples.The antimicrobial spectrum against more than twenty strains including VRE, MRSA,MRSE was measured with MIC method.The result showed that the microbial fermentation broth of strain I07-01349 has the activity against MRSA,MRSE,MSSA, SE and MSSE but not against VREF,E.faecalis;the microbial fermentation broth of strain I06-01113 has the activity against Candida albicans,Staphylococcus aureus, Aspergillus niger、MSSA,VREF,E.faecalis but not MRSA,the microbial fermentation broth of strain I03-3506 has the activity against Candida albicans,Staphylococcus aureus,Aspergillus niger,E.faecalis but not all the other strains including VREF;the microbial fermentation broth of strain I03-01347 has the activity against MSSA15、MRSA 05-3、SE、MRSE 05-1、MSSE 05-20,but not all the other strains including VREF; the microbial fermentation broth of strain I03-723 and I03-8772 have the activity against many clinical antibiotic resistant strains including VREF,MRSA,MRSE with the potent inhibitory activity against E.faecium PDF at the same time.These two strains were classified as two novel species of the genus Actinoplanes based on a polyphasic approach and the DNA-DNA relatedness between them was 47.0%.
Based on the consideration to enrichment the microbial resource for screening,103 strains were islated from the plant samples of 15 kinds of plants including 13 kinds of Mangrove plants and 2 kinds of medicinal plants.After dereplication and grouping by their cultural characteristics on different media,representatives of the isolates were phylogeneticly analyzed based on 16S rRNA genes.These strains were determined to fall within 5 genera:Streptomyces,Micromonospora,Nocardia,Lentzea,Saccharothrix. Above 90%of them were Streptomycetes.Lentzea and Saccharothrix were isolted from plant for the first time.8 of the representatives hold low 16S rRNA gene similarities with validly published species.3 strains of them(the similarities with the closest type species are all below 98.5%) are probably novel strains of genus Streptomycetes,Lentzea and Saccharothrix.Endophytic rare actinomycete isolate I07-01838 and I I08-00453 were picked out with the good inhibitory activiry again PDF,and I07-01838 was classified to Micromonospora carbonacea.Micromonospora carbonacea was isolated from plant and was found to produce PDF inhibitor for the first time.
In this study,some bioactive strains obtained from some other screening models in the national key laboratory were classified with polyphasic approach.Actinomycete strain I04-4776 which produced up-regulators of CLA-1 was identified a novel strain of genus Streptomyces,for which the name Streptomyces emeienseis sp.nov.is proposed. Its bioactive compound 4776B is obtained from Actinomcete metabolites.An other strain I04-5486 from this model was determined to Streptomyces kunmingensis.In an other screening model targeting the BMP-2 promoter,strain 104-5195 which produces Amicoumacin B(a compound which can enhance the expression of the BMP-2) was classified as a novel strain of genus Nocardia;the name Nocardia jinanensis sp.nov.is proposed.Amicoumacin B was previously reported to be produced by Bacillus pumilus, and I04-5195 was the first actinomycete identified toproduce amicoumacin B.
引文
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