靶向革兰氏阴性菌生物膜的糖苷水解酶挖掘及功能研究

Mining and functional study of glycoside hydrolases against biofilms of Gram-negative bacteria

目的挖掘对革兰氏阴性菌生物膜具有抑制和破坏作用,特别是具有跨种活性的糖苷水解酶。方法利用已有基因组数据,通过序列相似性比对及基因注释分析寻找新型糖苷水解酶,通过基因克隆和蛋白表达纯化获得一系列不同革兰氏阴性菌来源的糖苷水解酶。以靶向铜绿假单胞菌生物膜的糖苷水解酶PslG31-442作为阳性对照酶,构建靶向铜绿假单胞菌生物膜的药物筛选模型,并进一步构建靶向肺炎克雷伯杆菌生物膜的药物筛选模型。利用自行构建的筛选模型对上述不同糖苷水解酶进行活性筛选。结果筛选得到一种具有跨种活性的、来自克雷伯菌属糖苷水解酶19家族的糖苷水解酶KPGH3。KPGH3能够有效抑制铜绿假单胞菌PAO1、PA14菌株以及肺炎克雷伯ATCC 700603菌株生物膜的形成,EC_(50)分别为36.85、43.34和82.58 nmol/L;并能够在1 h内有效破坏3种菌株形成的成熟生物膜,EC_(50)分别为45.35、29.3和18.23 nmol/L。结论糖苷水解酶KPGH3不但能在纳摩尔级浓度抑制多种致病性革兰氏阴性菌生物膜的形成,并能有效破坏上述菌株已经形成的陈旧生物膜,具有潜在应用价值。

Objective To investigate glycoside hydrolases that can inhibit and disrupt Gram-negative bacteria biofilms,especially with cross-species activity.Methods A series of novel glycoside hydrolase genes from different Gram-negative bacteria was mined through sequence similarity comparisons and gene annotation analysis based on existing genomic data.Encoded proteins was obtained through gene cloning,protein expression and purification.The glycoside hydrolase PslG31-442 was expressed as a positive control to construct a screening model for the disruption of P.aeruginosa biofilms.Based on this,a drug screening model for disruption of K.pneumoniae biofilm was further constructed according to experimental requirements.Using a self-built screening model,the above-mentioned different glycoside hydrolases have been screened for activity.Results KPGH3,a member of glycoside hydrolases 19 family from Klebsiella with cross-kingdom activity,was obtained by screening.The study found that KPGH3 effectively inhibited the biofilm formation of P.aeruginosa strain PAO1,PA14 and K.pneumoniae ATCC 700603,with an EC_(50) of 36.85,43.34 and 82.58 nmol/L,respectively.More importantly,KPGH3 were capable of disrupting preexisting biofilms in 1 h with an EC_(50) of 45.35(PAO1),29.3(PA14),and 18.23 nmol/L(K.pneumoniae).Conclusion The glycoside hydrolase KPGH3 not only significantly inhibits the formation of a variety of pathogenic Gram-negative bacteria biofilms,but also disrupts the preexisting biofilms that have been formed by the above-mentioned strains,which has a significant potential application value.