高活性噬菌体解聚酶∆Dpo42对大肠杆菌生物被膜的抑制作用及机制研究

The formation of bacterial biofilms brings great difficulties to the treatment of infectious diseases. Some phages can encode depolymerases to inhibit the formation of bacterial biofilms, and then improve the sensitivity of antibiotics. Applicant has found a new phage-associated depolymerase Dpo42 which has a dose-dependent activity to prevent the biofilm formation of E. coli. However, the mechanism of inhibiting the formation of biofilm is unclear. A study has proved the residues 281-295 of PhaZ7 depolymerase act as the active site of the enzyme. Thus, we guess Dpo42 also has at least one key active site giving its anti-biofilm activity. But the active site of Dpo42 is not clear. In order to clarify the location of the active site and improve its activity, the project aims to establish a three-dimensional structure model of Dpo42 and predict its active site. Site directed mutagenesis was performed to verify the role of its active site. Reconstructing the catalytic active groups with low activity was reconstructed and the high activity mutant ∆Dpo42 was constructed. Taking advantage of the clinical isolation of drug-resistant E. coli, the anti-biofilm activity of ∆Dpo42 will be verified in vitro as well as in vivo, which providing the foundation of novel anti-biofilm drug of E. coli.

细菌生物被膜的形成给感染性疾病的治疗带来很大的困难。有些噬菌体可以编码解聚酶来抑制细菌生物被膜的形成，进而提高抗生素的敏感性。申请人前期研究发现一新的大肠杆菌噬菌体解聚酶Dpo42可抑制宿主菌生物被膜的形成，且具有浓度依赖性，但其抑制生物被膜形成的机制不清，有研究证实解聚酶PhaZ7的活性位点位于281-295氨基酸残基的位置，因此推测，Dpo42也存在至少一个关键的活性位点赋予了其抗生物被膜的活性。但Dpo42的活性位点尚不清楚，为了证实这一推断及明确其活性位点并提高其活性，本项目拟建立Dpo42的三维结构模型并预测其活性位点，通过点突变来验证其活性位点的作用，针对其活性较低的催化活性基团进行改造，构建高活性突变体∆Dpo42，以临床分离的耐药性大肠杆菌为研究对象，通过体内外实验来验证∆Dpo42抗生物被膜的作用，从而为探索新型抗大肠杆菌生物被膜的药物提供基础。

细菌生物被膜的形成给感染性疾病的治疗带来很大的困难。有些噬菌体可以编码解聚酶来抑制细菌生物被膜的形成，进而提高抗生素的敏感性。前期课题组研究获得一新的大肠杆菌噬菌体解聚酶Dpo42可抑制宿主菌生物被膜的形成，且具有浓度依赖性，但Dpo42的活性位点尚不清楚，本项目对大肠杆菌噬菌体vB_EcoM_ECOO78的解聚酶Dpo42进行了全面的研究，建立了Dpo42三维结构模型，并预测其活性位点，共预测到8个结合位点，其中预测最可能的蛋白结合位点活性位点为：位点5（ASN235、ASP236、LEU238、ILE239、PHE240、GLY262、PRO263、LEU264、LYS265、PHE266、PHE287、ASP288、ILE292），共包含13个氨基酸。进一步将解聚酶Dpo42截短为C186、C372、C558、N186、N372、N558，研究表明C558与Dpo42一样具有抗大肠杆菌生物被膜的活性，并且能够抑制2株具有强生物膜的能力的大肠杆菌HXM和E9生物被膜的形成，值得一提的是C558不影响人B淋巴母细胞的细胞存活率，无毒无害，且能够增加正常人血清抗大肠杆菌的能力，能够在防治大肠杆菌及其生物被膜方面具有应用价值。