Function and safety of bacterial alginate lyase for the therapy of biofilm-dependent infection of Pseudomonas aeruginosa

细菌藻酸盐裂解酶治疗铜绿假单胞菌生物膜依赖性感染的功能和安全性

基本信息

批准号：
10556017
负责人：
MURATA Kousaku
金额：
$ 5.57万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

In bacterial infections, typically found in the case of Pseudomonas aeruginosa infection, the infected bacterial cells synthesize and excrete highly viscous and spramolecular polysaccharides and multiply in the environments surrounded by the polymer. However, the biofilms has a property to inhibit transport of antimicrobes or function of phagocytes, thus makes it difficult to treat biofilm-dependent bacterial infection diseases. In order to overcome the difficulty and develop novel therapeutic means, we proposed the use of bacterial alginate lyase that liquefies and removes biofilms. However, before doing so, the antigenicity of the bacterial enzyme should be eliminated. Through biochemical and computer graphic analyses, we determined antigenic-epitope in the vicinity of the N-terminal region (27Ser-44Cys) of the enzyme. The region is on the enzyme surface and far from the active center. Among the amino acids in the region, 27Ser, 29Gln, 32Asp, and 40Lys were highly exposed to the surface of the enzyme, and they were converted to non-polar amino acid alanine by site-directed mutagenesis. The mutated enzymes (S27A, Q29A, D32A, and K40A) were expressed in Escherichia coli cells. The enzyme K40A formed firm inclusion body and was not renatured in active form. Other enzymes were purified and subjected to the antigenic study in mouse system. In addition to these studies, we determined the detailed active site structure through the analyses of enzyme/substrate complex.

在细菌感染中，通常在铜绿假单胞菌感染的情况下发现，感染的细菌细胞合成并排泄高度粘稠和涂抹分子多糖，并在聚合物包围的环境中繁殖。然而，生物膜具有抑制抗菌或吞噬细胞功能的运输的特性，因此很难治疗依赖生物膜的细菌感染疾病。为了克服困难并发展新的治疗手段，我们提出了使用液化和去除生物膜的细菌藻酸盐裂解酶的使用。但是，在这样做之前，应消除细菌酶的抗原性。通过生化和计算机图形分析，我们确定了酶的N末端区域（27ser-44Cys）附近的抗原 - ePitope。该区域位于酶表面，远离活性中心。在该地区的氨基酸中，27ser，29GLN，32ASP和40lys高度暴露于酶的表面，并通过定分定向的诱变将其转化为非极性氨基酸丙氨酸。突变的酶（S27a，Q29a，d32a和K40a）在大肠杆菌细胞中表达。酶K40a形成了牢固的包容体，并未以活性形式重新恢复。将其他酶纯化，并在小鼠系统中进行抗原研究。除了这些研究外，我们还通过酶/底物复合物的分析确定了详细的活性位点结构。