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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，通常见于铜绿假单胞菌感染，受感染的细菌细胞合成并分泌高粘性和超分子多糖，并在聚合物包围的环境中繁殖。然而，生物膜具有抑制抗菌剂转运或吞噬细胞功能的特性，因此使得生物膜依赖性细菌感染疾病的治疗变得困难。为了克服这一困难并开发新的治疗手段，我们提出使用细菌藻酸盐裂解酶来液化和去除生物膜。然而，在此之前，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 N 末端区域 (27Ser-44Cys) 附近的抗原表位。该区域位于酶表面，远离活性中心。该区域的氨基酸中，27Ser、29Gln、32Asp和40Lys高度暴露于酶表面，通过定点诱变将它们转化为非极性氨基酸丙氨酸。突变酶（S27A、Q29A、D32A 和 K40A）在大肠杆菌细胞中表达。酶K40A形成坚固的包涵体并且未以活性形式复性。其他酶被纯化并在小鼠系统中进行抗原研究。除了这些研究之外，我们还通过分析酶/底物复合物确定了详细的活性位点结构。