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

在细菌感染中，通常是在铜绿假单胞菌感染的情况下，受感染的细菌细胞合成并分泌高粘性和无规分子的多糖，并在聚合物周围的环境中繁殖。然而，生物膜具有抑制抗菌药物转运或抑制吞噬细胞功能的特性，从而使依赖生物膜的细菌感染性疾病的治疗变得困难。为了克服这一困难，开发新的治疗手段，我们提出了使用细菌藻酸盐裂解酶来液化和去除生物膜。然而，在这样做之前，细菌酶的抗原性应该被消除。通过生化和计算机图形分析，我们确定了该酶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。在该区域的氨基酸中，27Ser、29Gln、32Asp和40Lys高度暴露于酶的表面，并通过定点突变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。K40A酶形成了牢固的包涵体，没有以活性形式复性。对其他酶进行纯化，并在小鼠系统中进行抗原性研究。除了这些研究外，我们还通过酶/底物复合体的分析确定了详细的活性中心结构。

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.