Chan Pak-Ho, Chan Kwok-Chu, Liu Hong-Bing, Chung Wai-Hong, Leung Yun-Chung, Wong Kwok-Yin
Department of Applied Biology and Chemical Technology and Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
Anal Chem. 2005 Aug 15;77(16):5268-76. doi: 10.1021/ac0502605.
The increasing emergence of new bacterial beta-lactamases that can efficiently hydrolyze beta-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for beta-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled beta-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen beta-lactamases, including the Bacillus cereus beta-lactamase I (PenPC), B. cereus beta-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various beta-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible Omega-loop, which is close to the enzyme's active site, with a cysteine residue on a class A beta-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of beta-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial beta-lactamases and beta-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial beta-lactamases and beta-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of beta-lactamase-resistant antibiotics from beta-lactamase-sensitive ones in the screening of bacterial beta-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent beta-lactam antibiotics for treatment of bacterial infections.
新型细菌β-内酰胺酶不断涌现，它们能高效地将β-内酰胺抗生素水解为临床上无活性的羧酸，这给细菌感染的治疗带来了一个棘手的问题，因此迫切需要开发一种有用的工具，能够以高通量的方式快速筛选细菌中的β-内酰胺酶，以检测各种候选抗生素。本文描述了使用一种荧光素标记的β-内酰胺酶突变体（E166Cf）作为一种便捷的荧光工具来筛选β-内酰胺酶，包括蜡样芽孢杆菌β-内酰胺酶I（PenPC）、蜡样芽孢杆菌β-内酰胺酶II、地衣芽孢杆菌PenP、大肠杆菌TEM-1和阴沟肠杆菌P99，检测它们对各种β-内酰胺抗生素（青霉素G、青霉素V、氨苄西林、头孢呋辛、头孢西丁、莫西沙星、头孢菌素）的活性，使用的是96孔酶标仪。E166Cf突变体是通过将A类β-内酰胺酶（蜡样芽孢杆菌PenPC）中靠近酶活性位点的柔性Ω环上的Glu166替换为半胱氨酸残基构建而成，随后用硫醇反应性荧光素-5-马来酰亚胺标记该突变体。与野生型酶相比，这些修饰显著损害了E166Cf突变体的水解活性。在存在β-内酰胺抗生素的情况下，E166Cf突变体的荧光强度会增加。对于E166Cf突变体耐药的抗生素（头孢呋辛、头孢西丁、莫西沙星），荧光信号缓慢增加直至达到平台期。对于E166Cf突变体可缓慢水解的抗生素（青霉素G、青霉素V、氨苄西林），荧光信号迅速增加至平台期，长时间孵育后会下降。在存在细菌β-内酰胺酶和β-内酰胺酶耐药抗生素的情况下，E166Cf突变体保留其特征性的荧光信号模式。相反，在存在细菌β-内酰胺酶和β-内酰胺酶敏感抗生素的情况下，E166Cf突变体的荧光信号会降低。在针对一组候选抗生素筛选细菌β-内酰胺酶时，E166Cf突变体的荧光信号能够明确区分β-内酰胺酶耐药抗生素和β-内酰胺酶敏感抗生素。这种简单的方法可能为选择有效的β-内酰胺抗生素治疗细菌感染提供一种替代工具。
