群体感应淬灭酶克隆表达及其对铜绿假单胞菌的影响

Cloning and expression of quorum quenching enzyme affecting Pseudomonas aeruginosa

摘要:

【背景】由于抗生素的大量使用，导致细菌耐药性越来越强，寻找新的抗细菌感染药物成为研究热点。【目的】克隆表达群体感应淬灭酶，探究其对铜绿假单胞菌毒力及致病性的影响。【方法】利用PCR技术从产群体感应淬灭酶的芽孢杆菌QSI-1基因组DNA中克隆出aiiA基因，将其克隆到表达载体pET30a并导入大肠杆菌E. coli BL21(DE3)中进行诱导表达，通过镍柱亲和层析获得纯化的N-酰基高丝氨酸内酯酶。用不同浓度的淬灭酶作用于铜绿假单胞菌，检测其对铜绿假单胞菌毒力因子产生以及生物膜形成能力的影响；以秀丽隐杆线虫为模型，考察其对线虫感染铜绿假单胞菌存活率的影响。【结果】克隆表达出群体感应淬灭酶，该酶能显著抑制铜绿假单胞菌毒力因子产生和生物膜的形成，并能降低铜绿假单胞菌对感染线虫的致死率。【结论】群体感应淬灭酶可作为一种能高效抑制细菌致病性的物质，为临床治疗细菌性感染提供新的策略。

Abstract:

[Background] Due to the extensive use of antibiotics, antimicrobial resistance has become a big problem. Searching for new antibacterial drugs has become a research hotspot. [Objective] Cloning and expression of quorum quenching enzyme and investigate its effect on the pathogenicity of Pseudomonas aeruginosa. [Methods] Quorum quenching enzyme gene aiiA gene from quorum quenching bacterium Bacillus sp. QSI-1 was amplified by PCR methods. The aiiA gene was cloned into the expression vector pET30a and transformed into E. coli BL21(DE3). The expression AiiA protein was purified with a HiTrap Q Sepharose column. P. aeruginosa PAO1 was cultured in medium containing different concentration of quorum-quenching enzyme. The supernatant was used to detect the level of pyocyanin, rhamnolipid and total protease, and biofilm also been detected. The quorum-quenching enzyme was applied to Caenorhabditis elegans infected with P. aeruginosa, the survival rate of the nematode was calculated. [Results] We successfully cloned an N-acylhomoserine lactonase gene from Bacillus sp. QSI-1. The purified quorumquenching enzyme significantly inhibited the production of virulence factors and biofilm formation in P. aeruginosa and reduced the mortality of nematodes infected by P. aeruginosa. [Conclusion] As a substance that can effectively inhibit pathogenic bacteria, quorumquenching enzyme may become a new drug for clinical treatment of bacterial infections.