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

【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.coliBL21(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。

[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 pET30 a 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 quorum quenching 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, quorum quenching enzyme may become a new drug for clinical treatment of bacterial infections.