PDF(Pubmed)
Abstract：
Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine antibacterials with agents that decrease pathogen virulence via the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen Pseudomonas aeruginosa. Here we describe the characteristics of heterologously expressed YtnP lactonase from Bacillus paralicheniformis ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL-1 concentration reduced the amount of P. aeruginosa-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of P. aeruginosa PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in P. aeruginosa PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL-1 improved the survival of P. aeruginosa PAO1-infected zebrafish (Danio rerio), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of P. aeruginosa PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL-1 YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated P. aeruginosa infections.
摘要：
由于抗生素抗性菌株的出现,细菌感染变得越来越难以治疗。增加治疗功效的有希望的策略是将抗菌药物与通过调节群体感应（QS）降低病原体毒力的药物组合。内酯酶抑制革兰氏阴性细菌中酰化高丝氨酸内酯（AHL）介导的QS，包括主要的医院病原体铜绿假单胞菌。在这里，我们描述了使用培养富集方法从农业土壤中分离的副衣芽孢杆菌ZP1（YtnP-ZP1）异源表达的YtnP内酯酶的特征。纯化的YtnP-ZP1水解不同的AHLs，优先于具有长酰基残基的底物，如在使用生物传感器和HPLC的测定中评估的。该酶在较宽的温度范围内表现出良好的热稳定性和活性。50μgmL-1浓度的YtnP-ZP1使铜绿假单胞菌产生的长链AHLs的量减少了85％，同时水解50%的短链AHLs。铜绿假单胞菌PAO1与YtnP-ZP1的孵育降低了其成群运动和弹性分解活性，而没有杀菌作用。YtnP-ZP1在铜绿假单胞菌PAO1和多耐药临床菌株BR5H中引起生物膜形成的抑制和成熟生物膜的崩解，通过结晶紫染色可见。用浓度高于25μgmL-1的YtnP-ZP1处理可提高铜绿假单胞菌PAO1感染斑马鱼（Daniorerio）的存活率，拯救80%的胚胎，而与妥布霉素或庆大霉素联用的存活率提高到100%。用50μgmL-1YtnP-ZP1和2×MIC妥布霉素治疗感染的斑马鱼尾部伤口上的铜绿假单胞菌PAO1生物膜可在2天内清除感染。广泛的毒性研究证明YtnP-ZP1对人细胞和斑马鱼无毒。总之,新型YtnP-ZP1内酰胺酶具有有效的抗毒力活性，可用于提高临床批准的抗生素在清除全身性和生物膜相关的铜绿假单胞菌感染方面的功效。
