Molecular Modification Of AidH And Its Anti-biofilm Effect In Combination With PslG

Pseudomonas aeruginosa is an increasingly prevalent opportunistic human pathogen,causing a wide range of acute and chronic infections in patients with severe burns,metabolic diseases,and cystic fibrosis.However,with the abuse of broad-spectrum antibiotics and antimicrobial drugs,multi-drug resistant P.aeruginosa has emerged as a matter of great concern to mankind.Biofilm formation is one of the leading reasons of drug resistance in P.aeruginosa.Along with biofilm formation,P.aeruginosa persists by increasing tolerance to antimicrobial agents and immune defences.Therefore,targeting biofilms to treat P.aeruginosa infection is a promising strategy for therapeutic agents.Anti-biofilm enzymes have emerged as an attractive target area from which to source new anti-infection technology solutions.Considering the complexity of clinical treatment,a single-method approach likely cannot achieve good results across the entire biofilm development process.We propose a combined enzymes strategy to both inhibition of biofilm formation and eradication of preformed biofilms.For prevention,quorum sensing system is a key component of biofilm communication that can be used to inhibit developmental processes.This system is based on acyl-homoserine lactone molecules 3-oxo-C12-HSL and C4-HSL inactivated by the N-acyl homoserine lactonase AidH.The mutant enzyme AidH_A147Gwas obtained by rational design.Compared with wild type,the hydrolysis efficiency of mutant AidH_A147Gfor 3-Oxo-C12-HSL and C4-HSL was increased by 1.4 times and 2.2 times,respectively.For enzymatic properties,AidH_A147Gshowed the best activity at 40℃and p H=8.After 12 h incubation at 37℃,both AidH and the mutant enzyme AidH_A147Gmaintained approximately 50%of their relative activity.The result shows that AidH_A147Genzyme could exert maximum activity in infection treatment conditions.Compared to the wild type,the mutation has no reduction in stability.In conclusion,AidH_A147Gcan serve as an antivirulence drug against P.aeruginosa infections.To evaluate the quorum-quenching effect of AidH_A147Gon clinical pathogens,three P.aeruginosa pathogenic strains were obtained from infected wounds of burn patients（PA1）,upper extremity skin injury of patients with typeⅡdiabetes mellitus（PA2）and sputum of patients with bronchiectasis and infection（PA3）,respectively.Application of AidH_A147Gmodulated the expression of QS genes in both wild-type and clinical strains with the repression of secretion for virulence factors,such as pyocyanin,proteolytic enzymes,alginate,elastase and biofilms.In this case,the wild-type strains had superior quorum quenching effect than the clinical isolates.Quorum quenching only regulates the biofilm growth and development process.When antimicrobial therapy is delayed,allowing the biofilm to mature,a direct targeted destruction of biofilms is then required.Therefore,we propose combining anti-QS and biofilm dispersal to simultaneously prevent biofilm formation and break down established biofilms.Psl G,a self-produced glycosyl hydrolase,efficiently breaks up biofilms component Psl.Laser confocal results showed that for both wild type and clinical strains,the combined enzymes was more effective in inhibiting biofilms compared to using AidH_A147Gor Psl G alone.Biofilm-targeting enzymes dramatically prevent and break down the biofilm,neither inhibit growth of planktonic bacteria.Biofilm-targeting enzymes are thus used as auxiliary therapy in conjunction with antibiotics.Consistent with the anti-biofilm results,the combination strategy led to the most significant increase in antibiotic susceptibility.In this case,the use of the combined enzymes significantly improved the susceptibility of the aminoglycoside antibiotic amikacin and theβ-lactam antibiotic piperacillin to the wild-type strain PAO1.In addition,the combined-enzyme intervention reverts three clinical multidrug-resistant strains from a resistance state to an intermediate or sensitive state to the clinically used aminoglycosides,β-lactams,and quinolones antibiotics.The results demonstrated that the combined-enzyme approach to inhibit biofilm is a potential clinical treatment for P.aeruginosa infection.