Study On The Mechanisms Of Heat Shock And Hypoionic Shock Potentiate Aminoglycoside Antibiotics To Kill Bacteria

Antibiotic resistance and tolerance in bacterial pathogens pose a huge challenge to current treatment options for bacterial infections,and poses a serious threat to human health.Tolerance is an important factor to the evolution of resistance.Accordingly,viable methods for eliminating bacterial tolerance need to be developed and covering the mechanisms which they induce cell death,can provide new ideas for solving the problem of antibiotic resistance and treating repeated bacterial infections.In this paper,the mechanism of heat shock and hypoionic shock promoting the killing of EScherichia coli was studied.In this paper,we first explored the method of heat shock enhanced antibiotics in killing Escherichia coli.And we further explored the killing efficacy of this method in other bacterium,persisters and acute skin wound model.Finally,we explained the mechanism of killing bacteria from the level of biochemistry and molecular biology.The main results are as follows:1.Heat shock enhances aminoglycosides(tobramycin,gentamicin,streptomycin,kanamycin)against Escherichia coli,but not β-lactam(ampicillin)and quinolone antibiotics(ofloxacin);The enhancement effect is positively correlated with heat shock temperature,time and aminoglycoside antibiotic concentration.2.This method could potentiate killing of other Gram-negative bacteria(P.aeruginosa,S.flexneri,S.typhimurium,A.hydrophila),and even potentiates aminoglycosides killing of multiple drug resistant bacteria(K.pneumoniae,A.baumannii)at a concentration lower than MIC,but has no effect on grampositive bacteria.3.The method can effectively kill spontaneous Escherichia coli persisters and triggered Escherichia coli persisters which induced by starvation,nutrient shift and chemical drug.4.An acute skin wound model of P.aeruginosa was established to verify the killing effect of this method in vivo,and the results showed that the killing effect was significantly better than that of normal temperature treatment(P＜0.01).5.The membrane potential was increased which induced by heat shock,and enhanced antibiotic uptake.6.Heat shock combined with aminoglycoside antibiotics induces irreversible intracellular protein aggregation.Protein aggregation was identified by LCMS-MS,250 proteins were identified,26% of them are encoded by essential genes.The addition of chemical chaperones(betaine,trehalose,trimethylamine N-oxide,proline)significantly reduced synergy bactericidal efficacy in parallel with intracellular protein aggregation.These results suggest that the accumulation of a large number of irreversible proteins is an important factor leading to cell death which inteduced by heat shock combined with aminoglycoside antibiotics.In addition,heat shock protein knockout or overexpression and heat shock acclimation were used to support this conclusion.7.It was found that the addition of antioxidants(L-glutathione,vitamin C,ebuselenium,lipoic acid,N-acetyl-L-cysteine,and quercetin),overexpression of antioxidant enzymes or mutant strains with reduced NADH sources weaken the bactericidal efficacy of heat shock promoting aminoglycoside antibiotics.Antioxidants also reduce the the level of reactive oxygen species(ROS).Suggesting that ROS is an important factor leading to cell death.8.Further explored the relation of protein aggregat and ROS contributed by heat shock combined with aminoglycoside antibiotics.The results showed that addition chemical chaperones or antioxidants could reduce protein aggregate and reactive oxygen,espeally chemical chaperones.The result suggest that protein aggregate is an important source of ROS.In addition,the addition of the two also reduced the uptake of aminoglycoside antibiotics.The second part of this paper is to study the molecular mechanism of hypoionic shock to enhance bactericidal action of aminoglycosides by biochemical and molecular methods.The main findings are as follows:1.Based on the phenomenon that hypoionic shock can promote the opening of mechanosensitive channels.A △LKIOM(lacks the genes of msc L,msc K,yna I,ybi O and msc M)mutant was constructed by CRISPR-Cas9 method.△LKIOM and MJF612(lacks the genes of msc L,msc S,msc K,and ybd G)mutants were used to explored the relationship between the mechanosensitive channels and hypoionic shock enhances the bactericidal action of aminoglycosides.The results showed that the promotion of antibiotic uptake by hypoionic shock was related to mechanosensitive channels.Single channel complementary expression demonstrated that aminoglycoside antibiotics were uptake by mechanosensitive channels of Msc S-like.2.In order to determine the relationship between the Msc S channel and streptomycin,molecular docking was used to predict the binding site of streptomycin and Msc S channel protein,and the point mutation G101 S was constructed and complementary expression.The point mutation can lead to weakened bactericidal efficacy of streptomycin and reduced antibiotic uptake.The site not only targets streptomycin,but also has similar inhibitory effects on other aminoglycoside antibiotics.3.Hypoionic shock induced aminoglycoside potentiation is suppressed by the The addition of salt,and the uptake of antibiotics was inhibited,indicating that low ions are a prerequisite for enhance bactericidal uptake of antibiotics.In summary,this paper,we found a new method to kill tolerance cells which by heat shock combined with aminoglycoside antibiotics.The amplification effect of protein aggregation and ROS are the important cause of cell death.In addition,hypoionic shock promotes the bactericidal action of aminoglycosides is related to the Msc S channel.These results reveal the importance of Msc S-Like channel plays a role in aminoglycosides transport and protein aggregation and oxidative stress in mediated bacterial death.The findings Providing new insights for the study of antibiotic bactericidal mechanisms,and strategies for the treatment of bacterial infections.