Perturbation Of Bacterial Carbon Metabolism Inhibits Stress Mediated ROS Accumulation And Triggers Antimicrobial Tolerance

Although the mechanisms of commonly used antibiotics have long been extensively studied,the increasingly serious problems of bacterial tolerance and resistance to antimicrobials remain to be addressed.In recent years,the relationship between bacterial carbon metabolism and the lethal action of antibiotics has been widely reported.Antibiotics can stimulate bacterial carbon metabolic changes,and such changes can also affect the antibacterial efficacy of antibiotics.Many related investigations have led to a general consensus:after lethal antimicrobial exposure,bacterial TCA cycle is intensified and NADH level is increased.As large amounts of NADH feed electron transfer chain to produce energy,excessive intracellular reactive oxygen species(ROS)accumulate as by-products,leading to lethal oxidative damage to cells,which constitutes a commonly shared mechanism for different antibiotics to execute bactericidal effect.However,the bacterial carbon metabolic network is complex,and how different metabolic nodes in various carbon metabolic pathways respond to antimicrobial stimulation is unclear and thus needs to be further explored.To this end,we explored the relationship between bacterial carbon metabolism,ROS accumulation,and antibiotic efficacy.First,we found that exogenous addition of glucose to nutrition-rich medium inhibited the lethality of quinolone antibiotics against Escherichia coli and several common pathogenic bacterial species in a concentration-dependent manner;glucose addition also inhibited the accumulation of intracellular ROS in E.coli after ciprofloxacin treatment.Bacterial killing experiments on metabolic-related mutants of E.coli showed that after the ptsG or pgi gene was knocked out to block glucose uptake or phosphorylated fructose metabolism,exogenous glucose no longer mediated E.coli tolerance to ciprofloxacin;flow cytometry analysis also showed that exogenous glucose did not inhibit the accumulation of intracellular ROS with ptsG and pgi mutants after antibiotic stress.However,knocking out the zwf or pykF gene to block the metabolic flow of glycolysis to pentose phosphate pathway or TCA cycle did not affect exogenous glucose-mediated antibiotic tolerance.The above results revealed that the accumulation of phosphorylated fructose mediated by glycolytic process,rather than the changes of pentose phosphate pathway or TCA cycle,was the major cause for antibiotic tolerance mediated by exogenous glucose addition.RT-qPCR experiments showed that glucose addition also increased the expression of ppGpp synthetic genes in E.coli after ciprofloxacin stimulation.When ppGpp synthesis was inactivated by genetic knockout,glucose no longer inhibited intracellular ROS accumulation and bacterial cell death under lethal pressure by ciprofloxacin,indicating that glucose-mediated antibiotic tolerance is mediated by stringent responses and ROS levels.Glucose metabolism affects intracellular ATP or cAMP levels in bacteria,and decreases in ATP or cAMP levels have been reported to mediate antibiotic tolerance.But our results showed that exogenous addition of ATP or cAMP did not affect the protective effect of glucose on E.coli under lethal antibiotic pressure.However,addition of the metabolic intermediate sodium pyruvate did reverse glucose-mediated antibiotic tolerance.Second,we found that inactivation of Eda(a key protein of E.coli ED metabolic pathway)by genetic deletion caused a wide range of biocide tolerance and inhibited intracellular ROS accumulation after ciprofloxacin treatment.The inactivation mutants of Eda protein were obtained by CRISPR gene editing technology and were subjected to bacterial killing experiments.We found that the survival of strains with the active site mutation of KHG aldolase(E45Q、K133Q)after ciprofloxacin treatment was comparable to that of the eda deletion mutant;the survival of strain with the active site mutation of KDPG aldolase(S184D)was significantly lower than that of the eda deletion mutant,indicating that the antibiotic tolerance of the eda mutation was mainly mediated by the inactivation of KHG aldolase.Based on antibiotic tolerance analysis of the double mutant of other metabolism-related genes and eda,we found that after knocking out of aceK gene to inhibit the bypass flow of isocitric acid to glyoxylate shunt,which simultaneously increased the TCA flux,the eda mutant-mediated tolerance disappeared and antibiotic-stimulated intracellular ROS increase occurred,suggesting that disturbance of TCA-glyoxylate metabolism mediated the tolerance of eda mutants to antibiotics.Exogenous addition of sodium pyruvate was found to reduce eda mutant-mediated tolerance to ciprofloxacin and suppression of antimicrobial-stimulated ROS accumulation.Thus,inactivation of KHG aldolase can reduce the lethal effect of antibiotics by inhibiting intracellular ROS accumulation after antimicrobial exposure.The eda mutant showed higher spontaneous and antibiotic induced mutation frequency than the isogenic wild-type strain,and thus was more likely to develop drug resistance.Phylogenetic analysis showed that Eda is conserved among many bacterial species,which makes tolerance and elevated drug resistance frequency,elicited by the inactivation of KHG aldolase,highly possible to occur among a variety of bacterial pathogens.Third,the deficiency of Pta-AckA pathway triggered tolerance of E.coli to diverse types of biocides.Moreover,flow cytometry analysis showed that the single gene mutation of pta and ackA also inhibited the accumulation of intracellular ROS after ciprofloxacin treatment.The Pta-AckA pathway mainly mediates the conversion of acetyl-CoA to acetate;however,exogenous addition of acetate failed to reverse the tolerant phenotype of pta and ackA mutants.RNA-seq data revealed that in the absence of antibiotic pressure,the catalase gene katE was expressed more in the pta and the ackA mutants than in wild-type strain,suggesting a possible stronger anti-oxidative stress capacity with the mutants than with wild-type cells;after 90 min of ciprofloxacin treatment,the expression levels of metabolism-related genes in pta and ackA mutants were generally lower than those in wild-type strain,and the expression levels of genes mediating acetate to acetyl-CoA conversion and TCA cycle related genes were also lower than those in wild-type strain,suggesting that deficiencies in pta or ackA may inhibit the increase of TCA cycle flux after antibiotic treatment,suppress the generation of ROS,and confer antibiotic tolerance,although more specific details still need further investigation.Unlike the case of pta mutation,ackA mutation did not inhibit the expression of ATP synthesis related genes after ciprofloxacin stress.The ackA mutant may have stronger ability to synthesize ATP through substrate phosphorylation than the wild-type strain,which may,to a certain extent,further increase the ability of bacteria to tolerate antimicrobial killing.In the present work,three different metabolic processes were investigated to illustrate how perturbation of these processes affects ROS accumulation and bacterial tolerance to biocides.These studies expand the knowledge basis for further understanding the relationship between antibiotic lethality and carbon metabolism-mediated ROS accumulation.