A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics
Michael A.Kohanski,1,2,5,6Daniel J.Dwyer,1,3,6Boris Hayete,1,4Carolyn A.Lawrence,1,2
and James J.Collins1,2,3,4,*
1Center for BioDynamics and Center for Advanced Biotechnology
2Department of Biomedical Engineering
3Program in Molecular Biology,Cell Biology,and Biochemistry
4Bioinformatics Program
Boston University,Boston,MA02215,USA
5Boston University School of Medicine,Boston,MA02118,USA
6These authors contributed equally to this work.
*Correspondence:jcollins@bu.edu
阿尔山自驾游
DOI10.ll.2007.06.049
SUMMARY
Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria.Here we show that the three major classes of bactericidal antibiotics,regardless of drug-target interaction,stimulate the produc-tion of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death.We also show,in contrast,that bacteriostatic drugs do not produce hydroxyl radicals.We demon-strate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle,a transient depletion of NADH,destabili-zation of iron-sulfur clusters,and stimulation of the Fenton reaction.Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage,includ-ing proteins involved in triggering the DNA damage ,RecA.
INTRODUCTION
Current antimicrobial therapies,which cover a wide array of targets(Walsh,2003),fall into two general
categories: bactericidal drugs,which kill bacteria with an efficiency of>99.9%,and bacteriostatic drugs,which merely inhibit growth(Pankey and Sabath,2004).Antibacterial drug-target interactions are well studied and predominantly fall into three classes:inhibition of DNA replication and repair,inhibition of protein synthesis,and inhibition of cell-wall turnover(Walsh,2000).The bactericidal antibiotic
killing mechanisms are currently attributed to the class-specific drug-target interactions.However,our under-standing of many of the bacterial responses that occur as a consequence of the primary drug-target interaction remains incomplete(Davis,1987;Drlica and Zhao,1997;
Lewis,2000;Tomasz,1979).
Bacteriostatic drugs predominantly inhibit ribosome function,targeting both the30S(tetracycline family and aminocyclitol family)and50S(macrolide family and chlor-amphenicol)ribosome subunits(Chopra and Roberts, 2001;Poehlsgaard and Douthwaite,2005;Tenson et al., 2003;Weisblum and Davies,1968).The aminocyclitol group of30S inhibitors includes the bactericidal aminogly-coside family of drugs and the bacteriostatic drug specti-nomycin;the aminoglycoside family,excluding spectino-mycin,is the only class of ribosome inhibitors known to cause protein mistranslation(Davis,1
987;Weisblum and Davies,1968).With regard to other classes of bactericidal antibiotics,quinolones target DNA replication and repair by binding DNA gyrase complexed with DNA,which drives double-strand DNA break formation and cell death(Drlica and Zhao,1997).Cell-wall synthesis inhibitors(such as b-lactams),which interact with penicillin-binding proteins (Tomasz,1979)and glycopeptides that interact with pep-tidoglycan building blocks(Reynolds,1989),interfere with normal cell-wall synthesis and induce lysis and cell death.
With the alarming spread of antibiotic-resistant strains of bacteria(Walsh,2000,2003),a better understanding of the specific sequence of events leading to cell death from the wide range of bactericidal antibiotics is needed for future antibacterial drug advancement.
We have recently shown that bacterial gyrase inhibitors, including synthetic quinolone antibiotics and the native proteic toxin CcdB,induce a breakdown in iron regulatory dynamics,which promotes formation of reactive oxygen species that contribute to cell death(Dwyer et al.,2007).
boston universityHydroxyl radical formation utilizing internal iron and the Fenton reaction appears to be the most significant con-tributor to cell death among the reactive oxygen species formed.The Fenton reaction leads to the formation of Cell130,797–810,September7,2007ª2007Elsevier Inc.797
hydroxyl radicals through the reduction of hydrogen peroxide by ferrous iron(Imlay et al.,1988;Imlay and Linn,1986).We chose to investigate whether hydroxyl radical formation also contributes to antibiotic-induced cell death in bacteria among the other classes of antibi-otics.Here we report that the three major classes of bac-tericidal antibiotics,regardless of drug-target interaction, stimulate hydroxyl radical formation in bacteria.Further-more,we demonstrate that hydroxyl radical generation contributes to the killing efficiency of these lethal drugs. We also show,in contrast,that bacteriostatic drugs do not produce hydroxyl radicals.We demonstrate that all bactericidal drug classes utilize internal iron from iron-sulfur clusters to promote Fenton-mediated hydroxyl radical formation and show that these events appear to be mediated by the tricarboxylic acid(TCA)cycle and a transient depletion of NADH.We propose that there is a common mechanism of cellular death underlying all classes of bactericidal antibiotics whereby harmful hydroxyl radicals are formed as a function of metabo-lism-related NADH depletion,leaching of iron from iron-sulfur clusters,and stimulation of the Fenton reaction. RESULTS
Bactericidal Antibiotics Induce Hydroxyl Radical Formation
Using the dye hydroxyphenylfluorescein(HPF),which is oxidized by hydroxyl radicals with high specificity(Setsu-kinai et al.,2003),wefirst examined a concentration of hydrogen peroxide known to i
nduce hydroxyl radical formation via Fenton chemistry(Imlay et al.,1988).As expected(Imlay et al.,1988),we observed cellular death with1mM hydrogen peroxide(Figure1A)accompanied by an increase in HPFfluorescence(Figure1B).Addition-ally,we confirmed dye specificity for hydroxyl radicals by inhibiting the Fenton reaction and hydroxyl radical forma-tion with the iron chelator2,20-dipyridyl and by directly quenching Fenton-generated hydroxyl radicals with the hydroxyl radical scavenger thiourea(Figures1A and1B). Application of iron chelators is an established means of blocking Fenton reaction-mediated hydroxyl radical for-mation by sequestering unbound iron(Imlay et al.,1988). Thiourea is a potent hydroxyl radical scavenger that is an established means of mitigating the effects of hydroxyl radical damage in both eukaryotes and prokaryotes(No-vogrodsky et al.,1982;Repine et al.,1981;Touati et al., 1995).In this manner,we showed that HPFfluorescence is a reliable measure of hydroxyl radical formation in bacteria.
We next investigated hydroxyl radical formation follow-ing exposure to the three major classes of bactericidal antibiotics in Escherichia li)(Figures1C and 1D).Specifically,we examined killing by the quinolone (250ng/ml norfloxacin),b-lactam(5m g/ml ampicillin), and aminoglycoside(5m g/ml kanamycin)classes.We found that each of the three different classes of bacteri-cidal antibiotics induced hydroxyl radical formation (Figure1D);norfloxacin and ampicillin induced hydroxyl radical for
mation within1hr and kanamycin by2hr after addition of drug(see Figure S1in the Supplemental Data available with this article online).In contrast,thefive bacteriostatic drugs we tested(Figure1E),including four different classes of ribosome inhibitors(chloramphenicol, spectinomycin,tetracycline,and the macrolide erythro-mycin)as well as an inhibitor of RNA polymerase(rifamy-cin SV,referred to as rifamycin;Wehrli and Staehelin [1971]),did not stimulate hydroxyl radical production (Figure1F and Figure S2D).
Interestingly,in ampicillin-treated cultures,we observed a bimodal distribution of hydroxyl radical pro-duction at2and3hr post drug application(Figure1D and Figure S1C)that correlated with the onset of cell lysis (Figure S8A);the decline in the number of cells producing radicals between2and3hr is consistent with the ongoing cell lysis.In contrast,prior to lysis(1hr posttreatment), ampicillin application yielded a uniform increase in hydroxyl radical formation(Figures S1C and S8A).These results suggest a role for hydroxyl radicals in both the lethal and lytic effects of b-lactams.
We sought to demonstrate that Gram-positive,as well as Gram-negative,bacteria produce hydroxyl radicals in response to bactericidal antibiotics.We examined hydroxyl radical production for a bacteriostatic drug (chloramphenicol),a bactericidal drug(norfloxacin),and both lethal(5m g/ml)and sublethal(1m g/ml)concentra-tions of vancomycin(a Gram-positive specific bactericidal drug;Reynold
s[1989])in a wild-type strain of Staphylo-coccus aureus(S.aureus)(Figure S3A).We observed an increase in hydroxyl radical production for the norfloxacin treatment and for the lethal concentration of vancomycin (Figure S3).Conversely,we did not observe hydroxyl rad-ical production for the chloramphenicol treatment or the sublethal concentration of vancomycin,the latter of which had no effect on growth(Figure S3).Cumulatively,our hydroxyl radical results suggest that the genetic and biochemical changes that arise following application of le-thal doses of bactericidal antibiotics create an intracellular environment that promotes the formation of highly delete-rious oxidative radical species.
Hydroxyl Radical Formation for All Bactericidal Classes Involves the Fenton Reaction
and Intracellular Iron
To demonstrate that hydroxyl radical formation is an important component of norfloxacin-,ampicillin-,and kanamycin-mediated killing,we additionally treated drug-exposed wild-type E.coli with the iron chelator 2,20-dipyridyl.For the three classes of bactericidal drug treatments,we observed a significant increase in bacterial survival following addition of2,20-dipyridyl(Figures2A, 2C,and2E),confirming that hydroxyl radicals are involved in bactericidal antibioti
c-induced cell death.2,20-dipyridyl significantly reduced hydroxyl radical formation in norflox-acin-treated cultures(Figure2B),and there appeared to
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be some recovery from the norfloxacin-induced growth arrest and DNA damage between 2and 3hr into the treatment in the presence of 2,20-dipyridyl (Figure 2A).Similarly,killing by ampicillin and kanamycin was reduced to less than 0.5logs following application of the iron chelator (Figures 2C and 2E)and was accompanied by a significant reduction in hydroxyl radical formation (Fig-ures 2D and 2F).As expected,addition of the iron chelator to bacteriostatic drug-treated cultures,which do not pro-duce hydroxyl radicals,had no effect on the growth-arresting properties of these bacteriostatic classes of drugs (Figure S4A).We next sought to directly block the harmful effects of hydroxyl radicals generated via the Fenton reaction by adding thiourea to drug-treated cultures.We found that cultures treated with norfloxacin and thiourea showed a significant delay in cell death at 1hr and a near 1-log increase in survival at 3hr relative to norfloxacin treatment alone (Figure 2A).This increase in survival again correlated with a decrease in the detectable levels of hydroxyl radi-cals (Figure 2B).Thiourea was able to reduce ampicillin-mediated killing (Figure 2C)and hydroxyl radical formation (Figure 2D)to the same extent that 2,20-dipyridyl was.Thiourea was less efficient at mitigati
ng bacterial
cell
Figure 1.Hydroxyl Radical Production li by Hydrogen Peroxide and Antibiotics
(A,C,and E)Log change in colony-forming units per milliliter (cfu/ml).Black squares represent a no-drug control.In this and all other figures,error bars represent ±SD of the mean.
(B,D,and F)Generation of hydroxyl radicals.Representative measurements are shown and were taken 3hr following addition of drug.Gray diamonds represent time-zero baseline measurements.
(A and B)Survival (A)and hydroxyl radical formation (B)following 1mM H 2O 2treatment alone (green),plus 150mM thiourea (red),or plus 500m M 2,20-dipyridyl (blue).
蜕变的近义词(C and D)Survival (C)and hydroxyl radical generation (D)following exposure to bactericidal antibiotics (5m g/ml ampicillin [Amp],blue;5m g/ml kana-mycin [Kan],green;250ng/ml norfloxacin [Nor],red).
(E and F)Survival (E)and hydroxyl radical generation (F)following exposure to bacteriostatic drugs (600m g/ml erythromycin [Eryth],light blue;400m g/ml spectinomycin [Spect],yellow;15m g/ml chloramphenicol [Cam],pink;10m g/ml tetracycline [Tet],blue;500m g/ml rifamycin [Rif],red).
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death following kanamycin treatment (Figure 2E),which was reflected by the capacity of thiourea to reduce,but not eliminate,kanamycin-mediated hydroxyl radical for-mation (Figure 2F);this requires further investigation.Addition of the radical quencher to bacteriostatic drug-treated cultures had minimal effects on the growth-arrest-ing properties of these bacteriostatic classes of drugs (Figure S4B).
Our results with 2,20-dipyridyl and thiourea indicate that hydroxyl radical formation and the Fenton reaction play a critical role in effective killing by quinolones,b -lactams,and aminoglycosides.The ferrous iron required for hy-droxyl radical formation could come from extracellular sources,such as iron import,or from intracellular sources,such as iron storage proteins or iron-sulfur clusters.To determine whether disabling iron import would
Figure 2.Effect of Iron Chelation,Hydroxyl Radical Quenching,and Disabling of Iron-Sulfur Cluster Synthesis on the Killing Efficiency of Bactericidal Antibiotics
(A,C,and E)Log change in cfu/ml following exposure to 250ng/ml Nor (A),5m g/ml Amp (C),or 5m g/ml Kan (E).Changes in cfu/ml following addition of 500m M 2,20-dipyidyl (blue diamonds)or 150mM thiourea (red diamonds)to wild-type (li and an iron-sulfur cluster synthesis mutant,D iscS (yellow diamonds),are shown.In each panel,black squares represent a no-drug control and green diamonds represent li exposed to drug alone.
(B,D,and F)Generation of hydroxyl radicals following exposure to 250ng/ml Nor (B),5m g/ml Amp (D),or 5m g/ml Kan (F).Representative measure-ments are shown and were taken 3hr following addition of drug.The gray line represents time-zero baseline measurements,and the green line rep-resents li exposed to drug alone.Changes in hydroxyl radical formation following addition of 500m M 2,20-dipyidyl (blue line)or 150mM thiourea (red line)to li and an iron-sulfur cluster synthesis mutant,D iscS (yellow line),are shown.
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bactericidal drug lethality,we examined the efficacy of bactericidal antibiotics in a D tonB strain.TonB is a re-quired protein in the energy-dependent step of iron trans-port across the inner membrane li(Moeck and Coulton,1998),and a tonB knockout has previously been shown to have a protective effect following exposure to oxidant stress exogenously induced via application of hydrogen peroxide(Touati et al.,1995).Our data show that removal of tonB provided no protective effect against norfloxacin-,kanamycin-,or ampicillin-mediated killing (Figure S5).This suggests that the import of external iron does not play a significant role in effecting killing by bac-tericidal drugs.
To determine whether oxidative damage of iron-sulfur clusters is a key source of ferrous iron driving hydroxyl radical formation for bactericidal drugs,we examined the killing properties of the bactericidal drugs in a D iscS strain;the iscS knockout has been shown to significantly impair iron-sulfur cluster synthesis capabilities and result in a decrease in iron-sulfur cluster abundance(Djaman et al.,2004;Schwartz et al.,2000).In this strain,we ob-served a significant reduction in cell death following treat-ment with norfloxacin(Figure2A),ampicillin(Figure2C),or kanamycin(Figure2E).We found that the protective effect of D iscS is related to a reduction in hydroxyl radical forma-tion following treatment with norfloxacin(Figure2B),ampi-cillin(Figure2D),or kanamycin(Figure2F).These results imply that intracellular ferrous iron is a key source for Fen-ton-mediated hydroxyl radical formation by bactericidal drugs.
Catabolic NADH Depletion Is the Trigger for Hydroxyl Radical Formation
It is interesting to consider how functionally distinct bacte-ricidal drugs commonly stimulate damage to iron-sulfur clusters.The established mechanism underlying leaching of iron from iron-sulfur clusters predominantly occurs via superoxide(Imlay,2006;Keyer and Imlay,1996;Liochev and Fridovich,1999),and it is well accepted that the ma-jority of superoxide generation li occurs through oxidation of the respiratory electron transport chain driven by oxygen and the conversion of NADH to NAD+(Imlay and Fridovich,1991).We utilized gene expression micro-arrays and statistical analyses(see Experimental Proce-dures)tofind sets of genes commonly upregulated or downregulated by the bactericidal drugs norfloxacin,am-picillin,and kanamycin relative to the bacteriostatic drug spectinomycin(Table1).Interestingly,pathway enrich-ment(q value<0.05)using Gene Ontology(Ashburner et al.,2000;Camon et al.,2004)found NADH-coupled electron transport(NADH dehydrogenase I)to be a key upregulated pathway common to all three bactericidal drug classes(Table1).
We used a modified version(Leonardo et al.,1996)of the NAD+cycling assay developed by Bernofsky and Swan(1973)to monitor NAD+and NADH concentrations in li following treatment with norfloxacin, ampicillin,and kanamycin(Figure3A).For all three bacte-
ricidal drugs,we observed a>5-fold increase in the NAD+/ NADH ratio0.5hr after drug addition(Figure3A).This ratio returned to untreated levels by1hr(Figure3A).The in-crease in the NAD+/NADH ratio was predominantly due to a large relative drop in NADH accompanied by a modest surge in NAD+.This spike was not observed in an un-treated culture,where the NAD+/NADH ratio remained tightly bounded(Figure3A).More importantly,treatment with the bacteriostatic drug spectinomycin had no effect on the NAD+/NADH ratio relative to the untreated culture (Figure3A).A surge in NADH consumption upon exposure to bactericidal antibiotics likely induces a burst in super-oxide generation via the respiratory chain.Accordingly, these events may promote destabilization of iron-sulfur clusters,stimulation of the Fenton reaction,and cell death.
NADH is generated from NAD+during the TCA cycle (Cronan and Laporte,2006).Therefore,loss of TCA-cycle component genes should reduce the available pool of NADH,decrease superoxide generation,and lead to in-creased survival following exposure to bactericidal drugs.
Since NADH is produced at different points along the TCA cycle,the increase in survival should follow a gradient rel-ative to the number of NADH molecules produced.Loss of genes before production of thefirst reduced dinucleotide儿童绘画图
(e.g.,aconitase B[acnB]or isocitrate dehydrogenase
[icdA])should lead to larger increases in survival than loss of genes after the various NADH-producing steps in the TCA ,2-ketoglutarate dehydrogenase [sucB,sucA,lpdA]or malate dehydrogenase[mdh]) (Figure3B).We found that blocking the TCA cycle before the formation of thefirst reduced dinucleotide(D icdA and
D acnB)led to increased survival following norfloxacin
treatment,which had the largest increase in NAD+/ NADH ratio,whereas TCA-cycle knockouts after this point
(D sucB and D mdh)behaved like wild-type(Figure3C).
Blocking the TCA cycle through to the second NADH for-mation step(D acnB,D icdA,and D sucB)led to increased survival following ampicillin treatment,while blocking the last NADH formation step(D mdh)did not affect survival (Figure3D).Finally,each of the TCA-cycle knockout strains(D acnB,D icdA,D sucB,and D mdh)exhibited in-creased survival following exposure to kanamycin (Figure3E).
It is important to note that aconitase A(AcnA)and aco-nitase B are the two main forms of aconitase li: AcnB functions as the main catabolic enzyme in the TCA cycle,while AcnA responds to oxidati
ve stress(Cunning-ham et al.,1997).As expected,for all three classes of bac-tericidal drugs,we observed increased survival only with
D acnB;D acnA behaved like wild-type(Figures3C–3E).In-
蓬莱仙terestingly,one of thefirst mutants selected for resistance to low levels of nalidixic acid,a quinolone,was mapped to
a loss of isocitrate dehydrogenase(icdA)(Helling and Ku-
kora,1971),while later studies found that removing both acnA and acnB similarly conferred resistance(Gruer et al.,1997).The surge in NADH consumption induced by bactericidal drugs,coupled with the phenotypic results from the TCA-cycle knockouts,all point toward efficient Cell130,797–810,September7,2007ª2007Elsevier Inc.801
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