How antibiotics kill bacteria: from targets to networks
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REVIEWS
How antibiotics kill bacteria:
from targets to networks
Michael A. Kohanski*‡, Daniel J. Dwyer* and James J. Collins*‡§
Abstract | Antibiotic drug–target interactions, and their respective direct effects, are
generally well characterized. By contrast, the bacterial responses to antibiotic drug
treatments that contribute to cell death are not as well understood and have proven to be
complex as they involve many genetic and biochemical pathways. In this Review, we discuss
the multilayered effects of drug–target interactions, including the essential cellular
processes that are inhibited by bactericidal antibiotics and the associated cellular response
mechanisms that contribute to killing. We also discuss new insights into these mechanisms
that have been revealed through the study of biological networks, and describe how these
insights, together with related developments in synthetic biology, could be exploited to
create new antibacterial therapies.
Our understanding of how antibiotics induce bacte- structural integrity following treatment with inhibitors of
Bactericidal
Antimicrobial exposure that
rial cell death is centred on the essential bacterial cell cell wall synthesis6 , and with cellular energetics, ribosome
leads to bacterial cell death. function that is inhibited by the primary drug–target binding and protein mistranslation following treatment
interaction. Antibiotics can be classified based on the with inhibitors of protein synthesis7. In addition, recent
Bacteriostatic cellular component or system they affect, in addition to evidence points towards a common mechanism of cell
Antimicrobial exposure that
whether they induce cell death (bactericidal drugs) or death involving disadvantageous cell responses to drug-
inhibits growth with no loss of
viability. merely inhibit cell growth (bacteriostatic drugs). Most induced stresses that are shared by all classes of bacteri-
current bactericidal antimicrobials — which are the cidal antibiotics, which ultimately contributes to killing
Cell envelope focus of this Review — inhibit DNA, RNA, cell wall or by these drugs8. Specifically, treatment with lethal con-
Layers of the cell surrounding protein synthesis1. centrations of bactericidal antibiotics results in the pro-
the cytoplasm that include lipid
membranes and peptidoglycans.
Since the discovery of penicillin in 1929 (Ref. 2), other, duction of harmful hydroxyl radicals through a common
more effective antimicrobials have been discovered and oxidative damage cell death pathway that involves altera-
developed by elucidation of drug–target interactions and by tions in central metabolism (that is, in the tricarboxylic
*Howard Hughes Medical drug molecule modification. These efforts have greatly acid (TCA) cycle) and iron metabolism8–10.
Institute and the Department
of Biomedical Engineering,
enhanced our clinical armamentarium. Antibiotic- In this Review we describe our current knowledge of
Center for BioDynamics, mediated cell death, however, is a complex process that the drug–target interactions and the associated mecha-
and Center for Advanced begins with the physical interaction between a drug nisms by which the main classes of bactericidal antibiot-
Biotechnology, Boston molecule and its specific target in bacteria, and involves ics kill bacteria. We also describe recent efforts in network
University, 44 Cummington
alterations to the affected bacterium at the biochemical, biology that have yielded new mechanistic insights into
Street, Boston,
Massachusetts 02215, USA. molecular and ultrastructural levels. The increasing prev- how bacteria respond to lethal antibiotic treatments, and
‡
Boston University School of alence of drug-resistant bacteria3, as well as the increased discuss how these insights and related developments in
Medicine, 715 Albany Street, means of gaining resistance, has made it crucial to bet- synthetic biology could be used to develop new, effective
Boston, Massachusetts ter understand the multilayered mechanisms by which means to combat bacterial infections.
02118, USA.
§
Wyss Institute for
currently available antibiotics kill bacteria, as well as to
Biologically Inspired explore and find alternative antibacterial therapies. Inhibition of DNA replication by quinolones
Engineering, Harvard Antibiotic-induced cell death has been associated DNA synthesis, mRNA transcription and cell division
University, Boston, with the formation of double-stranded DNA breaks require the modulation of chromosomal supercoiling
Massachusetts 02115, USA.
following treatment with inhibitors of topoisomer- through topoisomerase-catalysed strand breakage and
Correspondence to J.J.C.
e‑mail: jcollins@bu.edu ase II (also known as DNA gyrase)4, with the arrest of rejoining reactions11–13. These reactions are exploited by
doi:10.1038/nrmicro2333 DNA-dependent RNA synthesis following treatment the synthetic quinolone class of antimicrobials, includ-
Published online 4 May 2010 with rifamycins5, with cell envelope damage and loss of ing the clinically relevant fluoroquinolones, which target
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Table 1 | Antibiotic targets and pathways
Drug type Drug Derivation Species range Primary target Pathways affected
Fluoroquinolones*
DNA synthesis Nalidixic acid, Synthetic Aerobic Gram-positive Topoisomerase II DNA replication, SOS
inhibitor ciprofloxacin, and Gram-negative (DNA gyrase), response, cell division,
levofloxacin and species, some topoisomerase IV ATP generation, TCA
gemifloxacin anaerobic cycle, Fe–S cluster
Gram-negative species synthesis, ROS formation,
(C. perfringes) and and envelope and
M. tuberculosis redox-responsive
two-component systems
Trimethoprim–sulfamethoxazole
DNA synthesis Co-trimoxazole Synthetic Aerobic Gram-positive Tetrahydrofolic acid Nucleotide biosynthesis
inhibitor (a combination of and Gram-negative synthesis inhibitors and DNA replication
trimethoprim and species
sulfamethoxazole
in a 1:5 ratio)
Rifamycins
RNA synthesis Rifamycins, rifampin Natural and semi-synthetic Gram-positive and DNA-dependent RNA transcription, DNA
inhibitor and rifapentine forms of ansamycins (derived Gram-negative species, RNA polymerase replication and SOS
from S. mediterranei) and M. tuberculosis response
β-lactams*
Cell wall Penicillins (penicillin, Natural and semi-synthetic Aerobic and anaerobic Penicillin-binding Cell wall synthesis, cell
synthesis ampicillin, oxacillin), forms of carbonyl lactam Gram-positive and proteins division, autolysin activity
inhibitors cephalosporins ring-containing azetidinone Gram-negative species (regulated by LytSR–VncRS
(cefazolin, cefoxitin molecules (from P. notatum, two-component system),
ceftriaxone, C. acremonium and S. cattleya) SOS response, TCA cycle,
cefepime) and Fe–S cluster synthesis,
carbapenems ROS formation,
(imipenem) and envelope and
redox-responsive
two-component systems
Glycopeptides and glycolipopeptides
Cell wall Vancomycin; Natural and semi-synthetic forms Gram-positive species Peptidoglycan Cell wall synthesis,
synthesis teicoplanin of amino sugar-linked peptide units (terminal transglycosylation,
inhibitor chains (for glycopeptides) or d-Ala-d-Ala transpeptidation and
of fatty acid-bearing, amino dipeptide) autolysin activation (VncRS
sugar-linked peptide chains (for two-component system)
glycolipopetides) derived from
actinobacteria
Lipopeptides
Cell wall Daptomycin and Natural and semi-synthetic Gram-positive Cell membrane Cell wall synthesis and
synthesis polymixin B forms of fatty acid-linked species (daptomycin), envelope two-component
inhibitors peptide chains (from Gram-negative species systems
S. roseosporus and B. polymyxa) (polymixins)
Aminoglycosides
Protein Gentamicin, Natural and semi-synthetic Aerobic Gram-positive 30S ribosome Protein translation
synthesis tobramycin, forms of amino sugars (-mycins and Gram-negative (mistranslation by tRNA
inhibitors streptomycin and from Streptomyces spp. and species, and mismatching), ETC, SOS
kanamycin -micins from Micromonospora M. tuberculosis response, TCA cycle, Fe–S
spp.) cluster synthesis, ROS
formation, and envelope
and redox-responsive
two-component systems
Tetracyclines
Protein Tetracycline and Natural and semi-synthetic Aerobic Gram-positive 30S ribosome Protein translation
synthesis doxycycline forms of four-ringed and Gram-negative (through inhibition of
inhibitors polyketides (from species aminoacyl tRNA binding
S. aureofaciens and S. rimosus) to ribosome)
Macrolides
Protein Erythromycin and Natural and semi-synthetic Aerobic and anaerobic 50S ribosome Protein translation
synthesis azythromycin forms of 14- and 16-membered Gram-positive and (through inhibition
inhibitors lactone rings (from S. erythraea Gram-negative species of elongation and
and S. ambofaciens) translocation steps) and
free tRNA depletion
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Table 1 (cont.) | Antibiotic targets and pathways
Drug type Drug Derivation Species range Primary target Pathways affected
Streptogramins
Protein Pristinamycin, Natural and semi-synthetic Aerobic and anaerobic 50S ribosome Protein translation
synthesis dalfopristin and forms of pristinamycin I (group B, Gram-positive and (through inhibition of
inhibitors quinupristin macrolactone ringed-peptides) and Gram-negative species‡ initiation, elongation
pristinamycin II (group A, and translocation
endolactone oxazole nucleus-bearing steps) and free tRNA
depsipeptides) (from Streptomyces depletion
spp.)
Phenicols
Protein Chloramphenicol Natural and semi-synthetic forms of Some Gram-positive and 50S ribosome Protein translation
synthesis dichloroacetic acid with an aromatic Gram-negative species, (through inhibition of
inhibitor nucleus and aminopropanediol chain including B. fragilis, elongation step)
(from S. venezuelae) N. meningitidis, H. influenzae
and S. pneumoniae
* Drug efficacy can vary across species range based on drug generation. ‡When used as a combination of pristinamycin I and pristinamycin II. B. fragilis, Bacillus fragilis;
B. polymyxa, Bacillus polymyxa; C. acremonium, Cephalosporium acremonium; ETC: electron transport chain; H. influenzae, Haemophilus influenzae; M. tuberculosis,
Mycobacterium tuberculosis; N. meningitidis, Neisseria meningitidis; P. notatum, Penicillum notatum; ROS, reactive oxygen species; S. ambofaciens, Streptomyces ambofaciens;
S. aureofaciens, Streptomyces aureofaciens; S. cattleya, Streptomyces cattleya; S. erythraea, Saccharopolyspora erythraea; S. mediterranei, Streptomyces mediterranei;
S. pneumoniae, Streptococcus pneumoniae; S. rimosus, Streptomyces rimosus; S. roseosporus, Streptomyces roseosporus; S. venezuelae, Streptomyces venezuelae; TCA,
tricarboxylic acid.
DNA–topoisomerase complexes4,14,15. Quinolones are is to generate double-stranded DNA breaks that are
derivatives of nalidixic acid, which was discovered as a trapped by covalently (but reversibly) linked topo-
byproduct of the synthesis of chloroquine (a quinine) isomerases, the function of which is compromised26–28.
and was introduced in the 1960s to treat urinary tract As a result of quinolone–topoisomerase–DNA complex
infections16. Nalidixic acid and other first generation formation, the DNA replication machinery becomes
quinolones (for example, oxolinic acid) are rarely used arrested at blocked replication forks, leading to inhi-
today owing to their toxicity 17. Second (ciprofloxacin), bition of DNA synthesis, which immediately leads
third (levofloxacin) and fourth (gemifloxacin) genera- to bacteriostasis and eventually cell death4 (fIG. 1). It
tion quinolone antibiotics (TABLe 1) can be classified on should be noted that the effects on DNA replication
the basis of their chemical structure and of qualitative that correlate with bacteriostatic concentrations of qui-
differences between the killing mechanisms they nolones are thought to be reversible4,29. Nonetheless,
use16,18. considering that topoisomerase II has been found to
The quinolone class of antimicrobials interferes with be distributed approximately every 100 kb along the
the maintenance of chromosomal topology by target- chromosome30, inhibition of topoisomerase function
ing topoisomerase II and topoisomerase Iv, trapping by quinolone antibiotics and the resulting formation of
these enzymes at the DNA cleavage stage and prevent- stable complexes with DNA have substantial negative
ing strand rejoining 4,19,20 (fIG. 1). Despite the general consequences for the cell in terms of its ability to deal
functional similarities between topoisomerase II and with drug-induced DNA damage31.
topoisomerase Iv, their susceptibility to quinolones
varies across bacterial species20 (TABLe 1). For exam- The role of protein expression in quinolone-mediated
ple, several studies have shown that topoisomerase Iv cell death. The introduction of double-stranded
is the primary target of quinolones in Gram-positive DNA breaks following topoisomerase inhibition by
bacteria (for example, Streptococcus pneumoniae 21), quinolones induces the DNA stress response ( SOS
whereas in Gram-negative bacteria (for example, response), in which RecA is activated by DNA dam-
Escherichia coli 13 and Neisseria gonorrhoea22) their pri- age and promotes self-cleavage of the lexA repres-
mary target is topoisomerase II (and topoisomerase Iv sor protein, inducing the expression of SOS response
is the secondary target). genes such as DNA repair enzymes32. Notably, several
studies have shown that preventing the induction of
Introduction of DNA breaks and replication fork arrest. the SOS response enhances killing by quinolones
The ability of quinolone antibiotics to kill bacteria is (except in the case of nalidixic acid)8,33. Preventing
a function of the stable interaction complex that is the activation of the SOS response has also been
SOS response
formed between drug-bound topoisomerases and shown to reduce the formation of drug-resistant
The DNA stress response
pathway in E. coli, the cleaved DNA4. On the basis of studies using DNA cleav- mutants by blocking the induction of error-prone
prototypical network of genes age mutants of topoisomerase II23 and topoisomerase DNA polymerases34, homologous recombination20 and
of which is regulated by the Iv24 that do not prevent quinolone binding, and stud- horizontal transfer of drug-resistance elements35,36.
transcriptional repressor LexA, ies that have shown that strand breakage can occur in Together with studies revealing that co-treatment with
and is commonly activated by
the co-regulatory protein RecA,
the presence of quinolones25, it is accepted that DNA quinolones and the protein synthesis inhibitor chloram-
which promotes LexA strand breakage occurs after the drug has bound to the phenicol inhibits the ability of quinolones to kill bacte-
self-cleavage when activated. enzyme. Therefore, the net effect of quinolone treatment ria19,37, there seems to be a clear relationship between the
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Quinolones
DNA polymerase Protein independent
complex
Replication Quinolones
fork
SOS DNA repair Cell death
Topoisomerase
Protein dependent
β-lactams
Gram negative Gram positive
Outer
membrane
PBP
β-lactam
Lysis and
cell death
Autolysin
Inner Periplasmic
membrane membrane
Aminoglycosides
Aminoglycoside Outer
30s membrane
mRNA
Cell death
Misfolded Inner
Ribosome
protein membrane
50s
Increased
aminoglycoside
uptake
Figure 1 | Drug-target interactions and associated cell death mechanisms. Quinolone antibiotics interfere
with changes in DNA supercoiling by binding to topoisomerase II or topoisomerase IV. This leads to the
Nature formation
Reviews of
| Microbiology
double-stranded DNA breaks and cell death in either a protein synthesis-dependent or protein synthesis-independent
manner. β-lactams inhibit transpeptidation by binding to penicillin-binding proteins (PBPs) on maturing peptidoglycan
strands. The decrease in peptidoglycan synthesis and increase in autolysins leads to lysis and cell death. Aminoglycosides
bind to the 30S subunit of the ribosome and cause misincorporation of amino acids into elongating peptides. These
mistranslated proteins can misfold, and incorporation of misfolded membrane proteins into the cell envelope leads
to increased drug uptake. This, together with an increase in ribosome binding, has been associated with cell death.
primary effects of quinolone–topoisomerase–DNA com- inhibition of DNA replication by quinolones, has a cata-
plex formation and the response of the bacteria (through strophic effect on prokaryotic nucleic acid metabolism
the stress-induced expression of proteins) to these effects and is a potent means of inducing bacterial cell death5.
in the bactericidal activity of quinolone antibiotics. For Rifamycins inhibit DNA-dependent transcription by sta-
example, the contribution of reactive oxygen species bly binding with high affinity to the β-subunit (encoded
(ROS) to quinolone-mediated cell death has recently been by rpoB) of a DNA-bound and actively transcribing
shown to occur in a protein synthesis-dependent man- RNA polymerase41–43 (TABLe 1). The β-subunit is located
ner38. Also, the chromosome-encoded toxin MazF has in the channel that is formed by the RNA polymerase–
been shown to be required under certain conditions for DNA complex, from which the newly synthesized RNA
efficient killing by quinolones owing to its ability to alter strand emerges44. Rifamycins uniquely require RNA
protein carbonylation39, a form of oxidative stress40. synthesis to not have progressed beyond the addition
of two ribonucleotides; this is attributed to the ability of
Inhibition of RNA synthesis by rifamycins the drug molecule to sterically inhibit nascent RNA
The inhibition of RNA synthesis by the rifamycin class strand initialization45. It is worth noting that rifamycins
of semi-synthetic bactericidal antibiotics, similarly to the are not thought to act by blocking the elongation step of
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RNA synthesis, although a recently discovered class ring) is an analogue of the terminal d-alanyl-d-alanine
of RNA polymerase inhibitors (based on the compound dipeptide of peptidoglycan and acts as a substrate for the
CBR703) could inhibit elongation by allosterically PBP during the acylation phase of cross link formation.
modifying the enzyme46. Penicilloylation of the PBP active site blocks the hydro-
Rifamycins were first isolated47 from the Gram-positive lysis of the bond created with the now ring-opened drug,
bacterium Amycolatopsis mediterranei (originally known thereby disabling the enzyme59,60.
as Streptomyces mediterranei) in the 1950s. Mutagenesis of By contrast, most actinobacterium-derived glyco-
this organism has led to the isolation and characterization peptide antibiotics (for example, vancomycin) inhibit
of more potent rifamycin forms48, including the clinically peptidoglycan synthesis by binding peptidoglycan units
relevant rifamycin Sv and rifampicin. Rifamycins are (at the d-alanyl-d-alanine dipeptide) and by blocking
considered bactericidal against Gram-positive bacteria transglycosylase and PBP activity 61. In this way, glyco-
and bacteriostatic against Gram-negative bacteria, a dif- peptides (whether free in the periplasm like vancomy-
ference that has been attributed to drug uptake and not to cin or membrane-anchored like teicoplanin62) generally
affinity of the drug with the RNA polymerase β-subunit 49. act as steric inhibitors of peptidoglycan maturation and
Notably, rifamycins are among the first-line therapies reduce the mechanical strength of the cell, although some
used against mycobacteria because they efficiently induce chemically modified glycopeptides have been shown to
mycobacterial cell death50, although rifamycins are often directly interact with the transglycosylase63. It is worth
used in combinatorial therapies owing to the rapid nature noting that β-lactams can be used to treat Gram-positive
of resistance development49,51. and Gram-negative bacteria, whereas glycopeptides are
Interestingly, an interaction between DNA and effective against only Gram-positive bacteria owing to
the hydroquinone moiety of RNA polymerase-bound low permeability (TABLe 1). In addition, antibiotics that
rifamycin has been observed52, and this interaction has inhibit the synthesis (for example, fosfomycin) and trans-
been attributed to the location of the rifamycin mol- port (for example, bacitracin) of individual peptidogly-
ecule in relation to DNA in the DNA–RNA polymerase can units are also currently in use, as are lipopeptides (for
complex 42. This proximity, coupled with the reported example, daptomycin), which affect structural integrity by
ability of rifamycin to cycle between a radical and non- inserting themselves into the cell membrane and inducing
radical form (rifamycin Sv and rifamycin S52,53), may membrane depolarization.
damage DNA through a direct drug–DNA interaction. Research into the mechanism of killing by peptido-
This hypothesis could account for the observation that glycan synthesis inhibitors has centred on the lysis event.
rifamycin Sv can induce the SOS DNA damage response Initially, it was thought that inhibition of cell wall syn-
in E. coli and that treatment of recA-mutant E. coli results in thesis by β-lactams caused cell death when internal pres-
cell death whereas treatment of wild-type E. coli leads sure built up owing to cell growth outpacing cell wall
to bacteriostasis8. expansion, resulting in lysis6. This unbalanced growth
hypothesis was based in part on the notion that active pro-
Inhibition of cell wall synthesis tein synthesis is required for lysis to occur following the
Lytic cell death. The bacterial cell is encased by layers of addition of β-lactams.
peptidoglycan (also known as murein), a covalently cross- The lysis-dependent cell death mechanism, however,
linked polymer matrix that is composed of peptide-linked has proven to be much more complex, involving many
β-(1-4)-N-acetyl hexosamine54. The mechanical strength active cellular processes. Seminal work showed that
afforded by this layer of the cell wall is crucial to a bacte- S. pneumoniae deficient in amidase activity (possessed by
rium’s ability to survive environmental conditions that can peptidoglycan hydrolase or autolysins) did not grow or die
alter prevailing osmotic pressures; of note, the degree of following treatment with a lysis-inducing concentration
Lysis peptidoglycan cross-linking correlates with the structural of a β-lactam, an effect known as antibiotic tolerance64.
Rupture of the cell envelope integrity of the cell55. Maintenance of the peptidoglycan Autolysins are membrane-associated enzymes that break
leading to the expulsion of
layer is accomplished by the activity of transglycosylases down bonds between and within peptidoglycan strands,
intracellular contents into the
surrounding environment with and penicillin-binding proteins (PBPs; also known as making them important during normal cell wall turnover
eventual disintegration of the transpeptidases), which add disaccharide pentapeptides and maintenance of cell shape55. Autolysins have also been
cell envelope. to extend the glycan strands of existing peptidoglycan shown to play a part in lytic cell death in bacterial species
molecules and cross-link adjacent peptide strands of that contain numerous peptidoglycan hydrolases, such as
Peptidoglycan hydrolase
An enzyme that introduces
immature peptidoglycan units, respectively 56. E. coli 65. In E. coli, a set of putative peptidoglycan hydro-
cuts between carbon–nitrogen β-lactams and glycopeptides are among the classes of lases (lytM domain factors) were shown to be important
non-peptide bonds while antibiotics that interfere with specific steps in homeostatic for rapid ampicillin-mediated lysis66. The discovery that
pruning the peptidoglycan cell wall biosynthesis. Successful treatment with a cell wall autolysins contribute to cell death expanded our under-
layer. It is important for
synthesis inhibitor can result in changes to cell shape and standing of lysis and showed that active degradation of
homeostatic peptidoglycan
turnover. size, induction of cell stress responses and ultimately cell the peptidoglycan layer by peptidoglycan hydrolases, in
lysis6 (fIG. 1). For example, β-lactams (including penicil- conjunction with inhibition of peptidoglycan synthesis by
Autolysin lins, carbapenems and cephalosporins) block the cross- a β-lactam antibiotic, triggers lysis64 (fIG. 1).
An enzyme that hydrolyses the linking of peptidoglycan units by inhibiting the peptide
β-linkage between the
monosaccharide monomers in
bond formation reaction that is catalysed by PBPs55,57,58. Non-lytic cell death. S. pneumoniae lacking peptidoglycan
peptidoglycan units and can This inhibition is achieved by penicilloylation of the PBP hydrolase activity can still be killed by β-lactams, but at
induce lysis when in excess. active site — the β-lactam (containing a cyclic amide a slower rate than autolysin-active cells, indicating that
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there is a lysis-independent mode of killing induced by ofloxacin or pefloxacin83. This suggests that peptido-
β-lactams64,67. evidence suggests that some of these non- glycan turnover and the SOS response could have a role
lytic pathways are regulated by bacterial two-component in antibiotic-mediated lytic killing responses.
systems68. For example, in S. pneumoniae, the vncSR two-
component system controls the expression of the autolysin Inhibition of protein synthesis
lytA and regulates tolerance to vancomycin and penicil- The process of mRNA translation occurs over three
lin through lysis-dependent69 and lysis-independent70 cell sequential phases (initiation, elongation and termina-
death pathways. tion) that involve the ribosome and a range of cytoplas-
In Staphylococcus aureus, the lytSR two-component mic accessory factors84. The ribosome is composed of
system can similarly affect cell lysis by regulating autolysin two ribonucleoprotein subunits, the 50S and 30S, which
activity 71. lytR activates the expression of lrgAB72, which assemble (during the initiation phase) following the
was found to inhibit autolysin activity and thereby lead formation of a complex between an mRNA transcript,
to antibiotic tolerance73. lrgA is similar to bacteriophage N-formylmethionine-charged aminoacyl tRNA, several
holin proteins73, which regulate the access of autolysins initiation factors and a free 30S subunit 85. Drugs that
to the peptidoglycan layer. Based on this information, inhibit protein synthesis are among the broadest classes
an additional holin-like system, cidAB, was uncovered of antibiotics and can be divided into two subclasses: the
in S. aureus and found to activate autolysins, render- 50S inhibitors and 30S inhibitors (TABLe 1).
ing S. aureus more susceptible to β-lactam-mediated 50S ribosome inhibitors include macrolides (for
killing 74,75. Complementation of cidA into a cidA-null example, erythromycin), lincosamides (for example,
strain reversed the loss of autolysin activity but did not clindamycin), streptogramins (for example, dalfopristin–
completely restore sensitivity to β-lactams74. quinupristin), amphenicols (for example, chlorampheni-
col) and oxazolidinones (for example, linezolid)86,87.
Role of the SOS response in cell death by β-lactams. 50S ribosome inhibitors work by physically blocking
Treatment with β-lactams leads to changes in cell mor- either initiation of protein translation (as is the case for
phology that are associated with the primary drug–PBP oxazolidinones88) or translocation of peptidyl tRNAs,
interaction. Generally speaking, PBP1 inhibitors cause which serves to inhibit the peptidyltransferase reac-
cell elongation and are potent triggers of lysis, PBP2 tion that elongates the nascent peptide chain. A model
inhibitors alter cell shape but do not cause lysis and for the mechanism by which these drugs act has been
PBP3 inhibitors influence cell division and can induce formulated by studies of macrolides, lincosamides and
filamentation76. Interestingly, β-lactam subtypes have dis- streptogramins. The model involves blocking the access
tinct affinities for certain PBPs, which correlate with the of peptidyl tRNAs to the ribosome (to varying degrees),
ability of these drugs to stimulate autolysin activity and subsequent blockage of the peptidyltransferase elonga-
induce lysis76,77. Accordingly, PBP1-binding β-lactams are tion reaction by steric inhibition and eventually trigger-
also the most effective inducers of peptidoglycan hydro- ing dissociation of the peptidyl tRNA89,90. This model
lase activity, and PBP2 inhibitors are the least proficient also accounts for the phenomenon that these classes of
autolysin activators77. drugs lose their antibacterial activity when elongation has
Filamentation can occur following the activation of the progressed beyond a crucial length91.
DNA damage-responsive SOS network of genes78 owing 30S ribosome inhibitors include tetracyclines and
to expression of SulA, a key component of the SOS net- aminocyclitols. Tetracyclines work by blocking the access
work that inhibits septation and leads to cell elongation of aminoacyl tRNAs to the ribosome92. The aminocycli-
by binding to and inhibiting polymerization of septation- tol class comprises spectinomycin and aminoglycosides
triggering FtsZ monomers79,80. Interestingly, β-lactams (for example, streptomycin, kanamycin and gentamicin),
that inhibit PBP3 and induce filamentation have been which bind the 16S rRNA component of the 30S ribos-
shown to stimulate the DpiAB two-component system, ome subunit. Spectinomycin interferes with the stability
which can activate the SOS response81. β-lactam lethality of peptidyl tRNA binding to the ribosome by inhibiting
can be enhanced by disrupting DpiAB signalling or by elongation factor-catalysed translocation, but does not
knocking out sulA. This indicates that SulA may protect cause protein mistranslation93–95. By contrast, the inter-
against β-lactam killing by shielding FtsZ and limiting a action between aminoglycosides and the 16S rRNA can
division ring interaction among PBPs and peptidoglycan induce an alteration in the conformation of the com-
hydrolases. In support of this idea, SulA expression lim- plex formed between an mRNA codon and its cognate
its the lysis observed in a strain of E. coli that expresses charged aminoacyl tRNA at the ribosome. This pro-
FtsZ84 (a mutant of FtsZ that is active only under certain motes tRNA mismatching, which can result in protein
temperatures and media conditions) and lacks PBP4 and mistranslation96–99.
PBP7 (Ref. 82). Among ribosome inhibitors, naturally derived
DNA-damaging antimicrobials that do not directly aminoglycosides are the only class that is broadly bac-
disrupt peptidoglycan turnover, such as quinolones, also tericidal. Macrolides, streptogramins, spectinomycin,
Two-component system cause filamentation by activating the SOS response4. tetracyclines, chloramphenicol and macrolides are typi-
A two-protein signal relay Interestingly, a mutant strain of E. coli that is deficient cally bacteriostatic; however, they can be bactericidal in a
system composed of a sensor
histidine kinase and a cognate
in diaminopimelic acid synthesis (E. coli W7), a key species- or treatment-specific manner. For example, chlo-
receiver protein, which is building block of peptidoglycan, undergoes lysis follow- ramphenicol has been shown to kill S. pneumoniae and
typically a transcription factor. ing treatment with the fluoroquinolone antimicrobials Neisseria meningitidis effectively 100, and chloramphenicol
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Box 1 | Drug synergy In E. coli aminoglycoside-mediated killing has
been linked with alterations to the cell membrane
Combinatorial antibiotic treatments can have diverse effects on bacterial survival. ultrastructure that ultimately increase drug uptake109,110.
Antibiotics can be more effective as a combination treatment displaying either an Aminoglycosides can affect membrane composition
additive effect (an effect equal to the sum of the treatments) or a synergistic effect
through the incorporation of mistranslated membrane
(an effect greater than the sum of the treatments). The combination can also be
proteins into the cytoplasmic membrane, thereby increas-
antagonistic — that is, the effect of the combination treatment is less than the
effect of the respective single-drug treatments136. Technological advances have ing cell permeability, which allows increased access of the
allowed high-throughput quantification of drug–drug interactions at the level of drug 103 (fIG. 1). Sufficient aminoglycoside uptake resulting
cell survival and target binding, thereby opening the door for the systematic study in increased ribosome inhibition and cell death could also
of synergistic and antagonistic drug combinations137. occur as a function of the changes in membrane integrity
The exploration of the survival fitness landscape between drug combinations has owing to the incorporation of mistranslated membrane
allowed the study of the mechanisms by which antibiotics work against bacteria138 and proteins103. An alteration in membrane permeability
has also allowed a study of the evolution of drug resistance137. Further study of the synergy owing to aminoglycoside-induced membrane damage is
or antagonism between antibiotics will provide additional insight into the underlying cell thought to be one of the mechanisms by which amino-
death mechanisms for the individual classes of antibiotics. For example, the suppressive
glycosides cooperate with β-lactams (see BOX 1 for more
interaction between protein synthesis inhibitors and DNA synthesis inhibitors has been
on drug synergy and antagonism).
shown to be due to non-optimal ribosomal RNA regulation by DNA-inhibiting drugs139.
The synergy between aminoglycosides and β-lactams has been attributed to Another consequence of mistranslated protein incorpo-
β-lactam-mediated membrane damage leading to increased uptake of aminoglycosides140. ration into the bacterial membrane is the activation of enve-
It will be interesting to see whether the synergy between these two drugs is also related lope (Cpx) and redox-responsive (Arc) two-component
to the induction of the envelope stress response that has been observed following systems. These intracellular signal relay systems regulate
treatment with aminoglycosides10. the expression of genes that are important for the main-
tenance of membrane integrity and composition111, and
and the macrolide azythromycin have exhibited bacteri- membrane-coupled energy generation112,113, respectively.
cidal activity against Haemophilus influenzae100,101. This Disruption of Cpx or Arc two-component system sig-
species-specific variability in ribosome inhibitor-medi- nalling (through a series of single-gene knockouts) has
ated cell death probably has to do with sequence differ- recently been shown to reduce the killing efficacy of
ences among bacterial species in the variable regions of aminoglycosides, a result associated with findings link-
the highly conserved ribosomal proteins and RNAs102. In ing bactericidal antibiotic-induced cell death with drug
addition, high concentrations of macrolides and combina- stress-induced changes in metabolism. Interestingly, dis-
tions of streptogramin group A and group B can behave in ruption of Cpx or Arc two-component system signalling
a bactericidal manner. For the rest of this section, however, was also shown to reduce the lethality of β-lactam and
we focus on aminoglycosides, which have the best-studied quinolone antibiotics10. Together, these findings point
mechanism of killing by ribosome inhibition. towards a broad role for the envelope stress-responsive
and redox-responsive two-component systems in killing
Aminoglycoside uptake and cell death. Binding of by bactericidal drugs (fIG. 2).
aminoglycosides to the ribosome does not bring trans-
lation to an immediate standstill. Instead, as noted Antibiotic network biology
above, this class of drugs promotes protein mistransla- As noted above, antibiotic-mediated cell death is a complex
tion through the incorporation of inappropriate amino process that only begins with the drug–target interaction
acids into elongating peptide strands96; this phenotype and the primary effects of these respective interactions.
is specific for aminoglycosides and contributes to cell The development of new antibiotics and the improvement
killing (fIG. 1). of current antibacterial drug therapies would benefit from
Respiration also has a crucial role in aminoglycoside a better understanding of the specific sequences of events
uptake and lethality 103. Following the initial step of drug beginning with the binding of a bactericidal drug to its
molecule adsorption (in Gram-negative species such target and ending in bacterial cell death.
as E. coli) through electrostatic interaction, changes in Bioinformatics approaches that use high-throughput
membrane potential allow aminoglycosides to access the genetic screening or gene expression profiling have
cell. Respiration-dependent uptake relies on the activity proven to be valuable tools to explore the response layers
of membrane-associated cytochromes and maintenance of of bacteria to different antibiotic treatments114. For exam-
the electrochemical potential through the quinone pool104,105. ple, recent screens for antibiotic susceptibility in a single-
Accordingly, under anaerobic conditions aminoglyco- gene deletion library of non-essential genes in E. coli 115
side uptake is severely limited in both Gram-positive and and a transposon mutagenesis library in P. aeruginosa116
Gram-negative bacteria106,107, although there is evidence have provided important insights into the numbers and
that aminoglycoside uptake can occur under certain types of genes that affect treatment efficiency (bactericidal
anaerobic conditions by a mechanism that is sensitive versus bacteriostatic effects), including those related to
to nitrate levels. In E. coli and Pseudomonas aeruginosa, drug molecule efflux, uptake or degradation. In addition,
aminoglycoside uptake can take place when nitrate is monitoring global changes in gene expression patterns,
Quinone pool used as an electron acceptor in place of oxygen, and or signatures, resulting from antibiotic treatment over
Membrane-associated cyclic
aromatic-based compounds
anaerobic bacteria that have quinones and cytochromes a range of conditions, has advanced our understanding
that shuttle electrons along the can take up aminoglycosides if sufficient anaerobic of the off-target effects elicited by primary drug–target
electron transport chain. electron transport occurs108. interactions114.
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Aminoglycoside
β-lactam
Quinolone
PBP
Topoisomerase
NADH
mdh
NAD+
Cpx and Arc two
Metabolic acnB component systems
feedback TCA cycle [Fe–S]
NADH NADP+ S S Disulphide
bond
HS HS formation
NAD+ NADPH
I II IV V
Hyperactivation of
electron transport chain Uq
O2– Superoxide
formation
NADH NAD+
ADP + Pi ATP
Damage
Fenton
reaction Fe–S cluster
•OH
Fe2+ damage
Hydroxyl radical
production Fe3+
Damage to DNA,
lipids and proteins
Cell death
Figure 2 | common mechanism of cell death induced by bactericidal antibiotics. The primary drug–target interactions
(aminoglycoside with the ribosome, quinolone with topoisomerase, and β-lactam with penicillin-binding proteins (PBPs))
stimulate the oxidation of NADH through the electron transport chain, which is dependent onNature
the tricarboxylic acid (TCA)
Reviews | Microbiology
cycle. Hyperactivation of the electron transport chain stimulates superoxide (O2–) formation. Superoxide damages Fe–S
clusters, making ferrous iron available for oxidation by the Fenton reaction. The Fenton reaction leads to the formation of
hydroxyl radicals (•OH), which damage DNA, lipids and proteins. This contributes to antibiotic-induced cell death.
Quinolones, β-lactams and aminoglycosides also trigger hydroxyl radical formation and cell death through the envelope
(Cpx) and redox-responsive (Arc) two-component systems. It is also possible that redox-sensitive proteins, such as those
containing disulphides, contribute in undetermined way to the common mechanism (dashed lines). Figure modified, with
permission, from Ref. 8 © (2007) Elsevier Science. acnb, aconitase b; mdh, malate dehydrogenase; uq, ubiquinone.
A need also exists for the application of network biol- connections and biochemical pathway classifications,
ogy methods to discern and resolve the potential interplay to functionally enrich datasets and predict relationships
between genes and proteins coordinating bacterial stress that exist among genes under tested conditions. As such,
response pathways. Typically, such methods incorporate biological network studies of drug-treated bacteria can
gene expression profiling data and the results of high- be used to advance our understanding of how groups of
throughput genetic screens, along with the contents of genes interact functionally, rather than in isolation, when
databases detailing experimentally identified regulatory cells react to antibiotic stress117.
430 | juNe 2010 | vOluMe 8 www.nature.com/reviews/micro
© 2010 Macmillan Publishers Limited. All rights reservedREVIEWS
Protein Outer membrane
DegP degradation
Inner
O2 NO3–
membrane
Uq I II Uq IV V
P PP P
CpxA
CpxA
ArcA
ArcB
ADP + Pi
SecA SecA
ATP
CpxR ArcA
Aminoglycoside
TCA cycle
ArcA P
CpxR P
Superoxide
formation
Mistranslation Envelope stress response and cell death
Figure 3 | Aminoglycosides trigger hydroxyl radical-mediated cell death. The interaction between aminoglycosides and
the ribosome causes mistranslation and misfolding of membrane proteins. Incorporation of mistranslated, misfolded proteins
Nature Reviews | Microbiology
into the cell membrane stimulates the envelope (Cpx) and redox-responsive (Arc) two-component systems. Activation of these
systems perturbs cell metabolism and the membrane potential, resulting in the formation of lethal hydroxyl radicals. Figure
modified, with permission, from Ref. 10 © (2008) Elsevier Science. TCA, tricarboxylic acid; uq, ubiquinone.
To help address this problem, researchers have reaction123. under these conditions, the Fenton reac-
developed methods to construct quantitative models of tion was found to be fuelled by superoxide-mediated
regulatory networks118–122 and have recently used these destabilization of Fe–S cluster catalytic sites, repair
reconstructed network models to identify the sets of of these damaged Fe–S clusters and related changes
genes, associated functional groups and biochemical path- in iron-related gene expression9.
ways that act in concert to mediate bacterial responses Building on this work, it was later shown that all
to antibiotics8–10,119. Below we highlight some mechanistic major classes of bactericidal antibiotics (including
insights that have been obtained from antibiotic network β-lactams, aminoglycosides and quinolones) pro-
biology, and discuss some opportunities and challenges mote the generation of lethal hydroxyl radicals in
for this emerging area of research. both Gram-negative and Gram-positive bacteria,
despite the stark differences in their primary drug–
A common mechanism for antibiotic-mediated cell target interactions8. Stress response network analysis
death. As an example of the utility of studying bacterial methods used in this study suggested that antibiotic-
stress responses at the systems level, biological network induced hydroxyl radical formation is the end prod-
analysis methods were recently employed to identify uct of a common mechanism, in which alterations in
new mechanisms that contribute to bacterial cell death central metabolism related to NADH consumption
following topoisomerase II inhibition by the fluo- (increased TCA cycle and respiratory activity) are
roquinolone antibiotic norfloxacin9. As noted above, crucial to superoxide-mediated iron–sulphur cluster
quinolones are known to induce cell death through destabilization and stimulation of the Fenton reaction.
the introduction of double-stranded DNA breaks fol- These predictions were validated by the results of addi-
lowing arrest of topoisomerase function4. To identify tional phenotypic experiments, biochemical assays and
additional contributions to cell death resulting from gene expression measurements, confirming that lethal
topoisomerase II poisoning, reconstruction of stress levels of bactericidal antibacterials trigger a com-
response networks was carried out following treatment mon oxidative damage cellular death pathway, which
of E. coli with lethal concentrations of norfloxacin. This contributes to killing by these drugs (fIG. 2).
work identified an oxidative damage-mediated cell Most recently, the study of antibiotic-induced stress
death pathway, which involves ROS generation and response networks has been aimed at determining
a breakdown in iron regulatory dynamics following exactly how the primary effect of a given bactericidal
norfloxacin-induced DNA damage. More specifically, triggers aspects of cell death that are common to all
Fenton reaction norfloxacin treatment was found to promote super- bactericidal drugs. For example, a comparative analy-
Reaction of ferrous iron (fe2+)
with hydrogen peroxide to
oxide generation soon after topoisomerase II poison- sis of stress response networks, reconstructed using
produce ferric iron (fe3+) and ing and to ultimately result in the generation of highly gene expression data from E. coli treated with amino-
a hydroxyl radical. destructive hydroxyl radicals through the fenton cyclitols (spectinomycin, gentamicin and kanamycin),
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Box 2 | Synthetic biology for antibacterial applications
The study of complex antibiotic-related cell death systems
can be aided by synthetic biology. Delivery of engineered
gene circuits that alter response network behaviour can serve
Synthetic
as a tool to experimentally examine antibiotic-mediated
perturbation
cell death pathways, as well as a means to enhance killing by
an antibiotic (see the figure).
Bacteriophages, which are bacterium-specific viruses, Antibiotic
show promise as an effective means to deliver network perturbed network
perturbations to bacteria to improve antibiotic
Novel
lethality141,142. Bacteriophages have been used to enhance drug Bacteriophage
killing of Escherichia coli by bactericidal antibiotics through targets
Network
the delivery of proteins that modify the oxidative stress reconstruction
response or inhibit DNA damage repair systems142. algorithms
Bacteriophages are species specific, so it may be possible
to use engineered bacteriophages to deliver antibiotic-
enhancing synthetic gene networks, therapeutic proteins
Antibiotics
or antimicrobial peptides that are highly specific for an Increased antibiotic
infecting organism. This would allow efficient treatment of susceptibility
a bacterial infection, while sparing the typical commensal
body flora (see the figure).
Nature Reviews | Microbiology
was used to identify the incorporation of mistrans- in the same way to antibiotic treatment. Network biol-
lated proteins into the cell membrane as the trigger ogy approaches, which provide the field of antibiotic
for aminoglycoside-induced oxidative stress10 (fIG. 3). research with an opportunity to view response mecha-
Interestingly, mistranslated membrane proteins were nisms of different bacterial species to various classes of
shown to stimulate radical formation by activating antibiotics, could be extended to the context of partic-
the Cpx and Arc two-component systems, ultimately ular infectious species, persistent infections or disease
altering TCA cycle metabolism; the TCA cycle had settings. As an example, it is generally accepted that
previously been implicated in bacterial susceptibility Gram-negative bacteria are not susceptible to the glyco-
to aminoglycosides8,124. peptide vancomycin or the depolarizing lipopeptide
The discovery of the common oxidative damage daptomycin; however, a single gene, yfgL, was recently
cellular death pathway has important implications for found that can make E. coli susceptible to glycolipid
the development of more effective antibacterial thera- derivatives of vancomycin132. Gene expression profil-
pies. Specifically, it indicates that all major classes of ing of daptomycin-treated S. aureus has revealed that
bactericidal drugs can be potentiated by inhibition daptomycin perturbs peptidoglycan synthesis through
of the DNA stress response network (that is, the SOS a mechanism involving the activation of cell wall stress
response), which plays a key part in the repair of systems and membrane depolarization133. Given these
hydroxyl radical-induced DNA damage. This may be findings, we might be able to combine our knowledge of
accomplished through the development of small mol- β-lactam- and aminoglycoside-induced gene signatures
ecules (for example, RecA inhibitors125) or synthetic with the results of high-throughput screens at various
biology approaches (BOX 2). drug doses to reconstruct drug-specific cell death net-
ROS, such as superoxide and hydroxyl radicals, are works that use Ygfl as a network anchor. Predicted func-
highly toxic and have deleterious effects on bacterial tional and regulatory relationships between enriched
physiology 123,126,127, even under steady-state conditions. genes could then be used to determine the secondary
There is still much to be learned about how oxidative effects of lipopeptide antibiotics and gain insight into
stress-related changes in bacterial physiology affect the different properties of this drug in Gram-negative
antibiotic-mediated cell death and the emergence of and Gram-positive bacteria.
resistance128,129. For example, it was recently discov- Moreover, the development of comparative net-
ered that endogenous nitric oxide produced by bac- work biology techniques will be essential to further
teria with nitric oxide synthases can protect against our understanding of how species-specific differ-
ROS-mediated cell death130. In addition, considering ences manifest themselves in divergent drug-specific
bacteria have developed mechanisms to avoid ROS cell death networks and variations in physiological
produced by phagocytes of the immune system 131, it responses. These methods could be particularly use-
will be interesting to explore, from a systems-level per- ful when examining pathogenic bacteria with sparse
spective, the relationship between immune-mediated systems-level data (such as Shigella or Salmonella spp.)
and drug-mediated cell death. that are closely related to well-studied bacteria (such
as E. coli). Through a greater understanding of the
Opportunities and challenges for antibiotic network biological networks that are related to an individual
biology. One of the more intriguing aspects of antibac- drug target, we eventually might be able to search for
terial therapies is that not all bacterial species respond meaningful network homologues among species in
432 | juNe 2010 | vOluMe 8 www.nature.com/reviews/micro
© 2010 Macmillan Publishers Limited. All rights reservedREVIEWS
Antimicrobial peptide
the same spirit as we currently search for gene homo- Concluding remarks
A short, naturally occurring logues. Network-based efforts could also lead to the Drug-resistant bacterial infections are becoming more
cationic peptide that has development of species-specific treatments, includ- prevalent and are a major health issue facing us today.
antibacterial properties ing synthetic biology-derived therapies (BOX 2), which This rise in resistance has limited our repertoire of
through its ability to interfere
could be useful in killing off harmful, invasive bacte- effective antimicrobials, creating a problematic situa-
with bacterial membranes.
ria, while leaving our normal bacterial flora intact. tion that has been exacerbated by the small number of
Finally, bacterial network analyses will also be use- new antibiotics introduced in recent years. The com-
ful in the study of non-classical antibacterial agents plex effects of bactericidal antibiotics discussed in this
that induce cell death. Antimicrobial peptides are short Review provide a large playing field for the develop-
cationic peptides that are thought to kill through ment of new antibacterial compounds, as well as adju-
interactions with the membrane that result in pore vant molecules and synthetic biology constructs, that
formation134,135. However, the mode of action of many could enhance the potency of current antibiotics. It
antimicrobial peptides could, in fact, be more complex, will be important to translate our growing understand-
and cell death networks uncovered for existing anti- ing of antibiotic mechanisms into new clinical treat-
biotics could be used as mechanistic templates to study ments and approaches so that we can effectively fight
cellular responses induced by antimicrobial peptides. the growing threat from resistant pathogens.
1. Walsh, C. Antibiotics: actions, origins, resistance (ASM 13. Gellert, M., Mizuuchi, K., O’Dea, M. H. & Nash, H. A. 25. Kampranis, S. C. & Maxwell, A. The DNA gyrase-
Press, Washington, D.C., 2003). DNA gyrase: an enzyme that introduces superhelical quinolone complex. ATP hydrolysis and the mechanism
2. Fleming, A. On antibacterial action of culture of turns into D.NA. Proc. Natl Acad. Sci. USA 73, of DNA cleavage. J. Biol. Chem. 273, 22615–22626
penicillium, with special reference to their use in 3872–3876 (1976). (1998).
isolation of B. influenzae. Br. J. Exp. Pathol. 10, 14. Sugino, A., Peebles, C. L., Kreuzer, K. N. & Cozzarelli, Reveals that quinolone antibiotic binding to the
226–236 (1929). N. R. Mechanism of action of nalidixic acid: topoisomerase II–DNA complex occurs before DNA
3. Taubes, G. The bacteria fight back. Science 321, purification of Escherichia coli nalA gene product and strand breakage and that DNA cleavage can occur,
356–361 (2008). its relationship to DNA gyrase and a novel nicking- albeit at a slower rate, in the presence of the drug
4. Drlica, K., Malik, M., Kerns, R. J. & Zhao, X. closing enzyme. Proc. Natl Acad. Sci. USA 74, based on the results of ATP hydrolysis and DNA
Quinolone-mediated bacterial death. Antimicrob. 4767–4771 (1977). cleavage assays.
Agents Chemother. 52, 385–392 (2008). 15. Gellert, M., Mizuuchi, K., O’Dea, M. H., Itoh, T. & 26. Yoshida, H., Bogaki, M., Nakamura, M. & Nakamura, S.
5. Floss, H. G. & Yu, T. W. Rifamycin-mode of action, Tomizawa, J. I. Nalidixic acid resistance: a second Quinolone resistance-determining region in the DNA
resistance, and biosynthesis. Chem. Rev. 105, genetic character involved in DNA gyrase activity. gyrase gyrA gene of Escherichia coli. Antimicrob.
621–632 (2005). Proc. Natl Acad. Sci. USA 74, 4772–4776 (1977). Agents Chemother. 34, 1271–1272 (1990).
6. Tomasz, A. The mechanism of the irreversible References 14 and 15 discuss the results of 27. Morais Cabral, J. H. et al. Crystal structure of the
antimicrobial effects of penicillins: how the beta- complementary in vivo and in vitro studies that breakage-reunion domain of DNA gyrase. Nature 388,
lactam antibiotics kill and lyse bacteria. Annu. Rev. characterized the genetic locus (nalA, later gyrA) 903–906 (1997).
Microbiol. 33, 113–137 (1979). and the basic mechanism of quinolone antibiotic 28. Heddle, J. & Maxwell, A. Quinolone-binding pocket of
This seminal review of β‑lactam‑mediated cell death action (prevention of DNA duplex strand DNA gyrase: role of GyrB. Antimicrob. Agents
discusses the intricacies of killing by various rejoining yielding double‑stranded DNA breaks), Chemother. 46, 1805–1815 (2002).
members of this antibiotic class in terms of the while postulating on the composition and 29. Goss, W. A., Deitz, W. H. & Cook, T. M. Mechanism of
specific drug‑inhibited protein targets and their energetic requirements of DNA gyrase activity. action of nalidixic acid on Escherichia coli.II. Inhibition
related cell wall maintenance functions. 16. Hooper, D. C. & Rubinstein, E. Quinolone of deoxyribonucleic acid synthesis. J. Bacteriol. 89,
7. Vakulenko, S. B. & Mobashery, S. Versatility of antimicrobial agents (ASM Press, Washington, D.C., 1068–1074 (1965).
aminoglycosides and prospects for their future. Clin. 2003). 30. Snyder, M. & Drlica, K. DNA gyrase on the bacterial
Microbiol. Rev. 16, 430–450 (2003). 17. Rubinstein, E. History of quinolones and their side chromosome: DNA cleavage induced by oxolinic acid.
8. Kohanski, M. A., Dwyer, D. J., Hayete, B., Lawrence, effects. Chemotherapy 47 (Suppl. 3), 3–8 (2001). J. Mol. Biol. 131, 287–302 (1979).
C. A. & Collins, J. J. A common mechanism of cellular 18. Lu, T. et al. Enhancement of fluoroquinolone activity 31. Cox, M. M. et al. The importance of repairing stalled
death induced by bactericidal antibiotics. Cell 130, by C-8 halogen and methoxy moieties: action against replication forks. Nature 404, 37–41 (2000).
797–810 (2007). a gyrase resistance mutant of Mycobacterium 32. Courcelle, J. & Hanawalt, P. C. RecA-dependent
Reveals that treatment of Gram‑positive and smegmatis and a gyrase-topoisomerase IV double recovery of arrested DNA replication forks. Annu. Rev.
Gram‑negative bacteria with lethal levels of mutant of Staphylococcus aureus. Antimicrob. Agents Genet. 37, 611–646 (2003).
bactericidal antibiotics induces the formation of Chemother. 45, 2703–2709 (2001). 33. Howard, B. M., Pinney, R. J. & Smith, J. T. Function of
hydroxyl radicals through a common mechanism 19. Chen, C. R., Malik, M., Snyder, M. & Drlica, K. DNA the SOS process in repair of DNA damage induced by
involving drug‑induced changes in NADH gyrase and topoisomerase IV on the bacterial modern 4-quinolones. J. Pharm. Pharmacol. 45,
consumption and central metabolism, notably the chromosome: quinolone-induced DNA cleavage. 658–662 (1993).
TCA cycle. J. Mol. Biol. 258, 627–637 (1996). 34. Cirz, R. T. et al. Inhibition of mutation and combating
9. Dwyer, D. J., Kohanski, M. A., Hayete, B. & Collins, Identifies topoisomerase IV as a second target of the evolution of antibiotic resistance. PLoS Biol. 3,
J. J. Gyrase inhibitors induce an oxidative damage fluoroquinolone antibiotics in Gram‑negative e176 (2005).
cellular death pathway in Escherichia coli. Mol. Syst. bacteria and characterizes subtle but crucial 35. Guerin, E. et al. The SOS response controls integron
Biol. 3, 91 (2007). differences in the mechanism of killing by various recombination. Science 324, 1034 (2009).
Describes the physiological responses of E. coli quinolone drugs. 36. Beaber, J. W., Hochhut, B. & Waldor, M. K. SOS
following inhibition of topoisomerase by a 20. Drlica, K. & Zhao, X. DNA gyrase, topoisomerase IV, response promotes horizontal dissemination of
fluoroquinolone and a peptide toxin, which include and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61, antibiotic resistance genes. Nature 427, 72–74
activation of the superoxide stress response and 377–392 (1997). (2004).
increased Fe–S cluster synthesis. These 21. Munoz, R. & De La Campa, A. G. ParC subunit of DNA 37. Lewin, C. S., Howard, B. M. & Smith, J. T. Protein- and
physiological changes result in hydroxyl radical topoisomerase IV of Streptococcus pneumoniae is a RNA-synthesis independent bactericidal activity of
production, which contributes to cell death. primary target of fluoroquinolones and cooperates ciprofloxacin that involves the A subunit of DNA
10. Kohanski, M. A., Dwyer, D. J., Wierzbowski, J., with DNA gyrase A subunit in forming resistance gyrase. J. Med. Microbiol 34, 19–22 (1991).
Cottarel, G. & Collins, J. J. Mistranslation of phenotype. Antimicrob. Agents Chemother. 40, 38. Wang, X., Zhao, X., Malik, M., & Drlica, K.
membrane proteins and two-component system 2252–2257 (1996). Contribution of reactive oxygen species to pathways of
activation trigger antibiotic-mediated cell death. Cell 22. Belland, R. J., Morrison, S. G., Ison, C. & Huang, quinolone-mediated bacterial cell death. J. Antimicrob.
135, 679–690 (2008). W. M. Neisseria gonorrhoeae acquires mutations in Chemother. 65, 520–524 (2010).
Shows that systems which facilitate membrane analogous regions of gyrA and parC in 39. Kolodkin-Gal, I., Sat, B., Keshet, A. & Engelberg-Kulka,
protein trafficking are central to aminoglycoside‑ fluoroquinolone-resistant isolates. Mol. Microbiol. 14, H. The communication factor EDF and the toxin-
induced oxidative stress and cell death. This occurs 371–380 (1994). antitoxin module mazEF determine the mode of action
by signalling through the redox‑ and the envelope 23. Critchlow, S. E. & Maxwell, A. DNA cleavage is not of antibiotics. PLoS Biol. 6, e319 (2008).
stress‑responsive two‑component systems. required for the binding of quinolone drugs to the 40. Dukan, S. et al. Protein oxidation in response to
11. Espeli, O. & Marians, K. J. Untangling intracellular DNA gyrase-DNA complex. Biochemistry 35, increased transcriptional or translational errors. Proc.
DNA topology. Mol. Microbiol. 52, 925–931 7387–7393 (1996). Natl Acad. Sci. USA 97, 5746–5749 (2000).
(2004). 24. Marians, K. J. & Hiasa, H. Mechanism of quinolone 41. Hartmann, G., Honikel, K. O., Knusel, F. & Nuesch, J.
12. Drlica, K. & Snyder, M. Superhelical Escherichia coli action. A drug-induced structural perturbation of the The specific inhibition of the DNA-directed RNA
DNA: relaxation by coumermycin. J. Mol. Biol. 120, DNA precedes strand cleavage by topoisomerase, IV. synthesis by rifamycin. Biochim. Biophys. Acta 145,
145–154 (1978). J. Biol. Chem. 272, 9401–9409 (1997). 843–844 (1967).
NATuRe RevIeWS | Microbiology vOluMe 8 | juNe 2010 | 433
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