Our understanding of how antibiotics induce bacte-rial cell death is centred on the essential bacterial cell function that is inhibited by the primary drug–target interaction. Antibiotics can be classified based on the cellular component or system they affect, in addition to whether they induce cell death (bactericidal drugs) or merely inhibit cell growth (bacteriostatic drugs). Most current bactericidal antimicrobials — which are the focus of this Review — inhibit DNA, RNA, cell wall or protein synthesis 1.
Since the discovery of penicillin in 1929 (Ref . 2), other, more effective antimicrobials have been discovered and developed by elucidation of drug–target interactions and by drug molecule modification. These efforts have greatly enhanced our clinical armamentarium. Antibiotic-mediated cell death, however, is a complex process that begins with the physical interaction between a drug molecule and its specific target in bacteria, and involves alterations to the affected bacterium at the biochemical, molecular and ultrastructural levels. The increasing prev-alence of drug-resistant bacteria 3, as well as the increased means of gaining resistance, has made it crucial to bet-ter understand the multilayered mechanisms by which currently available antibiotics kill bacteria, as well as to explore and find alternative antibacterial therapies.
Antibiotic-induced cell death has been associated with the formation of double-stranded DNA breaks following treatment with inhibitors of topoisomer-ase II (also known as DNA gyrase)4, with the arrest of D
NA-dependent RNA synthesis following treatment with rifamycins 5, with cell envelope damage and loss of
structural integrity following treatment with inhibitors of cell wall synthesis 6 , and with cellular energetics, ribosome binding and protein mistranslation following treatment with inhibitors of protein synthesis 7. In addition, recent evidence points towards a common mechanism of cell death involving disadvantageous cell responses to drug-induced stresses that are shared by all classes of bacteri-cidal antibiotics, which ultimately contributes to killing by these drugs 8. Specifically, treatment with lethal con-centrations of bactericidal antibiotics results in the pro-duction of harmful hydroxyl radicals through a common oxidative damage cell death pathway that involves altera-tions in central metabolism (that is, in the tricarboxylic
acid (TCA) cycle) and iron metabolism 8–10
.
In this Review we describe our current knowledge of the drug–target interactions and the associated mecha-nisms by which the main classes of bactericidal antibiot-ics kill bacteria. We also describe recent efforts in network biology that have yielded new mechanistic insights into how bacteria respond
to lethal antibiotic treatments, and discuss how these insights and related developments in synthetic biology could be used to develop new, effective means to combat bacterial infections.
Inhibition of DNA replication by quinolones
DNA synthesis, mRNA transcription and cell division require the modulation of chromosomal supercoiling through topoisomerase-catalysed strand breakage and rejoining reactions 11–13. These reactions are exploited by the synthetic quinolone class of antimicrobials, includ-ing the clinically relevant fluoroquinolones, which target
*Howard Hughes Medical Institute and the Department of Biomedical Engineering, Center for BioDynamics, and Center for Advanced Biotechnology, Boston
University, 44 Cummington Street, Boston,
Massachusetts 02215, USA.‡
Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.§
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston,
Massachusetts 02115, USA.Correspondence to J.J.C. e‑mail: jcollins@bu.edu doi:10.1038/nrmicro2333Published online 4 May 2010
Bactericidal
Antimicrobial exposure that leads to bacterial cell death.
Bacteriostatic
Antimicrobial exposure that inhibits growth with no loss of viability.
Cell envelope
Layers of the cell surrounding the cytoplasm that include lipid membranes and peptidoglycans.
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.
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DNA–topoisomerase complexes 4,14,15. Quinolones are derivatives of nalidixic acid, which was discovered as a byproduct of the synthesis of chloroquine (a quinine) and was introduced in the 1960s to treat urinary tract infections 16. Nalidixic acid and other first generation quinolones (for example, oxolinic acid) are rarely used today owing to their toxicity 17. Second (ciprofloxacin), third (levofloxacin) and fourth (gemifloxacin) genera-tion quinolone antibiotics (TABLe 1) can be classified on the basis of their chemical structure and of qualitative differences between the killing mechanisms they use 16,18.
The quinolone class of antimicrobials interferes with the maintenance of chromosomal topology by target-ing topoisomerase II and topoisomerase Iv , trapping these enzymes at the DNA cleavage stage and prevent-ing strand rejoining 4,19,20 (fIG. 1). Despite the general functional similarities between topoisomerase II and topoisomerase Iv, their susceptibility to quinolones varies across bacterial species 20 (TABLe 1). For exam-ple, several studies have shown that topo i somerase Iv is the primar
y target of quinolones in Gram-positive bacteria (for example, Streptococcus pneumoniae 21), whereas in Gram-negative bacteria (for example, Escherichia coli 13 and Neisseria gonorrhoea 22) their pri-mary target is topoisomerase II (and topoisomerase Iv is the secondary target).
Introduction of DNA breaks and replication fork arrest. The ability of quinolone antibiotics to kill bacteria is a function of the stable interaction complex that is formed between drug-bound topoisomerases and cleaved DNA 4. On the basis of studies using DNA cleav-age mutants of topoisomerase II 23 and topoisomerase Iv 24 that do not prevent quinolone binding, and stud-ies that have shown that strand breakage can occur in the presence of quinolones 25, it is accepted that DNA strand breakage occurs after the drug has bound to the enzyme. Therefore, the net effect of quinolone treatment
is to generate double-stranded DNA breaks that are trapped by covalently (but reversibly) linked topo-isomerases, the function of which is compromised 26–28. As a result of quinolone–topoisomerase–DNA complex formation, the DNA replication machinery becomes arrested at blocked replication forks, leading to inhi-bition of DNA synthesis, which immediately leads to bacteriostasis and eventually cell death 4 (fIG. 1). It should be noted that the effects on DNA replication that correlate with bacteriostatic concentrations of qui-nolones are thought to be reversible 4,29. Nonetheless, considering that topoisomerase II has been found to be distributed approximately every 100 kb along the chromosome
30, inhibition of topoisomerase function by quinolone antibiotics and the resulting formation of stable complexes with DNA have substantial negative consequences for the cell in terms of its ability to deal with drug-induced DNA damage 31.
The role of protein expression in quinolone-mediated cell death. The introduction of double-stranded DNA breaks following topoisomerase inhibition by quinolones induces the DNA stress response (SOS response ), in which RecA is activated by DNA dam-age and promotes self-cleavage of the lexA repres-sor protein, inducing the expression of SOS response genes such as DNA repair enzymes 32. Notably, several studies have shown that preventing the induction of the SOS response enhances killing by quinolones (except in the case of nalidixic acid)8,33. Preventing the activation of the SOS response has also been shown to reduce the formation of drug-resistant mutants by blocking the induction of error-prone DNA polymerases 34, homologous recombination 20 and
horizontal transfer of drug-resistance elements 35,36
.Together with studies revealing that co-treatment with quinolones and the protein synthesis inhibitor chloram-phenicol inhibits the ability of quinolones to kill bacte-ria 19,37, there seems to be a clear relationship between the
SOS response
The DNA stress response pathway in E. coli , the
prototypical network of genes of which is regulated by the transcriptional repressor LexA, and is commonly activated by the co-regulatory protein RecA, which promotes LexA
self-cleavage when activated.
Table 1 (cont.) |
Antibiotic targets and pathways 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.
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Nature Reviews | Microbiology
Protein dependent
Protein independent
β-lactams
primary effects of quinolone–topoisomerase–DNA com-plex formation and the response of the bacteria (through the stress-induced expression of proteins) to these effects in the bactericidal activity of quinolone antibiotics. For example, the contribution of reactive oxygen species (ROS) to quinolone-mediated cell death has recently been shown to occur in a protein synthesis-dependent man-ner 38. Also, the chromosome-encoded toxin MazF has been shown to be required under certain conditions for efficient killing by quinolones owing to its ability to alter protein carbonylation 39, a form of oxidative stress 40.
Inhibition of RNA synthesis by rifamycins The inhibition of RNA synthesis by the rifamycin class of semi-synthetic bactericidal antibiotics, similarly to the inhibition of DNA replication by quinolones, has a cata-strophic effect on prokaryotic nucleic acid metabolism and is a potent means of inducing bacterial cell death 5. Rifamycins inhibit DNA-dependent transcription by sta-bly binding with high affinity to the β-subunit (encoded by rpoB ) of a DNA-bound and actively transcribing RNA polymerase 41–43 (TABL
e 1). The β-subunit is located in the channel that is formed by the RNA polymerase–DNA complex, from which the newly synthesized RNA strand emerges 44. Rifamycins uniquely require RNA synthesis to not have progressed beyond the addition of two ribonucleotides; this is attributed to the ability of
the drug molecule to sterically inhibit nascent RNA strand initialization 45. It is worth noting that rifamycins are not thought to act by blocking the elongation step of
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 formation of 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.
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Lysis
Rupture of the cell envelope leading to the expulsion of intracellular contents into the surrounding environment with eventual disintegration of the cell envelope. Peptidoglycan hydrolase
An enzyme that introduces cuts between carbon–nitrogen non-peptide bonds while pruning the peptidoglycan layer. It is important for homeostatic peptidoglycan turnover.
Autolysin
An enzyme that hydrolyses the β-linkage between the monosaccharide monomers in peptidoglycan units and can induce lysis when in excess.RNA synthesis, although a recently discovered class
of RNA polymerase inhibitors (based on the compound
CBR703) could inhibit elongation by allosterically
modifying the enzyme46.
Rifamycins were first isolated47 from the Gram-positive
bacterium Amycolatopsis mediterranei (originally known
as Streptomyces mediterranei) in the 1950s. Mutagenesis of
this organism has led to the isolation and characterization
of more potent rifamycin forms48, including the clinically
relevant rifamycin Sv and rifampicin. Rifamycins are
considered bactericidal against Gram-positive bacteria
and bacteriostatic against Gram-negative bacteria, a dif-
ference that has been attributed to drug uptake and not to
affinity of the drug with the RNA polymerase β-subunit49.
Notably, rifamycins are among the first-line therapies
used against myco b acteria because they efficiently induce
mycobacterial cell death50, although rifamycins are often
used in combinatorial therapies owing to the rapid nature
of resistance development49,51.
Interestingly, an interaction between DNA and
the hydroquinone moiety of RNA polymerase-bound
rifamycin has been observed52, and this interaction has
been attributed to the location of the rifamycin mol-
ecule in relation to DNA in the DNA–RNA polymerase
complex42. This proximity, coupled with the reported
ability of rifamycin to cycle between a radical and non-
radical form (rifamycin Sv and rifamycin S52,53), may
damage DNA through a direct drug–DNA interaction.
This hypothesis could account for the observation that
rifamycin Sv can induce the SOS DNA damage response
in E. coli and that treatment of recA-mutant E. coli results in
cell death whereas treatment of wild-type E. coli leads
to bacteriostasis8.
Inhibition of cell wall synthesis
Lytic cell death. The bacterial cell is encased by layers of
peptidoglycan (also known as murein), a covalently cross-
linked polymer matrix that is composed of peptide-linked
β-(1-4)-N -acetyl hexosamine54. The mechanical strength
afforded by this layer of the cell wall is crucial to a bacte-
rium’s ability to survive environmental conditions that can
alter prevailing osmotic pressures; of note, the degree of
peptidoglycan cross-linking correlates with the structural
integrity of the cell55. Maintenance of the peptidoglycan
layer is accomplished by the activity of transglycosylases
and penicillin-binding proteins (PBPs; also known as
transpeptidases), which add disaccharide pentapeptides
to extend the glycan strands of existing peptidoglycan
molecules and cross-link adjacent peptide strands of
immature peptidoglycan units, respectively56.
β-lactams and glycopeptides are among the classes of
antibiotics that interfere with specific steps in homeostatic
cell wall biosynthesis. Successful treatment with a cell wall
synthesis inhibitor can result in changes to cell shape and
size, induction of cell stress responses and ultimately cell
lysis6(fIG. 1). For example, β-lactams (including penicil-
lins, carbapenems and cephalosporins) block the cross-
linking of peptidoglycan units by inhibiting the peptide
bond formation reaction that is catalysed by PBPs55,57,58.
This inhibition is achieved by penicilloylation of the PBP
active site — the β-lactam (containing a cyclic amide
ring) is an analogue of the terminal d-alanyl-d-alanine
dipeptide of peptidoglycan and acts as a substrate for the
PBP during the acylation phase of cross link formation.
Penicilloylation of the PBP active site blocks the hydro-
lysis of the bond created with the now ring-opened drug,
thereby disabling the enzyme59,60.
By contrast, most actinobacterium-derived glyco-
peptide antibiotics (for example, vancomycin) inhibit
peptidoglycan synthesis by binding peptidoglycan units
(at the d-alanyl-d-alanine dipeptide) and by blocking
transglycosylase and PBP activity61. In this way, glyco-
peptides (whether free in the periplasm like vancomy-
cin or membrane-anchored like teicoplanin62) generally
act as steric inhibitors of peptidoglycan maturation and
reduce the mechanical strength of the cell, although some
chemically modified glycopeptides have been shown to
directly interact with the transglycosylase63. It is worth
noting that β-lactams can be used to treat Gram-positive
and Gram-negative bacteria, whereas glycopeptides are
effective against only Gram-positive bacteria owing to
low permeability (TABLe 1). In addition, antibiotics that
inhibit the synthesis (for example, fosfomycin) and trans-
port (for example, bacitracin) of individual peptidogly-
can units are also currently in use, as are lipopeptides (for
example, daptomycin), which affect structural integrity by
inserting themselves into the cell membrane and inducing
membrane depolarization.
Research into the mechanism of killing by peptido-
glycan synthesis inhibitors has centred on the lysis event.
Initially, it was thought that inhibition of cell wall syn-
thesis by β-lactams caused cell death when internal pres-
sure built up owing to cell growth outpacing cell wall
expansion, resulting in lysis6. This unbalanced growth
hypo t hesis was based in part on the notion that active pro-
tein synthesis is required for lysis to occur following the
addition of β-lactams.
The lysis-dependent cell death mechanism, however,
has proven to be much more complex, involving many
active cellular processes. Seminal work showed that
S. pneumoniae deficient in amidase activity (possessed by
peptidoglycan hydrolase or autolysins) did not grow or die
following treatment with a lysis-inducing concentration
of a β-lactam, an effect known as antibiotic tolerance64.
Autolysins are membrane-associated enzymes that break
down bonds between and within peptidoglycan strands,
making them important during normal cell wall turnover
and maintenance of cell shape55. Autolysins have also been
shown to play a part in lytic cell death in bacterial species
that contain numerous peptidoglycan hydrolases, such as
E. coli65. In E. coli, a set of putative peptidoglycan hydro-
lases (lytM domain factors) were shown to be important
for rapid ampicillin-mediated lysis66. The discovery that
autolysins contribute to cell death expanded our under-
standing of lysis and showed that active degradation of
the peptidoglycan layer by peptidoglycan hydrolases, in
conjunction with inhibition of peptidoglycan synthesis by
a β-lactam antibiotic, triggers lysis64(fIG. 1).
Non-lytic cell death. S. pneumoniae lacking peptidoglycan
hydrolase activity can still be killed by β-lactams, but at
a slower rate than autolysin-active cells, indicating that
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T wo-component system
A two-protein signal relay system composed of a sensor histidine kinase and a cognate receiver protein, which is typically a transcription factor.there is a lysis-independent mode of killing induced by
β-lactams64,67. evidence suggests that some of these non-
lytic pathways are regulated by bacterial two-component
systems68. For example, in S. pneumoniae, the vncSR two-
component system controls the expression of the autolysin
lytA and regulates tolerance to vancomycin and penicil-
lin through lysis-dependent69 and lysis-independent70 cell
death pathways.
In Staphylococcus aureus, the lytSR two-component
system can similarly affect cell lysis by regulating autolysin
activity71. lytR activates the expression of lrgAB72, which
was found to inhibit autolysin activity and thereby lead
to antibiotic tolerance73. lrgA is similar to bacteriophage
to kill a mockingbirdholin proteins73, which regulate the access of autolysins
to the peptidoglycan layer. Based on this information,
an additional holin-like system, cidAB, was uncovered
in S. aureus and found to activate autolysins, render-
ing S. aureus more susceptible to β-lactam-mediated
killing74,75. Complementation of cidA into a cidA-null
strain reversed the loss of autolysin activity but did not
completely restore sensitivity to β-lactams74.
Role of the SOS response in cell death by β-lactams.
Treatment with β-lactams leads to changes in cell mor-
phology that are associated with the primary drug–PBP
interaction. Generally speaking, PBP1 inhibitors cause
cell elongation and are potent triggers of lysis, PBP2
inhibitors alter cell shape but do not cause lysis and
PBP3 inhibitors influence cell division and can induce
filamentation76. Interestingly, β-lactam subtypes have dis-
tinct affinities for certain PBPs, which correlate with the
ability of these drugs to stimulate autolysin activity and
induce lysis76,77. Accordingly, PBP1-binding β-lactams are
also the most effective inducers of peptidoglycan hydro-
lase activity, and PBP2 inhibitors are the least proficient
autolysin activators77.
Filamentation can occur following the activation of the
DNA damage-responsive SOS network of genes78 owing
to expression of SulA, a key component of the SOS net-
work that inhibits septation and leads to cell elongation
by binding to and inhibiting polymerization of septation-
triggering FtsZ monomers79,80. Interestingly, β-lactams
that inhibit PBP3 and induce filamentation have been
shown to stimulate the DpiAB two-component system,
which can activate the SOS response81. β-lactam lethality
can be enhanced by disrupting DpiAB signalling or by
knocking out sulA. This indicates that SulA may protect
against β-lactam killing by shielding FtsZ and limiting a
division ring interaction among PBPs and peptidoglycan
hydrolases. In support of this idea, SulA expression lim-
its the lysis observed in a strain of E. coli that expresses
FtsZ84 (a mutant of FtsZ that is active only under certain
temperatures and media conditions) and lacks PBP4 and
PBP7 (Ref. 82).
DNA-damaging antimicrobials that do not directly
disrupt peptidoglycan turnover, such as quinolones, also
cause filamentation by activating the SOS response4.
Interestingly, a mutant strain of E. coli that is deficient
in diaminopimelic acid synthesis (E. coli W7), a key
building block of peptidoglycan, undergoes lysis follow-
ing treatment with the fluoroquinolone antimicrobials
ofloxacin or pefloxacin83. This suggests that peptido-
glycan turnover and the SOS response could have a role
in antibiotic-mediated lytic killing responses.
Inhibition of protein synthesis
The process of mRNA translation occurs over three
sequential phases (initiation, elongation and termina-
tion) that involve the ribosome and a range of cytoplas-
mic accessory factors84. The ribosome is composed of
two ribonucleoprotein subunits, the 50S and 30S, which
assemble (during the initiation phase) following the
formation of a complex between an mRNA transcript,
N-formylmethionine-charged aminoacyl tRNA, several
initiation factors and a free 30S subunit85. Drugs that
inhibit protein synthesis are among the broadest classes
of antibiotics and can be divided into two subclasses: the
50S inhibitors and 30S inhibitors (TABLe 1).
50S ribosome inhibitors include macrolides (for
example, erythromycin), lincosamides (for example,
clindamycin), streptogramins (for example, dalfopristin–
quinupristin), amphenicols (for example, chlorampheni-
col) and oxazolidinones (for example, linezolid)86,87.
50S ribosome inhibitors work by physically blocking
either initiation of protein translation (as is the case for
oxazolidinones88) or translocation of peptidyl tRNAs,
which serves to inhibit the peptidyltransferase reac-
tion that elongates the nascent peptide chain. A model
for the mechanism by which these drugs act has been
formulated by studies of macrolides, lincosamides and
streptogramins. The model involves blocking the access
of peptidyl tRNAs to the ribosome (to varying degrees),
subsequent blockage of the peptidyltransferase elonga-
tion reaction by steric inhibition and eventually trigger-
ing dissociation of the peptidyl tRNA89,90. This model
also accounts for the phenomenon that these classes of
drugs lose their antibacterial activity when elongation has
progressed beyond a crucial length91.
30S ribosome inhibitors include tetracyclines and
amino c yclitols. Tetracyclines work by blocking the access
of aminoacyl tRNAs to the ribosome92. The aminocycli-
tol class comprises spectinomycin and aminoglycosides
(for example, streptomycin, kanamycin and gentamicin),
which bind the 16S rRNA component of the 30S ribos-
ome subunit. Spectinomycin interferes with the stability
of peptidyl tRNA binding to the ribosome by inhibiting
elongation factor-catalysed translocation, but does not
cause protein mistranslation93–95. By contrast, the inter-
action between aminoglycosides and the 16S rRNA can
induce an alteration in the conformation of the com-
plex formed between an mRNA codon and its cognate
charged aminoacyl tRNA at the ribosome. This pro-
motes tRNA mismatching, which can result in protein
mistranslation96–99.
Among ribosome inhibitors, naturally derived
aminoglycosides are the only class that is broadly bac-
tericidal. Macrolides, streptogramins, spectinomycin,
tetracyclines, chloramphenicol and macrolides are typi-
cally bacteriostatic; however, they can be bactericidal in a
species- or treatment-specific manner. For example, chlo-
ramphenicol has been shown to kill S. pneumoniae and
Neisseria meningitidis effectively100, and chloramphenicol
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