Review

Cite This: ACS Infect. Dis. 2019, 5, 1505−1517 pubs.acs.org/journal/aidcbc

(p)ppGpp and the Stringent Response: An Emerging Threat to

Antibiotic Therapy
Joanne K. Hobbs* and Alisdair B. Boraston*
Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Road, Victoria, BC V8P 5C2, Canada

ABSTRACT: In 1969, Cashel and Gallant first observed the presence

of (p)ppGppthe signaling molecule of the stringent responsein
starved bacterial cells. Fifty years later, (p)ppGpp and the stringent
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

response have emerged as essential master regulators of not only the

bacterial response to stress but also almost all aspects of bacterial
physiology, virulence, and immune evasion. More worryingly, a wealth
Downloaded via UNIV OF SAO PAULO on March 22, 2020 at 00:57:14 (UTC).

of data now indicate that (p)ppGpp and stringent response activation

pose a serious threat to the efficacy and clinical success of
antimicrobial therapy. Here, we focus on the central role that
(p)ppGpp and the stringent response play in the phenomenon of
antibiotic tolerance, as well as the acquisition, development, and
expression of antibiotic resistance. We review these consequences of
stringent response activation in relation to the main proteins involved
in (p)ppGpp production and control, in particular the complex interplay between monofunctional and bifunctional long RelA/
SpoT homologues (RSHs) and small alarmone synthetases (SASs). We also review the growing evidence to suggest that there
are multiple other indirect pathways of stringent response induction that can affect antibiotic efficacy. Finally, we summarize
recent studies that indicate the in vivo and clinical impact of (p)ppGpp production on antibiotic treatment outcomes. We
conclude by reviewing the progress to date in the search for novel therapeutics that target the stringent response.
KEYWORDS: stringent response, (p)ppGpp, antibiotic tolerance, antibiotic resistance, RelA/SpoT homologues,
small alarmone synthetase

viability5,15,16and that bacterial phyla, classes, and even

T his year marks the 50th anniversary of the discovery of
guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and
guanosine 5′-triphosphate 3-diphosphate (pppGpp)collec-
tively known as (p)ppGpp or “magic spot”in starved
bacterial cells.1 These “alarmones” are the effectors of the
stringent response, a universal stress response that is classically
induced by amino acid deprivation.2 Since its discovery,
(p)ppGpp has emerged as a master regulator of almost all
aspects of bacterial physiology, including growth rate control,
phase transition, sporulation, motility, competence, biofilm
formation, toxin production, and a multitude of other virulence
associations.3−5 There is also a growing body of evidence that
implicates (p)ppGpp and the stringent response in antibiotic
tolerance6−10 and resistance.11−13
In the classical description of stringent response activation in
Escherichia coli, amino acid starvation results in the presence of
uncharged tRNAs in the A site of ribosomes. The stalled
ribosomes are sensed by the protein RelA, which responds by
synthesizing (p)ppGpp from GTP/GDP and ATP; an
accompanying hydrolase protein, SpoT, degrades the
(p)ppGpp once the amino acid deprivation has been alleviated.
The overall effect of (p)ppGpp during the stringent response is
the downregulated transcription of most metabolic genes and
the upregulation of genes associated with amino acid
biosynthesis and stress responses.2 We now know that
(p)ppGpp production can be induced by a number of different
stimuli3,5,14on top of the basal levels required for
genera differ in (i) the mechanisms through which (p)ppGpp
affects transcription and translation,14 and (ii) the config-
uration and number of (p)ppGpp-producing enzymes they
possess.17
The RelA/SpoT homologue (RSH) superfamily contains
two types of characterized and functionally important enzyme
that synthesize and/or hydrolyze (p)ppGpp: long RSHs and
small alarmone synthetases (SASs).17 Small alarmone hydro-
lases (SAHs) also exist but are largely uncharacterized and are
sporadically distributed among bacteria; therefore, they will
not be discussed further here. Long RSHs contain both a
(p)ppGpp hydrolase domain and a (p)ppGpp synthetase
domain in the N-terminal half of the protein, while the C-
terminal half is involved in regulation and ribosome binding
(Figure 1).18 They can be further divided into three groups
according to activity and bacterial distribution, and herein, we
will be using the RelA/SpoT/Rel naming system.17,18 RelA
proteins are found in γ- and β-proteobacteria, such as E. coli
and Pseudomonas aeruginosa. The absence of a conserved
HDXXED motif in the hydrolase domain active site renders
this domain inactive.18 Therefore, RelA is a monofunctional
long RSH. The same classes of Proteobacteria also possess a
Received: May 31, 2019
Published: July 9, 2019

© 2019 American Chemical Society 1505 DOI: 10.1021/acsinfecdis.9b00204

ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
Figure 1. Domain structure of common RelA/SpoT homologue
(RSH) proteins. Long RSHs are composed of two regions: an N-
terminal enzymatic region and a C-terminal regulatory region. The
enzymatic region contains hydrolase and synthetase domains that
hydrolyze and synthesize (p)ppGpp, respectively. The regulatory
region contains a ThrRS, GTPase, and SpoT (TGS) domain, a
conserved helical domain (H), a zinc-finger or conserved cysteine
domain (ZFD/CC), and an RNA recognition motif or aspartate
kinase, chorismate, and TyrA (ACT) domain (RRM/ACT).18 In
RelA proteins, the hydrolase domain is inactive (indicated by *), and
in SpoT proteins, the synthetase domain exhibits only weak activity
(indicated by #). The small alarmone synthetases RelV, RelP, and
RelQ consist of only a synthetase domain that bears little sequence
identity to the synthetase domain of long RSHs.3
second long RSH, SpoT. SpoT proteins exhibit both
synthetase and hydrolase activity; however, the synthetase
function is weak and activated by different nutritional stimuli
to that of RelA.5 Finally, the most widely distributed long RSH
(and RSH in general) is Rel.17 Rel (also sometimes referred to
as Rsh or RelA) is a truly bifunctional RSH, exhibiting efficient
synthetase and hydrolase activity.19
Rel proteins are found in many important human pathogen-
containing phyla, including the Firmicutes (e.g., Staphylococcus
aureus, Enterococcus spp.) and Actinobacteria (e.g., Mycobacte-
rium tuberculosis). The two antagonistic enzymatic functions of
Rel are thought to be controlled through conformational shifts
and reciprocal regulation,20 with the synthetase-off/hydrolase-
on conformation being the resting state of the protein.21
Of the 12 subgroups of SAS that have been bioinformatically
identified, only three have been functionally characterized:
RelP, RelQ, and RelV.17 SASs consist of an isolated synthetase
domain that bears little sequence identity to the synthetase
domain of long RSHs.3 Due to the toxicity of high (p)ppGpp
levels,22 SASs are only found in bacteria that also possess Rel
or SpoT.17 SASs have also been identified in bacteriophage.23
The vast majority of Firmicutes members possess at least one
SAS, and important human pathogen-containing genera, like
Staphylococcus and Clostridium, contain both RelP and RelQ.17
RelV is only found in a small subset of γ-proteobacteria,
including Vibrio spp. While Rel/RelA is the main contributor
to (p)ppGpp production during the stringent response, these
accessory SASs are thought to play an important role in
bacterial homeostasis.5
In addition to their role in bacterial virulence,3 induction of
the stringent response and elevated (p)ppGpp levels have been
implicated in antibiotic resistance,11−13 tolerance,6−10 and
persistence.24 In this Review, we will employ the definitions of
Brauner et al.25 for these three phenomena. Antibiotic
resistance describes the inheritable ability of bacteria to grow
at high concentrations of an antibiotic for an indefinite period
of time. It can be readily detected as a minimum inhibitory
concentration (MIC) above that of a susceptible strain (and
above the resistance breakpoint, where one exists). In contrast,
antibiotic tolerance describes the ability of bacteria to survive
transient exposure to concentrations of antibiotic that would
1506
Review
otherwise be lethal, without exhibiting an increase in MIC.
Historically, tolerance was often referred to as “phenotypic
resistance” or “non-inherited resistance”;26−28 however, it is
now known that tolerance can also be genotypic.29,30 The
terms tolerance and persistence are also used interchangeably
in the literature31 but are actually distinct phenomena.
Tolerance can be detected in time-kill assays as a slower rate
of killing that applies to the whole population (Figure 2).
Figure 2. Schematic depiction of time-kill results obtained with
susceptible, tolerant, persistent and resistant strains. Tolerance can be
detected in time-kill assays as a slower rate of killing and greater
minimum duration for killing (MDK), for example, 99% of the
population compared with a susceptible strain, despite having the
same minimum inhibitory concentration (MIC). Persistence mani-
fests as a similar initial rate of killing and MDK99 to that of a
susceptible strain (and the same MIC), followed by the emergence of
a small persistent subpopulation that dies more slowly. A resistant
strain exhibits an MIC greatly above that of a susceptible strain and is
resistant to killing in a time-kill assay (at antibiotic concentrations that
kill the other strains).
Persistence, however, is a property displayed by a small
subpopulation of bacteria. The initial rate of killing for a
persistent strain is similar to that of its susceptible counterpart
and applies to the majority of the population; however, a
subpopulation (typically <1%) emerges that can survive
exposure to the antibiotic for longer.25 Therefore, tolerance
and persistence can be distinguished from each other in a time-
kill assay (Figure 2).32 The distinction between tolerance and
persistence is also complicated by the use of the term
“persistent” to describe difficult-to-treat infections. While
resistance is a well-known and recognized cause of antibiotic
treatment failure, tolerance is a poorly understood antibiotic
escape strategy and greatly underappreciated contributor to
persistent and relapsing infections.25,29,33
In the past decade, the clinical significance of the stringent
response has been realized through the isolation of two clinical
isolates (S. aureus and Enterococcus faecium) bearing mutations
in Rel that led to elevated (p)ppGpp levels.6,34 These isolates
were both associated with persistent infections that required
prolonged treatment with multiple antibiotics to clear. Here,
we review the wealth of evidence that now implicates the
stringent response and (p)ppGpp in the expression of
antibiotic tolerance and resistance. We also provide an
overview of recent attempts to design and discover inhibitors
of the stringent response.
■
THE STRINGENT RESPONSE AND ANTIBIOTIC
TOLERANCE
The molecular mechanisms behind antibiotic tolerance are
varied and not fully understood;26,29 however, the overarching
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases Review

Table 1. Associations between the Stringent Response and Antibiotic Tolerance

Organism (and RSH Genotype or induction (p)ppGpp
Category of induction proteins) method level Tolerance phenotype
Nutritional/chemical Escherichia coli (RelA, Isoleucine deprivation Higha Tolerance to ampicillin39
SpoT)
Escherichia coli (RelA, Lysine deprivation Higha Tolerance to most penicillins, cephalosporins and carbapenems38
SpoT)
Escherichia coli (RelA, Mupirocin treatment High41 Tolerance to ampicillin and norfloxacin41
SpoT)
Pseudomonas SHX treatment High7 Tolerance to ofloxacin7
aeruginosa (RelA,
SpoT)
Vibrio cholerae (RelA, SHX treatment High40 Tolerance to tetracycline, erythromycin and chloramphenicol40
SpoT, RelV)
Staphylococcus aureus Mupirocin treatment Higha Tolerance to vancomycin42
(Rel, RelP, RelQ)
Genetic manipulation of Escherichia coli (RelA, Overexpression of RelA High7 Tolerance to ampicillin,44 mecillinam46,47 and microcin J2548
one or more RSH SpoT)
Escherichia coli (RelA, ΔrelA Lowa Reduced tolerance to penicillin under amino acid deprivation37
SpoT)
Loss of tolerance to ampicillin39,41
Loss of tolerance to ampicillin, D-cycloserine and moenomycin
under amino acid deprivation43
Tolerance to ampicillin during regrowth in minimal medium with
glucose following prolonged stationary phase56
Escherichia coli (RelA, ΔrelA ΔspoT Nonea Loss of tolerance to ofloxacin under amino acid deprivation at
SpoT) 24 h51
Increased killing by ampicillin (in LB medium)41
Increased killing by ofloxacin and tobramycin in a biofilm7
Pseudomonas ΔrelA Low49 Increased killing by ofloxacin49
aeruginosa (RelA,
SpoT)
Pseudomonas ΔrelAΔspoT None7 Loss of tolerance to gentamicin, ofloxacin and meropenem in
aeruginosa (RelA, stationary phase50
SpoT)
Increased killing by ofloxacin,7,52 meropenem,7 colistin,7 and
gentamicin7 in a biofilm
Increased killing by ofloxacin in stationary phase7
Vibrio cholerae (RelA, ΔrelAΔspoT High40 Tolerance to tetracycline, erythromycin and chloramphenicol40
SpoT, RelV)
Vibrio cholerae (RelA, ΔrelAΔspoTΔrelV None40 Loss of tolerance to tetracycline, erythromycin and
SpoT, RelV) chloramphenicol in stationary phase40
Enterococcus faecalis Δrel High8 Tolerance to vancomycin8
(Rel, RelQ)
Enterococcus faecalis ΔrelQ Low8 Increased killing by vancomycin8
(Rel, RelQ)
Enterococcus faecalis ΔrelΔrelQ None8 Increased killing by vancomycin,8 norfloxacin,15 and ampicillin15
(Rel, RelQ)
Staphylococcus aureus ΔrelPΔrelQ Lowa Increased killing by ampicillin and vancomycin22
(Rel, RelP, RelQ)
Staphylococcus aureus ΔrelP or ΔrelQ Normala No change in killing by ampicillin or vancomycin22
(Rel, RelP, RelQ)
Staphylococcus aureus ΔrelΔrelPΔrelQ Nonea Increased killing by ampicillin and vancomycin22
(Rel, RelP, RelQ)
Mycobacterium Δrel None53 Loss of tolerance to isoniazid under nutrient starvation54
tuberculosis (Rel)
Staphylococcus aureus Gln360Stop mutation in Higha Tolerance to ciprofloxacin57
(Rel, RelP, RelQ) rel
Staphylococcus aureus Leu61Phe mutation in rel Higha Tolerance to daptomycin58
(Rel, RelP, RelQ)
Enterococcus faecium Leu152Phe mutation in rel High6 Tolerance to vancomycin, linezolid and daptomycin in a biofilm6
(Rel, RelQ)
Genetic manipulation of Escherichia coli (RelA, ΔdksA Normala Increased killing by fluoroquinolones, streptomycin and
an accessory protein SpoT) ampicillin59
Pseudomonas ΔdksA Moderateb49 Tolerance to ciprofloxacin and ofloxacin in stationary phase49
aeruginosa (RelA,
SpoT)
Vibrio cholerae (RelA, ΔdksA Normal40 Loss of tolerance to tetracycline, erythromycin, and
SpoT, RelV) chloramphenicol in stationary phase40
Staphylococcus aureus ΔrsgA or rsgA mutation Normala Tolerance to β-lactams, vancomycin, and ciprofloxacin9
(Rel, RelP, RelQ)

1507 DOI: 10.1021/acsinfecdis.9b00204

ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases Review

Table 1. continued
Organism (and RSH Genotype or induction (p)ppGpp
Category of induction proteins) method level Tolerance phenotype
Escherichia coli (RelA, Overexpression of HipA High10,60 Tolerance to carbenicillin,10 ampicillin,10,61 ciprofloxacin,31,61
SpoT) norfloxacin,60 cefotaxime,62 ofloxacin,62 and mitomycin C62
Escherichia coli (RelA, Overexpression of HipA in Lowa Loss of tolerance to ampicillin conferred by HipA
SpoT) ΔrelA overexpression10
Escherichia coli (RelA, Overexpression of HipA Normala Reduced tolerance to cefotaxime, ofloxacin and mitomycin C
SpoT) mutants conferred by wildtype HipA62
Escherichia coli (RelA, argS, alaS, pheS, leuS, thrS, High46 Tolerance to mecillinam,46,63 ciprofloxacin,57,63 rifampicin,63
SpoT) glnS or aspS mutation chloramphenicol,63 ampicillin,63 and trimethoprim63
Escherichia coli (RelA, argS, alaS, leuS or aspS Low46 Loss of tolerance to mecillinam46 or ciprofloxacin63 conferred by
SpoT) mutation and ΔrelA an aminoacyl tRNA synthetase mutation
Staphylococcus aureus ileS mutation Higha Tolerance to vancomycin, imipenem, and daptomycin42
(Rel, RelP, RelQ)
a
Not experimentally determined; theoretical level based on genotype and/or induction method. bDetermined during the stationary phase of
growth. Abbreviations: RelA/SpoT homologue (RSH), serine hydroxamate (SHX)

Figure 3. Induction and control of the stringent response in γ/β-proteobacteria and most other classes of bacteria. The cascade of (p)ppGpp
production, interaction, and degradation is shown. Step 1: Deprivation of one of more amino acid leads to an uncharged tRNA entering the A site
of the ribosome. Step 2: The stalled ribosome is sensed by RelA (2a) or Rel (2b), which responds by synthesizing (p)ppGpp. Step 3: (p)ppGpp
acts to downregulate transcription/translation either by binding to RNA polymerase (RNAP) in conjunction with DksA (3a), or binding to, and
inhibiting, GTPases such as RsgA that are required for proper ribosome assembly (3b). Many other mechanisms of (p)ppGpp-mediated
transcriptional/translational control exist; only those that have been experimentally associated with antibiotic tolerance are shown. Step 4: When
the amino acid deprivation has been alleviated, SpoT (4a) or Rel (4b) degrades (p)ppGpp; Rel catalyzes (p)ppGpp hydrolysis off of the ribosome.
Additional contributors to (p)ppGpp production and stringent response control include: (i) tetrameric small alarmone synthetases and (ii) tRNA
charging enzymes. Most members of the Firmicutes and Vibrio spp. possess accessory synthetases that synthesize (p)ppGpp; SpoT also possesses
weak (p)ppGpp synthetase activity (shown in gray). Inactivation or reduced activity of tRNA charging enzymes can indirectly induce the stringent
response. The protein kinase HipA phosphorylates the glutamyl-tRNA synthetase GltX, thereby inactivating it and preventing the charging of
glutamyl-tRNAs. Mutations in aminoacyl tRNA synthetase genes (such as ileS) can reduce their activity and lead to stringent response activation in
a similar manner. Boxes indicate enzymes and processes that are specific to that given species, genus, or phylum. It should be noted that stringent
response-activating mutations in aminoacyl tRNA synthetase genes and homologues of RsgA are also found in γ/β-proteobacteria.

feature is slow growth. Known mechanisms of slow growth that
lead to tolerance include defects in electron transport or
thymidine biosynthesis, as seen in small colony variants
(SCVs), and biofilms.28 As the majority of clinically relevant
antibiotics target active metabolic processes, a reduction in
1508
metabolism and growth rate leads to multidrug tolerance.35,36
Classical activation of the stringent response leads to the
shutdown of almost all metabolic processes and a state of
quiescence in cells. In the clinical setting, however, it is now
clear that the stringent response is not binary in its control and
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
can be partially activated.6,34 This results in cells that exhibit
reduced, rather than abolished, metabolism and growth.5 As
such, these cells are still able to proliferate and cause
infections,6,34 but benefit from the antibiotic tolerance afforded
by slow growth.33,35,36 A variety of different methods to
modulate levels of (p)ppGpp in different bacterial species have
been employed to investigate the association between the
stringent response and antibiotic tolerance. They can be
separated into three categories: chemical/nutritional, genetic
manipulation of RSHs, and genetic manipulation of an
accessory protein (Table 1).
Early Studies with E. coli. The first association between
the stringent response and antibiotic tolerance was made
almost 40 years ago. Following the observation that amino acid
starvation renders E. coli tolerant to the lytic effects of
penicillin, this tolerance was found to be largely dependent on
the presence of RelA.37 Amino acid deprivation is now a
commonly employed method of stringent response induction
in tolerance studies, and it can be achieved either through the
use of defined medium that lacks one or more amino acid,37,38
the addition of excess valine leading to isoleucine starvation,39
or through the inclusion of an amino acid analog such as serine
hydroxamate (SHX).7,40 The effects of isoleucine starvation
can also be mimicked by the addition of mupirocin
(pseudomonic acid),41,42 which is an isoleucyl tRNA
synthetase inhibitor. Following the initial association between
amino acid starvation, RelA, and penicillin tolerance, the
stringent response was linked to the tolerance of starved E. coli
to other inhibitors of cell wall biosynthesis.38,43 Reversal of this
tolerance in relA+ strains, via apparent “relaxation” of the
stringent response with a protein synthesis inhibitor, also
provided support for the role of RelA in multidrug tolerance.44
However, the use of amino acid deprivation to induce
(p)ppGpp production was not considered an ideal model
system as it causes global effects outside of increased
(p)ppGpp.45 Therefore, Rodionov and Ishiguro used over-
expression of RelA via a tac promoter and variable inducer
concentrations to demonstrate the direct relationship between
(p)ppGpp and ampicillin tolerance.44 A similar genetic system
was also subsequently used to demonstrate the relationship
between (p)ppGpp concentration and tolerance to the β-
lactam mecillinam and the peptide antibiotic microcin
J25.46−48 In fact, Joseleau-Petit et al. identified a threshold
concentration of (p)ppGpp above which tolerance was
exhibited.47 Later studies have since revealed that the tolerance
conferred by elevated (p)ppGpp levels extends beyond cell
wall synthesis inhibitors to antibiotics with diverse mechanisms
of action; e.g., refs 7 and 49. These early experiments with E.
coli formed the basis of our understanding of the relationship
between the stringent response and tolerance; however, the
complement of RelA/SpoT RSHs found in E. coli and other γ-
proteobacteria is not typical of the vast majority of human
pathogens and bacteria in general.
Complex Control of (p)ppGpp Levels. With the advent
of the genomic era, the full repertoire of bacterial RSH proteins
was realized and with it the complexity of (p)ppGpp control in
different pathogenic bacteria (Figure 3). By far the most
common method employed in the study of the stringent
response and tolerance is genetic manipulation of one or more
RSH (Table 1). In β- and γ-proteobacteria, elevated (p)ppGpp
levels can be achieved through the overexpression of
RelA.44,46−48 In bacteria that possess the bifunctional RSH
Rel, or a combination of Rel and SASs, deciphering the effects
1509
Review
of gene knockouts on (p)ppGpp levels is more complicated.
Double knockouts of relA and spoT in E. coli and P. aeruginosa
are unable to synthesize (p)ppGpp, even when stimulated;
therefore, they exhibit a loss of tolerance or increased
sensitivity to killing by an antibiotic in the stationary
phase,50 under amino acid deprivation,51 or in a biofilm7,52
(Table 1). In contrast, a ΔrelAΔspoT mutant of Vibrio cholerae
exhibits tolerance to inhibitors of protein synthesis.40 This
discordance is due to the presence of RelV in V. cholerae, which
in the absence of SpoT produces unregulated amounts of
(p)ppGpp (Figure 3). As expected, the tolerance phenotype of
V. cholerae is abolished in a triple ΔrelAΔspoTΔrelV mutant.
In bacteria that possess a bifunctional Rel, deletion of the rel
gene has varying effects. The M. tuberculosis genome encodes
for only a single RSH, Rel; therefore, a rel knockout mutant
possesses no detectable (p)ppGpp53 and does not exhibit the
usual tolerance to isoniazid induced by starvation conditions.54
In comparison, deletion of rel in Enterococcus faecalis has the
same ultimate effect as deletion of both relA and spoT in V.
cholerae (i.e., high [p]ppGpp)8 because E. faecalis also
possesses an accessory SAS, RelQ. Production of (p)ppGpp
by RelQ in the absence of a hydrolase results in tolerance to
the cell wall-active antibiotic vancomycin.8 This is despite the
fact that in response to mupirocin the rel mutant does not
mount the canonical stringent transcriptional response or
appear to accumulate large amounts of (p)ppGpp.8,55 There-
fore, the authors concluded that (p)ppGpp-mediated tolerance
in E. faecalis occurs at relatively low (p)ppGpp concentrations
and below those associated with typical induction of the
stringent response. In agreement with the proposed
importance of basal, RelQ-mediated (p)ppGpp levels for
tolerance in this bacterium, a ΔrelQ mutant is more readily
killed by vancomycin under nonstressed conditions than the
wildtype.8 As expected, a double ΔrelΔrelQ strain possesses no
(p)ppGpp and is killed faster than the wildtype and single
mutants by multiple antibiotics.15
S. aureus represents one of the most complex systems of
(p)ppGpp control as it contains Rel and two accessory SASs
(RelP and RelQ; Table 1). Like RelQ in E. faecalis, RelP and
RelQ have been strongly implicated in antibiotic tolerance in S.
aureus, specifically tolerance to cell wall biosynthesis inhibitors.
Transcription of relP and relQ, but not rel, is induced by
exposure to ampicillin and vancomycin. Furthermore, a double
ΔrelPΔrelQ mutant is more readily killed by these agents (but
not ciprofloxacin, a fluoroquinolone DNA synthesis inhibitor)
than the wildtype.22 Interestingly, single deletion mutants of
relP or relQ exhibit similar time-kill kinetics to the wildtype. In
contrast to E. faecalis, a rel deletion mutant of S. aureus could
not be generated and characterized in terms of tolerance due to
the essentiality of the hydrolase domain.22 This essentiality was
shown to apply only when both RelP and RelQ were present.
Therefore, the authors concluded that a single SAS (in
addition to rel) is sufficient to confer tolerance to cell wall-
active antibiotics but does not lead to a toxic accumulation of
(p)ppGpp. This raises the question of why S. aureus, and many
other bacteria, possess multiple SASs.
Indirect Induction and Control of the Stringent
Response. Variations in the complement of RSHs are not
the only differences in stringent response induction and
control between bacterial species that affect tolerance (Figure
3). Broadly speaking, the transcriptional and translational
effects of (p)ppGpp occur via two distinct mechanisms in
Proteobacteria and other bacterial phyla.5 In Proteobacteria,
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
(p)ppGpp binds to RNA polymerase to bring about the
transcriptional changes that are the hallmark of the stringent
response.45 This binding, and the downstream effects
associated with induction of the stringent response, are
critically dependent on the transcription factor DksA (Figure
3).64 The effect of dksA deletion on (p)ppGpp-mediated
antibiotic tolerance has been investigated in E. coli, P.
aeruginosa, and V. cholerae (Table 1). Deletion of dksA in E.
coli led to an increased rate of killing by ampicillin, two
fluoroquinolones, and a protein synthesis inhibitor compared
with the wildtype.59 A similar effect was observed in V.
cholerae, whereby a ΔdksA mutant exhibited a loss of tolerance,
usually conferred by the stationary phase, to three different
protein synthesis inhibitors.40 These findings are in agreement
with the inability of ΔdksA mutants to facilitate the
downstream effects of (p)ppGpp production64 and thus
tolerance. Paradoxically, a dksA deletion mutant of P.
aeruginosa exhibited a slightly elevated (p)ppGpp level
compared with the wildtype and significant tolerance to
fluoroquinolones in the stationary phase.49 In this instance, the
authors noted that the ΔdksA mutant exhibited higher
antibiotic tolerance than a ΔspoT mutant, despite containing
considerably less (p)ppGpp. Therefore, they speculated that
DksA may negatively regulate the expression of a putative gene
involved in tolerance in this bacterium.
In most bacterial phyla, (p)ppGpp does not bind to RNA
polymerase, and its effects on transcription and translation
occur indirectly through the modulation of intracellular GTP
levels.5,14,16 There are a multitude of mechanisms through
which (p)ppGpp affects GTP synthesis, including inhibition of
the GTPase RsgA.9 RsgA binds to the 30S ribosomal subunit
and is thought to play a chaperoning role in proper ribosome
assembly.65 Corrigan et al. have shown that (p)ppGpp binds to
RsgA and inhibits its GTPase activity, thereby disrupting
ribosome assembly and leading to slow growth.9 Deletion of
rsgA, or introduction of a mutated version with reduced
GTPase, mimics the effects of (p)ppGpp in S. aureus. Both the
deletion and mutated strains exhibit tolerance to β-lactams,
vancomycin, and ciprofloxacin (Table 1). Given that RsgA is
only one of many binding partners of (p)ppGpp, there are a
great number of other indirect mechanisms through which
(p)ppGpp-mediated tolerance could be achieved.
DksA and RsgA represent crucial facilitators in the
downstream effects of stringent response activation once
(p)ppGpp has been synthesized in response to uncharged
tRNAs. Several indirect mechanisms of stringent response
induction that lead to the occurrence of uncharged tRNAs
have also been associated with tolerance. HipA is a protein
kinase found in E. coli, and some other Gram-negative
bacteria,66 that phosphorylates the glutamyl-tRNA synthetase
enzyme (GltX; Figure 3).31,60 This phosphorylation inhibits
aminoacylation, resulting in the presence of uncharged
tRNAGlu and induction of the stringent response. Over-
expression of wildtype HipA in E. coli results in tolerance to
β-lactams and fluoroquinolones (Table 1).10,31,60−62,67 Some
studies have referred to this phenotype as “persistence,” but
using the definitions of Brauner et al.,25 we conclude this to be
tolerance. It should, however, be noted that HipA was
originally associated with bona f ide persistence.68 HipA-
mediated persistence has been directly linked to induction of
the stringent response through studies with the hipA7 allele.66
This allele contains two amino acid changes that enable HipA
to be expressed nontoxically without its antitoxin partner
1510
Review
(HipB) and confer high level persistence. Deletion of relA led
to a considerable reduction in the high persistence phenotype
conferred by hipA7, and deletion of both relA and spoT
returned the persistence rate to that of the wildtype hipA
strain.66
HipA induces the stringent response by inactivating the
tRNA synthetase GltX. Nonsynonymous mutations in the
genes encoding for other tRNA synthetases can also reduce
their activity, activate the stringent response, and confer
tolerance.42,46,57,63 Numerous mutations that confer tolerance
to the β-lactam mecillinam or ciprofloxacin in E. coli were
mapped to argS, alaS, and leuS, which encode for arginyl-,
alanyl-, and leucyl-tRNA synthetase, respectively.46,63 Other
tolerant mutants were found to bear mutations in the genes
encoding for different tRNA synthetases but not characterized
further.46,63 The biochemical effects of the argS, alaS, and leuS
mutations on tRNA synthetase activity were not tested.
However, the mutants exhibited slow growth and/or high
(p)ppGpp levels; therefore, it is likely that the mutations
reduced tRNA synthetase activity leading to the presence of
uncharged tRNAs. The tolerance conferred by the tRNA
synthetase mutations was also found to be either partially or
entirely dependent on the presence of relA. Similar effects of
tRNA synthetase mutations have been observed in S.
aureus.42,57 Nonsynonymous mutations in leuS and the ileS
gene encoding for isoleucyl-tRNA synthetase have been
identified in slow-growing, ciprofloxacin- or vancomycin-
tolerant mutants. These mutants exhibited the classic hallmarks
of tolerance: an MIC similar to the parental strain and far
below the resistance breakpoint, but a >2-fold higher minimum
duration for killing (MDK)32 required to kill 99.9% of the
population. In agreement with the multidrug tolerance
phenotype observed with other methods of stringent response
induction, the ileS mutations also conferred tolerance to the
membrane-damaging antibiotic daptomycin69 and the β-lactam
imipenem.42
■
THE STRINGENT RESPONSE AND ANTIBIOTIC
RESISTANCE
While tolerance is emerging as an important contributor to
antibiotic treatment failure in its own right, it also acts as a
precursor to the development of resistance.30,70 Antibiotic
resistance represents an ever-growing threat to our ability to
cure bacterial infections, and methicillin-resistant S. aureus
(MRSA) is of particular concern.71 In recent years, (p)ppGpp
production has been linked with the complex expression of
methicillin resistance in S. aureus, as well as the ability of
bacteria to acquire resistance genes or mutate antibiotic
targets.
(p)ppGpp and the Expression of β-Lactam Resist-
ance. Methicillin resistance is a term used to refer to
resistance to the β-lactam family of antibiotics, and it is
typically conferred by the presence of the mecA gene (either
chromosomal or on a plasmid), which encodes for the
alternate, low-affinity penicillin-binding protein PBP2A.71 In
most clinical isolates of MRSA, β-lactam resistance is expressed
heterogeneously; the population as a whole exhibits relatively
low level resistance, with an MIC barely above the
susceptibility breakpoint, but there exist subpopulations that
exhibit very high levels of resistance.72 The stringent response
has been implicated in the mechanism of heterogeneous β-
lactam resistance, specifically in the transition from hetero- to
homogeneous resistance. Mwangi et al. used whole genome
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
sequencing (WGS) to compare two populations (low and high
resistance) within an engineered heterogeneous MRSA and
identified two rel mutations in the highly resistant cells.12
These were the only mutations found genome-wide. The
primary mutation introduced a premature stop codon at the
end of the synthetase domain, thereby generating a truncated
Rel that lacks the regulatory region (Figure 1). The second rel
mutation was located in the now-deleted region. Subsequently,
a further two truncating mutations have been identified in the
regulatory region of Rel in highly resistant subpopulations of
clinical MRSA isolates,73,74 and yet another truncation
mutation has been linked to ciprofloxacin tolerance.57 The
original truncated Rel mutant exhibited homogeneous high-
level β-lactam resistance, slow growth, and elevated (p)ppGpp
consistent with partial activation of the stringent response.12
There are three ways in which deletion of the regulatory
domain of Rel could function to activate the stringent
response: promote synthetase activity, reduce hydrolase
activity, or a combination of both. Biochemical and whole-
cell studies with S. aureus have shown that the synthetase
function of Rel is negatively affected by the regulatory region
and truncation increases synthetase activity; however, the
increase in (p)ppGpp synthesis is not enough to counteract the
dominant hydrolase activity of Rel (which is apparently
unaffected by the regulatory domain).21 Therefore, the
molecular details behind stringent response activation via Rel
truncation is unknown. Nevertheless, the ability of stringent
response induction to convert heterogeneous MRSA isolates to
homogeneous, highly resistant forms has now been demon-
strated in many clonal types through the simple addition of
mupirocin or SHX to the growth medium.11,12,73,75−78 These
stringent response-activated strains exhibit higher levels of
PBP2A than their “relaxed” heterogeneous counterparts.
Interestingly, the resistance expression profile of MRSA strains
that do not possess an alternate PBP like PBP2A, and are
instead resistant to β-lactams by virtue of mutations in the
native PBP, does not change in the presence of mupirocin.11,78
Given the complexity of (p)ppGpp control and the different
routes of stringent response induction reviewed here in
relation to tolerance, it is not surprising that heterogeneous
expression of β-lactam resistance has also been associated with
mutations in genes other than rel. A partial revertant of the
original Rel truncation mutant was found to bear a non-
synonymous mutation in relQ.12 This mutant exhibited an
intermediate level of β-lactam resistance and increased colony
size. The relQ mutation can only be assumed to have reduced
basal production of (p)ppGpp to partially counter the effect of
the Rel truncation. Similarly, several relQ truncation mutations
have been reported to reverse small colony size and high-level
vancomycin resistance in slow-growing S. aureus.79,80 A more
detailed study into the effects of RelP and RelQ on β-lactam
resistance and mecA expression was recently performed.81
Deletion of relQ was found to greatly reduce the level of β-
lactam resistance through reduced mecA expression, while
deletion of relP led to higher mecA expression than the
wildtype (in response to β-lactam exposure) and an increased
MIC. Promoter analysis of both relQ and relP revealed that the
relQ promoter is 5-fold stronger than that controlling relP and
is significantly induced by deletion of relP; however, the relP
promoter is not responsive to relQ deletion. Therefore,
deletion of relP is more than compensated for by increased
expression of relQ, leading to the increased resistance level
observed. In the case of the ΔrelQ mutant, resistance could be
1511
Review
restored to the parental level by the presence of mupirocin
(presumably through induction of rel-mediated (p)ppGpp
synthesis). Therefore, it appears that high level β-lactam
resistance can be induced either through Rel or RelQ, but not
RelP.
Alternate routes of stringent response induction have also
been linked to the expression of β-lactam resistance. WGS of
six naturally occurring heteroresistant MRSA strains revealed
many mutations associated with the highly resistant sub-
populations, including nonsynonymous mutations in five
different tRNA synthetase genes.73 As reviewed earlier,
mutations in these (or analogous) genes have already been
shown to lead to elevated (p)ppGpp levels. Other mutations
associated with homogeneous, high-level β-lactam resistance
were identified in genes related to guanine metabolism,
transcription, and translation, and hypothesized to potentially
activate the stringent response indirectly.73,74,77 Mutations in
several of these genes have also been proposed to play a role in
the natural homogeneous β-lactam resistance profile of MRSA
COL, a strain that exhibits slow growth and elevated
(p)ppGpp.78
Mutation Rates and Resistance Acquisition under the
Stringent Response. There are two mechanisms through
which bacteria can become resistant to an antibiotic:
endogenous mutation or the acquisition of foreign DNA.82
Experimental evidence indicates a role for the stringent
response in both of these mechanisms. Stress responses, like
the stringent response, have long been associated with
increased bacterial mutability.83 Deletion of relA/rel from E.
coli or Bacillus subtilis leads to lower reversion (i.e., mutation)
rates in a variety of amino acid auxotrophs, and (p)ppGpp
concentration has been shown to be positively correlated with
the mutation rate in these genes.84−86 However, this
(p)ppGpp-mediated rise in mutation rate is thought to be
due to the increase in transcription rate of these genes,84,85 and
the vast majority of genes in the bacterial genome are actually
downregulated under the stringent response.2 Nevertheless,
deletion of relA and spoT has been reported to suppress the
emergence of fluoroquinolone-resistant colonies in stationary
phase P. aeruginosa.7 Furthermore, activation of the stringent
response via the hipA7 allele has recently been shown to
increase the emergence of resistant colonies in the presence of
four different antibiotics.70 This effect is thought to be linked
to the tolerance/persistence phenotype of the hipA7 strain.
Tolerance mutations are known to precede resistance
mutations in E. coli exposed to ampicillin.30 Interestingly, the
main tolerance mutations observed in this study occurred in
genes associated with indirect stringent response activation
(metG; methionyl-tRNA synthetase) and/or linked to high
level β-lactam resistance (prsA).73,74,78 A nonsynonymous rel
mutation was also identified in a laboratory strain of B. subtilis
that had been passaged in the presence of daptomycin until it
developed resistance.87 The rel mutation itself did not confer
daptomycin resistance, and we can only speculate that it may
have arisen prior to, and potentially encouraged the emergence
of, the bona f ide resistance mutation. The B. subtilis rel
mutation is distinct from one recently reported in several S.
aureus cultures following serial passage in the presence of
daptomycin (Table 1).58 The S. aureus mutation conferred
tolerance to daptomycin; any potential effect on subsequent
resistance development was not investigated.
In terms of the role of the stringent response in resistance
acquisition, the presence of relA and spoT has been implicated
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
in the increased expression of an E. coli integron integrase in
biofilms.13 Integrases catalyze the insertion of incoming gene
cassettes, such as resistance cassettes, into integrons that can
then express them. Biofilms are associated with high levels of
resistance gene transfer, and E. coli biofilms exhibit elevated
expression of a class 1 integron integrase compared with
planktonic growth.13 Deletion of relA and spoT abrogated this
effect and returned biofilm integrase expression to planktonic
levels (in the absence of any effect on biofilm density). The
explicit effect of relA-spoT deletion on the transfer of resistance
genes in biofilms, and other bacterial populations, remains to
be tested.
In Vivo Effects of (p)ppGpp on Antibiotic Efficacy. It is
clear that the stringent response and (p)ppGpp are important
contributors to antibiotic tolerance and resistance in vitro.
There is also now growing evidence of the in vivo importance
of these relationships. Here, we use the term “in vitro” to refer
to studies of bacteria in isolation (i.e., in test tube
experiments), and the term “in vivo” to describe animal
model studies. Murine infection models of M. tuberculosis and
P. aeruginosa have been used with RSH knockouts to
demonstrate the contribution of the stringent response to
antibiotic tolerance in vivo. During the chronic phase of
infection, wildtype M. tuberculosis is naturally tolerant to the
antibiotic isoniazid; a Δrel mutant was able to establish a
chronic infection in mice but was susceptible to significant
killing by isoniazid.54 Similarly, the fluoroquinolone ofloxacin
was relatively ineffective in mice infected with stationary phase
P. aeruginosa, but survival rates in mice infected with a
ΔrelAΔspoT mutant and treated with ofloxacin were
significantly higher.7 Deletion of relA and spoT also increased
the efficacy of ofloxacin in a murine biofilm model. These
studies provide a direct link between the stringent response
and antibiotic efficacy in vivo.
By far, most of the studies reviewed here have exploited gene
knockouts or methods of specific nutritional deprivation to
induce/prevent (p)ppGpp production and study the effects on
antibiotic efficacy. However, does this provide a biologically-
and clinically relevant model of stringent response activation
and its implications for therapy? In 2010, Gao et al. published
the first report of a clinical isolate in which the stringent
response had been partially activated.34 This MRSA isolate was
associated with a case of persistent and recurrent bacteremia
that required >100 days of antibiotic treatment to cure. During
the course of treatment, bacterial culture revealed that the
original isolate had evolved a SCV phenotype. WGS of the
parental and SCV isolates identified four nonsynonymous
mutations, including one in the rel gene. This Phe128Tyr
mutation occurred in the hydrolase domain of Rel and
conferred elevated (p)ppGpp levels, reduced growth rate, and
small colonies, presumably due to reduced hydrolase activity.
This discovery provided the first evidence of the clinical
relevance of stringent response activation. In addition to the rel
mutation, the SCV also possessed three mutations associated
with antibiotic resistance or reduced susceptibility. For two of
these mutations, it is not known whether they arose before or
after the rel mutation; however, the mutation that led to
reduced susceptibility to linezolid occurred after the
emergence of the SCV phenotype. The rel mutation was not
investigated for its ability to confer antibiotic tolerance.
Recently, a second clinical isolate bearing a stringent
response-activating mutation has been identified.6 An isolate
of Enterococcus faecium, again associated with a case of
1512
Review
persistent bacteremia, was found by WGS to bear a Leu152Phe
mutation in the hydrolase domain of Rel. This mutation arose
after only 3 days of antibiotic therapy and was the only
consistent mutation identified during the 28-day infection (the
parental isolate already possessed numerous resistance genes/
mutations). The rel mutation was shown to confer elevated
(p)ppGpp levels but no significant tolerance to daptomycin or
linezolid in planktonic culture. However, in biofilm culture the
rel mutant could not be eradicated by high concentrations of
daptomycin, linezolid, or vancomycin (unlike the wildtype).
This study is the first example of the clinical significance of the
stringent response in relation to antibiotic therapy.
It should be noted that, while there are only two reports to
date of clinical isolates bearing stringent response-activating
mutations, the stringent response is almost certainly activated
under many infection states and a contributor to persistent
infections. Bacteria experience a range of nutrient and other
stresses when transitioning through the human body,88 and the
difficulty in treating many susceptible (by MIC) infections is
strongly suspected to lie in the presence of slow-growing,
tolerant populations of bacteria.33,89 In fact, several studies
have demonstrated the heterogeneity of bacterial growth rates
within host tissues, as well as the positive correlation between
growth rate and antibiotic killing.90,91 We suspect that, with
further research, the clinical significance of the stringent
response will prove to have been greatly underappreciated.
■ THE SEARCH FOR STRINGENT RESPONSE
INHIBITORS
The overwhelming evidence reviewed here that links
(p)ppGpp and the stringent response to antibiotic tolerance
and resistance, as well as virulence,3 has led to growing interest
in the development of stringent response inhibitors. There are
currently two approaches being taken: (i) inhibition of
(p)ppGpp synthesis by Rel/RelA using substrate analogs and
(ii) indirect abrogation of (p)ppGpp synthesis and accumu-
lation through the use of protein synthesis inhibitors or
cationic peptides.
Design and Discovery of (p)ppGpp Synthetase
Inhibitors. The first reported inhibitors of (p)ppGpp
synthetase activity were a series of ppGpp analogs, the most
potent of which bore bis-phosphonate substitutions at
positions 5′ and 3′ of the ribose ring in place of the
pyrophosphate moieties.92 Further development of ppGpp
analogs led to relacin (Figure 4).93 Relacin is a 2′-
deoxyguanosine-based analog of ppGpp in which the
pyrophosphate moieties have been replaced with glycyl-glycine
dipeptides linked to the sugar ring by a carbamate bridge. It
was rationally designed based on the structure of Rel from
Streptococcus dysgalactiae,20 and modeling indicates that it
occupies much of the synthetase active site and forms a range
of hydrogen bonds and hydrophobic interactions. In vitro and
in vivo data suggest that relacin inhibits (p)ppGpp synthesis by
both Rel and RelA, but millimolar concentrations are required.
At the cellular level, relacin led to the death of B. subtilis (and
other bacteria) with an estimated IC50 of 200 μM and
prevented spore and biofilm formation. Prior to bacterial
death, relacin also induced a prolonged exponential phase, a
feature that has been observed previously in a spoT knockout
of Helicobacter pylori.94 Further evidence for the specificity of
relacin was provided by the resistance of a B. subtilis rel
knockout to the bactericidal effect of relacin. Given the
structural differences between long RSHs and SASs,20,95,96 it is
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
Figure 4. Substrates and chemical inhibitors of RelA/Rel. Shown are
the chemical structures of natural, rationally designed and screened
inhibitors of RelA/Rel identified to date. RelA/rel substrates (boxed)
ppGpp and GDP are shown for comparison. Relacin is a rationally
designed analog of ppGpp, whereas vitamin C is a natural inhibitor
that resembles GDP. Compounds AC and AB are designed acetylated
and acetylated-benzoylated derivatives of guanosine, respectively.
Compound X9 is the lead candidate inhibitor from a high-throughput
screen against Rel.
not surprising that relacin and other related ppGpp analogs
have no inhibitory effect against RelQ from E. faecalis.97,98
Several modifications to relacin have since improved its
potency. Replacement of the glycyl-glycine dipeptides with
glutamyl-glutamine dipeptides led to compound 2d.99 This
compound inhibited the (p)ppGpp synthetase activity of E. coli
RelA with greater potency than relacin; however, millimolar
concentrations were still required. Based on the studies of
relacin and other ppGpp analogs, functionalization of the
amine group at the C-2 position of the guanine moiety in
guanosine led to compounds AC and AB (Figure 4).100 These
compounds exhibited IC50 values in the range of 40 μM against
a mycobacterial Rel. Compound AB was found to be the more
potent of the two agents, but both compounds had a negative
impact on long-term survival and biofilm formation in
Mycobacterium smegmatis. Interestingly, Vitamin C exhibits
potent bactericidal activity against mycobacterial species and
has been proposed as a possible scaffold for future Rel
inhibitors by virtue of its structural analogy with GDP (Figure
4).101 Vitamin C itself binds with weak affinity to Rel from M.
smegmatis, and millimolar concentrations are required to
inhibit (p)ppGpp synthesis in vitro.
An auxotrophy-based high-throughput screen has been
developed in an attempt to identify novel Rel inhibitors.102
This screen employs a ΔrelPΔrelQ knockout of B. subtilis so
that effects of any inhibitors on (p)ppGpp production by Rel
are not masked by SAS-mediated compensation. The screen
relies on a key phenotypic difference between cells that do and
do not possess the ability to synthesize (p)ppGpp, namely,
differential growth when starved of lysine or valine. Wildtype
and (p)ppGpp null mutants can grow equally well in the
absence of lysine, but growth of a (p)ppGpp null mutant is
strictly dependent on valine.103 Therefore, specific inhibition of
Rel can be detected as inhibition of growth of the ΔrelPΔrelQ
mutant on valine but not lysine. A library of 17,500
compounds were screened using this approach, and five
potential inhibitors were identified. Unfortunately, further
1513
Review
testing revealed all of these to be nonspecific antibacterials.
Relacin was also put through this screen as a benchmark, but
failed to inhibit the growth of the ΔrelPΔrelQ mutant in either
growth medium at 2 mM. A more recent set of ppGpp analogs
also had no effect on growth in this system, despite exhibiting
IC50 values as low as 50 μM against E. coli RelA.98
An alternate high-throughput screening approach for
inhibitors of Rel that proved more successful was recently
reported.54 This screen employed a C-terminal truncated
version of Rel from M. tuberculosis and a novel (p)ppGpp
synthetase assay based on detection of released AMP. From a
library of 2 million compounds, 791 hits were obtained and
further screened in Rel-specific whole-cell assays, leading to the
identification of compound X9 as the lead candidate (Figure
4). X9 exhibited an IC50 of ∼15 μM against purified Rel and 2
μM against nutrient-starved M. tuberculosis in vitro. At 4 μM, it
also enhanced the susceptibility of nutrient-starved M.
tuberculosis to killing by isoniazid. This synergy (and the
activity of X9 alone against M. tuberculosis) could be abolished
by overexpression of Rel, thereby providing further evidence
that Rel is the target of X9. The molecular details of how X9
binds to, and inhibits, Rel is not known. Nevertheless, X9
displays the most potent activity of any Rel inhibitor to date.
Indirect Strategies To Prevent or Reverse Stringent
Response Induction. In addition to targeting the synthetase
activity of Rel/RelA directly, indirect methods aimed at
preventing the synthesis and/or accumulation of (p)ppGpp
have been investigated. It was first noted more than 20 years
ago that the protein synthesis inhibitor chloramphenicol
prevents (p)ppGpp accumulation (and concomitant ampicillin
tolerance) in E. coli.44 More recently, the protein synthesis
inhibitor thiostrepton was shown to inhibit the synthetase
function of RelA in a reconstituted biochemical system.41
Furthermore, three different protein synthesis inhibitors
(chloramphenicol, thiostrepton, and tetracycline) inhibited
(p)ppGpp accumulation in E. coli and B. subtilis exposed to
mupirocin.41 The general ability of protein synthesis inhibitors
to abrogate (p)ppGpp accumulation is thought to stem from
interference in the association between Rel/RelA and stalled
ribosomes (specifically the unacylated A site) and/or the
increase in tRNA aminoacylation that results from the
inhibition of translation. Unfortunately, neither chloramphe-
nicol or tetracycline was able to resensitize mupirocin-treated
cultures to ampicillin.41
An alternate strategy aimed at reversing stringent response
activation is the promotion of (p)ppGpp degradation.
(p)ppGpp and the stringent response have been linked to
biofilm formation in a range of pathogenic bacteria,3 and
biofilms are often used as an experimental model of an
activated stringent response.7,52 A number of antibiofilm
cationic peptides have been identified and proposed to act
via disruption of the stringent response.104,105 These peptides
display synergy with conventional antibiotics against in vitro
biofilms and in animal models of infection.105,106 1018 is a
small, synthetic, L-amino acid peptide derived from a bovine
host defense peptide. It exhibits potent activity against Gram-
positive and Gram-negative bacteria in biofilms, but not in
planktonic culture, and overproduction of (p)ppGpp (either
through overexpression of RelA or addition of SHX) leads to
resistance to 1018.104 Furthermore, 1018 prevents (p)ppGpp
accumulation in a range of bacteria and appears to eliminate it
postaccumulation in P. aeruginosa. A distinct and more potent
D-amino acid peptide, DJK-5, exhibits similar activity against
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
(p)ppGpp.105 The mechanism of action of these peptides to
prevent accumulation, and promote degradation, of (p)ppGpp
is proposed to occur via a direct interaction with (p)ppGpp.104
In coprecipitation and NMR spectroscopy experiments, 1018
exhibited greater binding to ppGpp than other nucleotides.
The disappearance of (p)ppGpp from 1018- and DJK-5-
treated cells (under detection conditions that dissociate
peptide-ppGpp complexes) led the authors to suggest that
these peptides do not sequester (p)ppGpp, but instead
promote its degradation by the cell.104 A mechanism for how
these peptides promote (p)ppGpp degradation has not been
proposed.
It should be noted that the (p)ppGpp-specific activity of
1018 has been questioned by Andresen et al.102,107 First, 1018
was put through the auxotrophy screen for Rel inhibitors and
found to inhibit growth of the ΔrelPΔrelQ mutant under lysine
or valine deprivation with similar potency. 1018 also exhibited
similar potency against a (p)ppGpp null mutant of B. subtilis,
which lacks any (p)ppGpp. In a separate study, a ΔrelAΔspoT
mutant of E. coli was actually slightly sensitized to, rather than
protected from, 1018 inhibition compared with the wild-
type.107 Second, under different experimental conditions to
those employed previously, planktonic and biofilm cultures of
P. aeruginosa were seemingly equally inhibited by 1018 (and a
control peptide in which the amino acid sequence of 1018 had
been reversed). Therefore, the authors concluded that the
antimicrobial effect of 1018 and related peptides does not rely
on specific recognition of (p)ppGpp. This area requires further
investigation; however, it may be, as suggested by Andresen et
al.,107 that peptide exposure stimulates indirect degradation of
(p)ppGpp in much the same way that certain protein synthesis
inhibitors are known to “relax” the stringent response.41,44
■ CONCLUSIONS
As reviewed here, there is now overwhelming evidence that
implicates (p)ppGpp and activation of the stringent response
in the ability of bacteria to evade and resist the action of many
antibiotics. This ability is not only mediated by the canonical
long RSHs, but also accessory SASs and the wider network of
potential stringent response inducers (e.g., tRNA synthetases,
HipA) and downstream facilitators (e.g., DksA, RsgA).
Furthermore, it should be noted that antibiotic tolerance has
also been linked to (p)ppGpp production outside of the
classical stringent response.15,22 This recent appreciation of the
complexities and extent of (p)ppGpp control leads to two
considerations in relation to antibiotic efficacy and treatment
strategies. First, attempts to target (p)ppGpp production with
novel therapeutics need to consider the RSH makeup of the
target organism(s). As we have seen with deletion mutants
(which mimic the effects of enzyme inhibition to some extent),
abrogating the activity of one RSH can actually lead to
stringent response activation via the remaining RSHs8,40,81
(Table 1). In terms of the ongoing search for inhibitors of the
bifunctional Rel enzyme, which is the most widespread RSH,
what has yet to be addressed is whether inhibition of
synthetase activity also inhibits hydrolase function (e.g., by
“locking” Rel in the synthetase-on/hydrolase-off conforma-
tion). In many pathogenic bacteria this would lead to an
overproduction of (p)ppGpp by SASs. As such, therapeutic
strategies that promote the degradation of (p)ppGpp (as
proposed for cationic peptides) may be more useful. Second,
the two stringent response-activated clinical isolates reported
to date both possessed mutations in rel; however, it is now
1514
Review
clear that there is a vast number of genes that could induce
(p)ppGpp production if mutated. This will likely make genetic
identification of stringent response activation in clinical isolates
more complicated. Furthermore, current methods of
(p)ppGpp quantitation (e.g., thin layer chromatography with
32
P-labeled material) are laborious and not suited to screening
of clinical isolates. An alternative route to identifying stringent
response activation would be through the detection of its
effects (e.g., tolerance). The current gold standard for
detection of tolerance is the time-kill assay. We strongly
advocate the use of time-kills to distinguish between tolerance
and persistence in the literature; however, these assays are not
well suited to high-throughput clinical use. A number of
recently developed agar-based assays for tolerance57,108 may
facilitate the identification of tolerance (related to the stringent
response or otherwise) among clinical isolates. Overall, more
research is required to assess the extent of the problem posed
by (p)ppGpp and the stringent response in the clinic.
■
AUTHOR INFORMATION
Corresponding Authors
*Phone: 250-721-7084. Fax: 250-721-8855. E-mail: jhobbs@
uvic.ca.
*Phone: 250-472-4168. Fax: 250-721-8855. E-mail: boraston@
uvic.ca.
ORCID
Alisdair B. Boraston: 0000-0001-6417-0592
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The writing of this review article was supported by a grant
from the British Columbia Lung Association awarded to J.K.H.
and A.B.B., and a Canadian Institutes of Health Research
operating grant awarded to A.B.B. (PJT 159786).
■
ABBREVIATIONS
ppGpp guanosine 5′-diphosphate 3′-diphosphate; pppGpp
guanosine 5′-triphosphate 3-diphosphate; MDK minimum
duration for killing; MIC minimum inhibitory concentration;
MRSA methicillin-resistant Staphylococcus aureus; PBP pen-
icillin-binding protein; RSH RelA/SpoT homologue; SAH
small alarmone hydrolases; SAS small alarmone synthetase;
SCV small colony variant; SHX serine hydroxamate; WGS
whole genome sequencing
■ REFERENCES
(1) Cashel, M., and Gallant, J. (1969) Two compounds implicated in
the function of the RC gene of Escherichia coli. Nature 221 (5183),
838−841.
(2) Cashel, M., Gentry, D., Hernandez, V., and Vinella, D. (1996)
The stringent response. In Escherichia coli and Salmonella: Cellular and
Molecular Biology (Neidhardt, F. C., Curtiss, R. I., Ingraham, J. L., Lin,
E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M.,
Schaechter, M., and Umbarger, H. E., Eds.) pp 1458−1496, ASM
Press, Washington, D.C.
(3) Dalebroux, Z. D., Svensson, S. L., Gaynor, E. C., and Swanson,
M. S. (2010) ppGpp conjures bacterial virulence. Microbiol. Mol. Biol.
Rev. 74 (2), 171−199.
(4) Boutte, C. C., and Crosson, S. (2013) Bacterial lifestyle shapes
stringent response activation. Trends Microbiol. 21 (4), 174−180.
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
(5) Gaca, A. O., Colomer-Winter, C., and Lemos, J. A. (2015) Many
means to a common end: the intricacies of (p)ppGpp metabolism and
its control of bacterial homeostasis. J. Bacteriol. 197 (7), 1146−1156.
(6) Honsa, E. S., Cooper, V. S., Mhaissen, M. N., Frank, M., Shaker,
J., Iverson, A., Rubnitz, J., Hayden, R. T., Lee, R. E., Rock, C. O.,
Tuomanen, E. I., Wolf, J., and Rosch, J. W. (2017) RelA mutant
Enterococcus faecium with multiantibiotic tolerance arising in an
immunocompromised host. mBio 8 (1), No. e02124-16.
(7) Nguyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi,
O., Beer, K., McKay, G., Siehnel, R., Schafhauser, J., Wang, Y.,
Britigan, B. E., and Singh, P. K. (2011) Active starvation responses
mediate antibiotic tolerance in biofilms and nutrient-limited bacteria.
Science 334 (6058), 982−986.
(8) Abranches, J., Martinez, A. R., Kajfasz, J. K., Chávez, V., Garsin,
D. A., and Lemos, J. A. (2009) The molecular alarmone (p)ppGpp
mediates stress responses, vancomycin tolerance, and virulence in
Enterococcus faecalis. J. Bacteriol. 191 (7), 2248−2256.
(9) Corrigan, R. M., Bellows, L. E., Wood, A., and Gründling, A.
(2016) ppGpp negatively impacts ribosome assembly affecting growth
and antimicrobial tolerance in Gram-positive bacteria. Proc. Natl.
Acad. Sci. U. S. A. 113 (12), E1710−E1719.
(10) Bokinsky, G., Baidoo, E. E. K., Akella, S., Burd, H., Weaver, D.,
Alonso-Gutierrez, J., García-Martín, H., Lee, T. S., and Keasling, J. D.
(2013) HipA-triggered growth arrest and β-lactam tolerance in
Escherichia coli are mediated by relA-dependent ppGpp synthesis. J.
Bacteriol. 195 (14), 3173−3182.
(11) Aedo, S., and Tomasz, A. (2016) Role of the stringent stress
response in the antibiotic resistance phenotype of methicillin-resistant
Staphylococcus aureus. Antimicrob. Agents Chemother. 60 (4), 2311−
2317.
(12) Mwangi, M. M., Kim, C., Chung, M., Tsai, J., Vijayadamodar,
G., Benitez, M., Jarvie, T. P., Du, L., and Tomasz, A. (2013) Whole-
genome sequencing reveals a link between β-lactam resistance and
synthetases of the alarmone (p)ppGpp in Staphylococcus aureus.
Microb. Drug Resist. 19 (3), 153−159.
(13) Strugeon, E., Tilloy, V., Ploy, M.-C., and Re, S. Da. (2016) The
stringent response promotes antibiotic resistance dissemination by
regulating integron integrase expression in biofilms. mBio 7 (4),
No. e00868-16.
(14) Hauryliuk, V., Atkinson, G. C., Murakami, K. S., Tenson, T.,
and Gerdes, K. (2015) Recent functional insights into the role of
(p)ppGpp in bacterial physiology. Nat. Rev. Microbiol. 13 (5), 298−
309.
(15) Gaca, A. O., Kajfasz, J. K., Miller, J. H., Liu, K., Wang, J. D.,
Abranches, J., and Lemos, J. A. (2013) Basal levels of (p)ppGpp in
Enterococcus faecalis: the magic beyond the stringent response. mBio 4
(5), No. e00646-13.
(16) Kriel, A., Bittner, A. N., Kim, S. H., Liu, K., Tehranchi, A. K.,
Zou, W. Y., Rendon, S., Chen, R., Tu, B. P., and Wang, J. D. (2012)
Direct regulation of GTP homeostasis by (p)ppGpp: a critical
component of viability and stress resistance. Mol. Cell 48 (2), 231−
241.
(17) Atkinson, G. C., Tenson, T., and Hauryliuk, V. (2011) The
RelA/SpoT homolog (RSH) superfamily: distribution and functional
evolution of ppGpp synthetases and hydrolases across the tree of life.
PLoS One 6 (8), No. e23479.
(18) Irving, S. E., and Corrigan, R. M. (2018) Triggering the
stringent response: signals responsible for activating (p)ppGpp
synthesis in bacteria. Microbiology 164 (3), 268−276.
(19) Mechold, U., Murphy, H., Brown, L., and Cashel, M. (2002)
Intramolecular regulation of the opposing (p)ppGpp catalytic
activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus
equisimilis. J. Bacteriol. 184 (11), 2878−2888.
(20) Hogg, T., Mechold, U., Malke, H., Cashel, M., and Hilgenfeld
(2004) Conformational antagonism between opposing active sites in a
bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism
during the stringent response. Cell 117 (1), 57−68.
(21) Gratani, F. L., Horvatek, P., Geiger, T., Borisova, M., Mayer, C.,
Grin, I., Wagner, S., Steinchen, W., Bange, G., Velic, A., Maček, B.,
1515
Review
and Wolz, C. (2018) Regulation of the opposing (p)ppGpp
synthetase and hydrolase activities in a bifunctional RelA/SpoT
homologue from Staphylococcus aureus. PLoS Genet. 14 (7),
No. e1007514.
(22) Geiger, T., Kästle, B., Gratani, F. L., Goerke, C., and Wolz, C.
(2014) Two small (p)ppGpp synthases in Staphylococcus aureus
mediate tolerance against cell envelope stress conditions. J. Bacteriol.
196 (4), 894−902.
(23) Jimmy, S., Saha, C. K., Stavropoulos, C., Garcia-Pino, A.,
Hauryliuk, V., and Atkinson, G. C. (2019) Discovery of small
alarmone synthetases and their inhibitors as toxin-antitoxin loci.
bioRxiv, DOI: 10.1101/575399.
(24) Maisonneuve, E., and Gerdes, K. (2014) Molecular
mechanisms underlying bacterial persisters. Cell 157 (3), 539−548.
(25) Brauner, A., Fridman, O., Gefen, O., and Balaban, N. Q. (2016)
Distinguishing between resistance, tolerance and persistence to
antibiotic treatment. Nat. Rev. Microbiol. 14 (5), 320−330.
(26) Corona, F., and Martinez, J. L. (2013) Phenotypic resistance to
antibiotics. Antibiotics (Basel, Switz.) 2 (2), 237−255.
(27) Levin, B. R., and Rozen, D. E. (2006) Non-inherited antibiotic
resistance. Nat. Rev. Microbiol. 4 (7), 556−562.
(28) Kahl, B. C., Becker, K., and Löffler, B. (2016) Clinical
significance and pathogenesis of staphylococcal small colony variants
in persistent infections. Clin. Microbiol. Rev. 29 (2), 401−427.
(29) Meylan, S., Andrews, I. W., and Collins, J. J. (2018) Targeting
antibiotic tolerance, pathogen by pathogen. Cell 172 (6), 1228−1238.
(30) Levin-Reisman, I., Ronin, I., Gefen, O., Braniss, I., Shoresh, N.,
and Balaban, N. Q. (2017) Antibiotic tolerance facilitates the
evolution of resistance. Science 355 (6327), 826−830.
(31) Germain, E., Castro-Roa, D., Zenkin, N., and Gerdes, K. (2013)
Molecular mechanism of bacterial persistence by HipA. Mol. Cell 52
(2), 248−254.
(32) Brauner, A., Shoresh, N., Fridman, O., and Balaban, N. Q.
(2017) An experimental framework for quantifying bacterial
tolerance. Biophys. J. 112 (12), 2664−2671.
(33) Tuomanen, E., Durack, D. T., and Tomasz, A. (1986)
Antibiotic tolerance among clinical isolates of bacteria. Antimicrob.
Agents Chemother. 30 (4), 521−527.
(34) Gao, W., Chua, K., Davies, J. K., Newton, H. J., Seemann, T.,
Harrison, P. F., Holmes, N. E., Rhee, H.-W., Hong, J.-I., Hartland, E.
L., Stinear, T. P., and Howden, B. P. (2010) Two novel point
mutations in clinical Staphylococcus aureus reduce linezolid suscept-
ibility and switch on the stringent response to promote persistent
infection. PLoS Pathog. 6, No. e1000944.
(35) Tuomanen, E., Cozens, R., Tosch, W., Zak, O., and Tomasz, A.
(1986) The rate of killing of Escherichia coli by β-lactam antibiotics is
strictly proportional to the rate of bacterial growth. Microbiology 132
(5), 1297−1304.
(36) Eng, R. H., Padberg, F. T., Smith, S. M., Tan, E. N., and
Cherubin, C. E. (1991) Bactericidal effects of antibiotics on slowly
growing and nongrowing bacteria. Antimicrob. Agents Chemother. 35
(9), 1824−1828.
(37) Goodell, W., and Tomasz, A. (1980) Alteration of Escherichia
coli murein during amino acid starvation. J. Bacteriol. 144 (3), 1009−
1016.
(38) Tuomanen, E., and Tomasz, A. (1986) Induction of autolysis in
nongrowing Escherichia coli. J. Bacteriol. 167 (3), 1077−1080.
(39) Rodionov, D. G., Pisabarro, A. G., de Pedro, M. A., Kusser, W.,
and Ishiguro, E. E. (1995) Beta-lactam-induced bacteriolysis of amino
acid-deprived Escherichia coli is dependent on phospholipid synthesis.
J. Bacteriol. 177 (4), 992−997.
(40) Kim, H. Y., Go, J., Lee, K.-M., Oh, Y. T., and Yoon, S. S. (2018)
Guanosine tetra- and pentaphosphate increase antibiotic tolerance by
reducing reactive oxygen species production in Vibrio cholerae. J. Biol.
Chem. 293 (15), 5679−5694.
(41) Kudrin, P., Varik, V., Oliveira, S. R. A., Beljantseva, J., Del Peso
Santos, T., Dzhygyr, I., Rejman, D., Cava, F., Tenson, T., and
Hauryliuk, V. (2017) Subinhibitory concentrations of bacteriostatic
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
antibiotics induce relA-dependent and relA-independent tolerance to
β-lactams. Antimicrob. Agents Chemother. 61 (4), No. e02173-16.
(42) Singh, M., Matsuo, M., Sasaki, T., Morimoto, Y., Hishinuma, T.,
and Hiramatsu, K. (2016) In vitro tolerance of drug-naive Staph-
ylococcusaureus strain FDA209P to vancomycin. Antimicrob. Agents
Chemother. 61 (2), No. e01154-16.
(43) Kusser, W., and Ishiguro, E. E. (1985) Involvement of the relA
gene in the autolysis of Escherichia coli induced by inhibitors of
peptidoglycan biosynthesis. J. Bacteriol. 164 (2), 861−865.
(44) Rodionov, D. G., and Ishiguro, E. E. (1995) Direct correlation
between overproduction of guanosine 3′,5′-bispyrophosphate
(ppGpp) and penicillin tolerance in Escherichia coli. J. Bacteriol. 177
(15), 4224−4229.
(45) Traxler, M. F., Summers, S. M., Nguyen, H.-T., Zacharia, V. M.,
Hightower, G. A., Smith, J. T., and Conway, T. (2008) The global,
ppGpp-mediated stringent response to amino acid starvation in
Escherichia coli. Mol. Microbiol. 68 (5), 1128−1148.
(46) Vinella, D., D’Ari, R., Jaffé, A., and Bouloc, P. (1992) Penicillin
binding protein 2 is dispensable in Escherichia coli when ppGpp
synthesis is induced. EMBO J. 11 (4), 1493−1501.
(47) Joseleau-Petit, D., Thévenet, D., and D’Ari, R. (1994) ppGpp
concentration, growth without PBP2 activity, and growth-rate control
in Escherichia coli. Mol. Microbiol. 13 (5), 911−917.
(48) Pomares, M. F., Vincent, P. A., Farías, R. N., and Salomón, R.
A. (2008) Protective action of ppGpp in microcin J25-sensitive
strains. J. Bacteriol. 190 (12), 4328−4334.
(49) Viducic, D., Ono, T., Murakami, K., Susilowati, H., Kayama, S.,
Hirota, K., and Miyake, Y. (2006) Functional analysis of spoT, relA
and dksA genes on quinolone tolerance in Pseudomonas aeruginosa
under nongrowing condition. Microbiol. Immunol. 50 (4), 349−357.
(50) Martins, D., McKay, G., Sampathkumar, G., Khakimova, M.,
English, A. M., and Nguyen, D. (2018) Superoxide dismutase activity
confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 115 (39),
9797−9802.
(51) Fung, D. K. C., Chan, E. W. C., Chin, M. L., and Chan, R. C. Y.
(2010) Delineation of a bacterial starvation stress response network
which can mediate antibiotic tolerance development. Antimicrob.
Agents Chemother. 54 (3), 1082−1093.
(52) Khakimova, M., Ahlgren, H. G., Harrison, J. J., English, A. M.,
and Nguyen, D. (2013) The stringent response controls catalases in
Pseudomonas aeruginosa and is required for hydrogen peroxide and
antibiotic tolerance. J. Bacteriol. 195 (9), 2011−2020.
(53) Primm, T. P., Andersen, S. J., Mizrahi, V., Avarbock, D., Rubin,
H., and Barry, C. E. (2000) The stringent response of Mycobacterium
tuberculosis is required for long-term survival. J. Bacteriol. 182 (17),
4889−4898.
(54) Dutta, N. K., Klinkenberg, L. G., Vazquez, M.-J., Segura-Carro,
D., Colmenarejo, G., Ramon, F., Rodriguez-Miquel, B., Mata-Cantero,
L., Porras-De Francisco, E., Chuang, Y.-M., Rubin, H., Lee, J. J., Eoh,
H., Bader, J. S., Perez-Herran, E., Mendoza-Losana, A., and
Karakousis, P. C. (2019) Inhibiting the stringent response blocks
Mycobacterium tuberculosis entry into quiescence and reduces
persistence. Sci. Adv. 5 (3), No. eaav2104.
(55) Gaca, A. O., Abranches, J., Kajfasz, J. K., and Lemos, J. A.
(2012) Global transcriptional analysis of the stringent response in
Enterococcus faecalis. Microbiology 158 (Pt8), 1994−2004.
(56) Varik, V., Oliveira, S. R. A., Hauryliuk, V., and Tenson, T.
(2016) Composition of the outgrowth medium modulates wake-up
kinetics and ampicillin sensitivity of stringent and relaxed Escherichia
coli. Sci. Rep. 6 (1), 22308.
(57) Matsuo, M., Hiramatsu, M., Singh, M., Sasaki, T., Hishinuma,
T., Yamamoto, N., Morimoto, Y., Kirikae, T., and Hiramatsu, K.
(2019) Genetic and transcriptomic analyses of ciprofloxacin-tolerant
Staphylococcus aureus isolated by the replica plating tolerance isolation
system (REPTIS). Antimicrob. Agents Chemother. 63 (2), No. e02019-
18.
(58) Berti, A. D., Shukla, N., Rottier, A. D., McCrone, J. S., Turner,
H. M., Monk, I. R., Baines, S. L., Howden, B. P., Proctor, R. A., and
1516
Review
Rose, W. E. (2018) Daptomycin selects for genetic and phenotypic
adaptations leading to antibiotic tolerance in MRSA. J. Antimicrob.
Chemother. 73 (8), 2030−2033.
(59) Hansen, S., Lewis, K., and Vulić, M. (2008) Role of global
regulators and nucleotide metabolism in antibiotic tolerance in
Escherichia coli. Antimicrob. Agents Chemother. 52 (8), 2718−2726.
(60) Kaspy, I., Rotem, E., Weiss, N., Ronin, I., Balaban, N. Q., and
Glaser, G. (2013) HipA-mediated antibiotic persistence via
phosphorylation of the glutamyl-tRNA-synthetase. Nat. Commun. 4
(1), 3001.
(61) Falla, T. J., and Chopra, I. (1998) Joint tolerance to beta-lactam
and fluoroquinolone antibiotics in Escherichia coli results from
overexpression of hipA. Antimicrob. Agents Chemother. 42 (12),
3282−3284.
(62) Correia, F. F., D’Onofrio, A., Rejtar, T., Li, L., Karger, B. L.,
Makarova, K., Koonin, E. V., and Lewis, K. (2006) Kinase activity of
overexpressed HipA is required for growth arrest and multidrug
tolerance in Escherichia coli. J. Bacteriol. 188 (24), 8360−8367.
(63) Garoff, L., Huseby, D. L., Praski Alzrigat, L., and Hughes, D.
(2018) Effect of aminoacyl-tRNA synthetase mutations on suscept-
ibility to ciprofloxacin in Escherichia coli. J. Antimicrob. Chemother. 73
(12), 3285−3292.
(64) Paul, B. J., Barker, M. M., Ross, W., Schneider, D. A., Webb, C.,
Foster, J. W., and Gourse, R. L. (2004) DksA: a critical component of
the transcription initiation machinery that potentiates the regulation
of rRNA promoters by ppGpp and the initiating NTP. Cell 118 (3),
311−322.
(65) Jomaa, A., Stewart, G., Martín-Benito, J., Zielke, R., Campbell,
T. L., Maddock, J. R., Brown, E. D., and Ortega, J. (2011)
Understanding ribosome assembly: the structure of in vivo assembled
immature 30S subunits revealed by cryo-electron microscopy. RNA 17
(4), 697−709.
(66) Korch, S. B., Henderson, T. A., and Hill, T. M. (2003)
Characterization of the hipa7 allele of Escherichia coli and evidence
that high persistence is governed by (p)ppGpp synthesis. Mol.
Microbiol. 50 (4), 1199−1213.
(67) Germain, E., Roghanian, M., Gerdes, K., and Maisonneuve, E.
(2015) Stochastic induction of persister cells by HipA through
(p)ppGpp-mediated activation of mRNA endonucleases. Proc. Natl.
Acad. Sci. U. S. A. 112 (16), 5171−5176.
(68) Moyed, H. S., and Bertrand, K. P. (1983) hipA, a newly
recognized gene of Escherichia coliK-12 that affects frequency of
persistence after inhibition of murein synthesis. J. Bacteriol. 155 (2),
768−775.
(69) Hobbs, J. K., Miller, K., O’Neill, A. J., and Chopra, I. (2008)
Consequences of daptomycin-mediated membrane damage in Staph-
ylococcus aureus. J. Antimicrob. Chemother. 62 (5), 1003−1008.
(70) Windels, E. M., Michiels, J. E., Fauvart, M., Wenseleers, T., Van
den Bergh, B., and Michiels, J. (2019) Bacterial persistence promotes
the evolution of antibiotic resistance by increasing survival and
mutation rates. ISME J. 13 (5), 1239−1251.
(71) Gajdács, M. (2019) The continuing threat of methicillin-
resistant Staphylococcus aureus. Antibiotics 8 (2), 52.
(72) Tomasz, A., Nachman, S., and Leaf, H. (1991) Stable classes of
phenotypic expression in methicillin-resistant clinical isolates of
staphylococci. Antimicrob. Agents Chemother. 35 (1), 124−129.
(73) Dordel, J., Kim, C., Chung, M., Pardos de la Gándara, M.,
Holden, M. T. J., Parkhill, J., de Lencastre, H., Bentley, S. D., and
Tomasz, A. (2014) Novel determinants of antibiotic resistance:
identification of mutated loci in highly methicillin-resistant sub-
populations of methicillin-resistant Staphylococcus aureus. mBio 5 (2),
No. e01000.
(74) Pardos de la Gándara, M., Borges, V., Chung, M., Milheiriço,
C., Gomes, J. P., de Lencastre, H., and Tomasz, A. (2018) Genetic
determinants of high-level oxacillin resistance in methicillin-
resistantStaphylococcus aureus. Antimicrob. Agents Chemother. 62 (6),
No. e00206-18.
(75) Kim, C., Mwangi, M., Chung, M., Milheirço, C., de Lencastre,
H., and Tomasz, A. (2013) The mechanism of heterogeneous beta-
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517
ACS Infectious Diseases
lactam resistance in MRSA: key role of the stringent stress response.
PLoS One 8 (12), No. e82814.
(76) Chung, M., Kim, C. K., Conceiçaõ , T., Aires-De-Sousa, M., de
Lencastre, H., and Tomasz, A. (2016) Heterogeneous oxacillin-
resistant phenotypes and production of PBP2A by oxacillin-
susceptible/mecA-positive MRSA strains from Africa. J. Antimicrob.
Chemother. 71 (10), 2804−2809.
(77) Milheiriço, C., de Lencastre, H., and Tomasz, A. (2017) Full-
genome sequencing identifies in the genetic background several
determinants that modulate the resistance phenotype in methicillin-
resistant Staphylococcus aureus strains carrying the novel mecC gene.
Antimicrob. Agents Chemother. 61 (3), No. e02500-16.
(78) Kim, C. K., Milheiriço, C., de Lencastre, H., and Tomasz, A.
(2017) Antibiotic resistance as a stress response: recovery of high-
level oxacillin resistance in methicillin-resistant Staphylococcus aureus
“auxiliary” ( fem) mutants by induction of the stringent stress
response. Antimicrob. Agents Chemother. 61 (8), No. e00313-17.
(79) Matsuo, M., Hishinuma, T., Katayama, Y., and Hiramatsu, K.
(2015) A mutation of RNA polymerase β’ subunit (rpoC) converts
heterogeneously vancomycin-intermediate Staphylococcus aureus
(hVISA) into “slow VISA. Antimicrob. Agents Chemother. 59 (7),
4215−4225.
(80) Matsuo, M., Yamamoto, N., Hishinuma, T., and Hiramatsu, K.
(2019) Identification of a novel gene associated with high-level β-
lactam resistance in heterogeneous vancomycin-intermediate Staph-
ylococcus aureus strain Mu3 and methicillin-resistant S. aureus strain
N315. Antimicrob. Agents Chemother. 63 (2), No. e00712-18.
(81) Bhawini, A., Pandey, P., Dubey, A. P., Zehra, A., Nath, G., and
Mishra, M. N. (2019) RelQ mediates the expression of β-lactam
resistance in methicillin-resistant Staphylococcus aureus. Front. Micro-
biol. 10, 339.
(82) Munita, J. M., and Arias, C. A. (2016) Mechanisms of antibiotic
resistance. Microbiol. Spectr. 4 (2), 481.
(83) Foster, P. L. (2007) Stress-induced mutagenesis in bacteria.
Crit. Rev. Biochem. Mol. Biol. 42 (5), 373−397.
(84) Wright, B. E. (1996) The effect of the stringent response on
mutation rates in Escherichia coliK-12. Mol. Microbiol. 19 (2), 213−
219.
(85) Wright, B. E., and Minnick, M. F. (1997) Reversion rates in a
leuB auxotroph of Escherichia coliK-12 correlate with ppGpp levels
during exponential growth. Microbiology 143 (3), 847−854.
(86) Rudner, R., Murray, A., and Huda, N. (1999) Is there a link
between mutation rates and the stringent response in Bacillus subtilis?
Ann. N. Y. Acad. Sci. 870, 418−422.
(87) Hachmann, A.-B., Sevim, E., Gaballa, A., Popham, D. L.,
Antelmann, H., and Helmann, J. D. (2011) Reduction in membrane
phosphatidylglycerol content leads to daptomycin resistance in
Bacillus subtilis. Antimicrob. Agents Chemother. 55 (9), 4326−4337.
(88) Fang, F. C., Frawley, E. R., Tapscott, T., and Vázquez-Torres,
A. (2016) Bacterial stress responses during host infection. Cell Host
Microbe 20, 133−143.
(89) Handwerger, S., and Tomasz, A. (1985) Antibiotic tolerance
among clinical isolates of bacteria. Annu. Rev. Pharmacol. Toxicol. 25
(1), 349−380.
(90) Claudi, B., Spröte, P., Chirkova, A., Personnic, N., Zankl, J.,
Schürmann, N., Schmidt, A., and Bumann, D. (2014) Phenotypic
variation of Salmonella in host tissues delays eradication by
antimicrobial chemotherapy. Cell 158 (4), 722−733.
(91) Kopf, S. H., Sessions, A. L., Cowley, E. S., Reyes, C., Van
Sambeek, L., Hu, Y., Orphan, V. J., Kato, R., and Newman, D. K.
(2016) Trace incorporation of heavy water reveals slow and
heterogeneous pathogen growth rates in cystic fibrosis sputum.
Proc. Natl. Acad. Sci. U. S. A. 113 (2), E110−E116.
(92) Wexselblatt, E., Katzhendler, J., Saleem-Batcha, R., Hansen, G.,
Hilgenfeld, R., Glaser, G., and Vidavski, R. R. (2010) ppGpp
analogues inhibit synthetase activity of Rel proteins from Gram-
negative and Gram-positive bacteria. Bioorg. Med. Chem. 18 (12),
4485−4497.
1517
Review
(93) Wexselblatt, E., Oppenheimer-Shaanan, Y., Kaspy, I., London,
N., Schueler-Furman, O., Yavin, E., Glaser, G., Katzhendler, J., and
Ben-Yehuda, S. (2012) Relacin, a novel antibacterial agent targeting
the stringent response. PLoS Pathog. 8 (9), No. e1002925.
(94) Zhou, Y. N., Coleman, W. G., Yang, Z., Yang, Y., Hodgson, N.,
Chen, F., and Jin, D. J. (2008) Regulation of cell growth during serum
starvation and bacterial survival in macrophages by the bifunctional
enzyme SpoT in Helicobacter pylori. J. Bacteriol. 190 (24), 8025−8032.
(95) Manav, M. C., Beljantseva, J., Bojer, M. S., Tenson, T., Ingmer,
H., Hauryliuk, V., and Brodersen, D. E. (2018) Structural basis for
(p)ppGpp synthesis by the Staphylococcus aureus small alarmone
synthetase RelP. J. Biol. Chem. 293 (9), 3254−3264.
(96) Steinchen, W., Schuhmacher, J. S., Altegoer, F., Fage, C. D.,
Srinivasan, V., Linne, U., Marahiel, M. A., and Bange, G. (2015)
Catalytic mechanism and allosteric regulation of an oligomeric
(p)ppGpp synthetase by an alarmone. Proc. Natl. Acad. Sci. U. S. A.
112 (43), 13348−13353.
(97) Gaca, A. O., Kudrin, P., Colomer-Winter, C., Beljantseva, J.,
Liu, K., Anderson, B., Wang, J. D., Rejman, D., Potrykus, K., Cashel,
M., Hauryliuk, V., and Lemos, J. A. (2015) From (p)ppGpp to
(pp)pGpp: characterization of regulatory effects of pGpp synthesized
by the small alarmone synthetase of Enterococcus faecalis. J. Bacteriol.
197 (18), 2908−2919.
(98) Beljantseva, J., Kudrin, P., Jimmy, S., Ehn, M., Pohl, R., Varik,
V., Tozawa, Y., Shingler, V., Tenson, T., Rejman, D., and Hauryliuk,
K. (2017) Molecular mutagenesis of ppgpp: turning a rela activator
into an inhibitor. Sci. Rep. 7 (1), 41839.
(99) Wexselblatt, E., Kaspy, I., Glaser, G., Katzhendler, J., and Yavin,
E. (2013) Design, synthesis and structure−activity relationship of
novel relacin analogs as inhibitors of Rel proteins. Eur. J. Med. Chem.
70, 497−504.
(100) Syal, K., Flentie, K., Bhardwaj, N., Maiti, K., Jayaraman, N.,
Stallings, C. L., and Chatterji, D. (2017) Synthetic (p)ppGpp
analogue is an inhibitor of stringent response in mycobacteria.
Antimicrob. Agents Chemother. 61 (6), No. e00443-17.
(101) Syal, K., Bhardwaj, N., and Chatterji, D. (2017) Vitamin C
targets (p)ppGpp synthesis leading to stalling of long-term survival
and biofilm formation in Mycobacterium smegmatis. FEMS Microbiol.
Lett. 364 (1), No. fnw282.
(102) Andresen, L., Varik, V., Tozawa, Y., Jimmy, S., Lindberg, S.,
Tenson, T., and Hauryliuk, V. (2016) Auxotrophy-based high
throughput screening assay for the identification of Bacillus subtilis
stringent response inhibitors. Sci. Rep. 6 (1), 35824.
(103) Kriel, A., Brinsmade, S. R., Tse, J. L., Tehranchi, A. K., Bittner,
A. N., Sonenshein, A. L., and Wang, J. D. (2014) GTP dysregulation
in Bacillussubtilis cells lacking (p)ppGpp results in phenotypic amino
acid auxotrophy and failure to adapt to nutrient downshift and
regulate biosynthesis genes. J. Bacteriol. 196 (1), 189−201.
(104) de la Fuente-Núñez, C., Reffuveille, F., Haney, E. F., Straus, S.
K., and Hancock, R. E. W. (2014) Broad-spectrum anti-biofilm
peptide that targets a cellular stress response. PLoS Pathog. 10 (5),
No. e1004152.
(105) de la Fuente-Núñez, C., Reffuveille, F., Mansour, S. C.,
Reckseidler-Zenteno, S. L., Hernández, D., Brackman, G., Coenye, T.,
and Hancock, R. E. W. (2015) D-enantiomeric peptides that eradicate
wild-type and multidrug-resistant biofilms and protect against lethal
Pseudomonas aeruginosa infections. Chem. Biol. 22 (2), 196−205.
(106) Pletzer, D., Mansour, S. C., and Hancock, R. E. W. (2018)
Synergy between conventional antibiotics and anti-biofilm peptides in
a murine, sub-cutaneous abscess model caused by recalcitrant
ESKAPE pathogens. PLoS Pathog. 14 (6), No. e1007084.
(107) Andresen, L., Tenson, T., and Hauryliuk, V. (2016) Cationic
bactericidal peptide 1018 does not specifically target the stringent
response alarmone (p)ppGpp. Sci. Rep. 6 (1), 36549.
(108) Gefen, O., Chekol, B., Strahilevitz, J., and Balaban, N. Q.
(2017) TDtest: easy detection of bacterial tolerance and persistence
in clinical isolates by a modified disk-diffusion assay. Sci. Rep. 7,
41284.
DOI: 10.1021/acsinfecdis.9b00204
ACS Infect. Dis. 2019, 5, 1505−1517