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

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(p)ppGpp and the Stringent Response: An Emerging Threat to Antibiotic Therapy Joanne K Hobbs, and Alisdair B. Boraston ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00204 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Infectious Diseases

(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 *Corresponding authors: Joanne K. Hobbs ([email protected]); Alisdair B. Boraston ([email protected])

Stringent response, (p)ppGpp, antibiotic tolerance, antibiotic resistance, RelA/SpoT homologs, small alarmone synthetase 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 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 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 homologs (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.

This year marks the 50th anniversary of the discovery of guanosine 5’-diphosphate 3’-diphosphate (ppGpp) and guanosine 5’-triphosphate 3-diphosphate (pppGpp) – collectively 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 viability5,15,16 – and that bacterial phyla, classes and even genera differ in: i) the mechanisms through which (p)ppGpp affects transcription and translation,14 and ii) the configuration and number of (p)ppGpp-producing enzymes they possess.17 The RelA/SpoT homolog (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 hydrolases (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

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ACS Infectious Diseases 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 inactive18. Therefore, RelA is a monofunctional long RSH. The same classes of Proteobacteria also possess a 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 Enzymatic

Long RSHs

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Synthetase

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RRM/ACT

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Hydrolase

Synthetase

TGS

H

ZFD/CC

RRM/ACT

C

Synthetase

C

RelA

Rel RelV/P/Q

N

N

Figure 1. Domain structure of common RelA/SpoT homolog (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

Rel proteins are found in many important human pathogen-containing phyla, including the Firmicutes (e.g. Staphylococcus aureus, Enterococcus spp.) and Actinobacteria (e.g. Mycobacterium 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 γproteobacteria, 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

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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 tolerance6–10 and persistence24. 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 below 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 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 literature,31 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). Persistence, on the other hand, 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 2-fold higher minimum duration for killing (MDK)32 required to kill 99.9 % of the population. In agreement with the multi-drug tolerance phenotype observed with other methods of stringent response induction, the ileS


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mutations also conferred tolerance to the membranedamaging 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 methicillinresistant 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 resistance. 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 homogenous resistance. Mwangi et al. used whole genome 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, highlyresistant forms has now been demonstrated in many

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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 nonsynonymous 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 highlevel 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 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 hetero-resistant MRSA strains revealed many mutations associated with the highly-resistant subpopulations, 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 βlactam 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:


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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; methionyltRNA 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 fide 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 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. But 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 persistent bacteremia, was found by WGS to bear a Leu152Phe mutation in the hydrolase domain of Rel. This mutation arose after only three 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 responseactivating 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 accumulation 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 glycylglycine 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 modelling 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 which 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 SASs20,95,96, it is 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

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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 and in vivo.

GDP ppGpp

Relacin

Compound AC

Vitamin C

Compound AB

Compound X9

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 of against Rel.

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 testing revealed all of these to be non-specific antibacterials. Relacin was also put through this screen as a benchmark, but failed to


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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 chloramphenicol 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 anti-biofilm 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 defence 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 post-accumulation in P. aeruginosa. A distinct and more potent D-amino acid peptide, DJK-5, exhibits similar activity against (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 co-precipitation 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 Firstly, 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 wildtype.107 Secondly, 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. Firstly, 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 synthetaseon/hydrolase-off conformation). 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. Secondly, the two stringent response-activated clinical isolates reported to date both possessed mutations in rel; however, it is now 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 32P-labelled 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 * Joanne K. Hobbs. Phone: 250-721-7084. Fax: 250-721-8855. E-mail: [email protected]. * Alisdair B. Boraston. Phone: 250-472-4168. Fax: 250-7218855. E-mail: [email protected].

Funding Sources The writing of this review article was supported by a grant from the British Columbia Lung Association awarded to JKH and ABB, and a Canadian Institutes of Health Research operating grant awarded to ABB (PJT 159786).

ABBREVIATIONS Guanosine 5’-diphosphate 3’-diphosphate, ppGpp; guanosine 5’-triphosphate 3-diphosphate, pppGpp; minimum duration for killing, MDK; minimum inhibitory concentration, MIC; methicillin-resistant Staphylococcus aureus, MRSA; penicillinbinding protein, PBP; RelA/SpoT homolog, RSH; small alarmone hydrolases, SAH; small alarmone synthetase, SAS; small colony variant, SCV; serine hydroxamate, SHX; whole genome sequencing, WGS.

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Graphical abstract

GTP/GDP + ATP

Rel/RelA

RelP/Q/V

(p)ppGpp Susceptible Tolerant Resistant

Tolerance

Resistance

Duration of antibiotic exposure (hours)


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

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