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35 ABSTRACT
36 Antibiotic failure is not only due to the development of resistance by pathogens, but it can also
37 often be explained by persistence and tolerance. Persistence and tolerance can be included in
38 the “persistent phenotype”, with high relevance for clinics. Two of the most important
39 molecular mechanisms involved in tolerance and persistence are toxin-antitoxin (TA) modules
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55 1. Introduction
56 Antimicrobial resistance crisis is a serious health problem worldwide. During the past fifty
57 years, very few new anti-infective molecules have been discovered (1). Hence, microbial
58 pathogens have been able to accumulate molecular mechanisms enabling them to counteract
59 antibiotics.
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89 There are several molecular mechanisms involved in bacterial persistence and tolerance to
90 antibiotics, reviewed by Trastoy and colleagues (2018), which include: (p)ppGpp network;
91 toxin-antitoxin (TA) system; the quorum sensing (QS) system; drug efflux pumps; reactive
92 oxygen species (ROS); the SOS response; and RpoS (sigma factor of stationary phase) (7).
93 (p)ppGpp orchestrates the stringent response (SR) in bacteria, thus it is produced during
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124 transcription causing a global re-wiring of the gene expression profile. Taken together, all
125 these changes lead to dormancy or slow growth for most cells (Figure 1) (31, 32).
126 Most Gram-positive bacteria possess only one long RSH, named Rel, which has both (p)ppGpp
127 synthetase and hydrolase activities (33, 34) together with a short RSH, named SAS (Small
128 Alarmone Synthetase) or SAH (Small Alarmone Hydrolase) (35). Nonetheless, the existence of a
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159 normal state where (p)ppGpp is present (52). In 2015, Yamaguchi and colleagues found that
160 elevated levels of (p)ppGpp led to inhibition of bacterial growth by interfering with the FtsZ
161 protein assembly in Salmonella paratyphi A (53). FtsZ is a protein essential for the prokaryotic
162 cell division that needs to form linear structures and has a GTP binding site; however,
163 increased levels of (p)ppGpp (20-fold higher than the required GTP levels) causes FtsZ to form
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194 increasing 1,000- to 10,000-fold the frequency of persisters. Moreover, in this study they also
195 demonstrated that relA knockouts diminished the high persistent phenotype in hipA7 mutants,
196 and that relA spoT knockouts completely eliminated this high persistence, suggesting that
197 hipA7 facilitates the establishment of the persistent state by inducing (p)ppGpp synthesis (65).
198 This result was confirmed in 2011 by Nguyen and colleagues in P. aeruginosa, where a relA
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229 phosphorylate GltX and inhibits charging of tRNA-glu. When uncharged tRNAs enter the
230 ribosomal A site, RelA-dependent synthesis of (p)ppGpp is triggered (Table 1 and Figure 1)
231 (42). Even though this model became very influential, the authors retracted the key articles in
232 2018, claiming that the apparent inhibition of persistence in the multiple-deletion strain was
233 due to an inadvertent lysogenisation with the bacteriophage Φ80, a contaminant that caused
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264 Some years later, in 2018, Ge and colleagues described that a glucose/galactose transporter of
265 H. pylori, Hp1174, functions as an efflux pump and is highly expressed in biofilm-forming and
266 MDR H. pylori strains. This transporter, encoded by the gluP gene, is upregulated by SpoT (78).
267 A H. pylori mutant lacking gluP gene and its product Hp1174 constituted an unstructured
268 biofilm whose matrix was damaged. As this study revealed that SpoT enzyme upregulates
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299 absence of oxygen. For both strains, they observed a 100-fold increase of ampicillin survival in
300 absence of oxygen compared to the strain under aerobic conditions. This study concludes that
301 the damage that ROS cause in the DNA was regulated by cAMP, a negative regulator of
302 persistence in uropathogenic E. coli (82).
303 5. PRDP: (p)ppGpp ribosome dimerization persister model
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334 that there are currently two approaches: (i) inhibition of (p)ppGpp synthetases by using
335 (p)ppGpp analogs and (ii) inhibition of (p)ppGpp accumulation by using protein inhibitors (90).
336 i. Inhibition of (p)ppGpp synthetases by using (p)ppGpp analogs
337 Several in vitro studies using double relA spoT null mutants in E. coli have shown that this
338 bacterium lacks the (p)ppGpp and therefore has significantly reduced persistence to
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369 1018 marks (p)ppGpp for degradation, exhibiting potent activity against biofilms produced by
370 Gram-positive (S. aureus) and Gram-negative bacteria (E. coli, P. aeruginosa, K. pneumoniae or
371 A. baumannii), but not in planktonic cultures (94). They also observed that the 1018 peptide
372 prevented the biofilm formation and degraded the pre-formed biofilm (as old as 2 days). As an
373 overproduction of (p)ppGpp leads to resistance to the 1018 peptide, the authors suggested
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404 conserved network in many phyla, and all microbes use it to protect themselves against
405 different types of stress.
406 The relevance of the role of the ATP in the antibiotic persistence is revealed as tolerant cells
407 slow down their metabolism and persistent cells are quiescent. Pontes and Groisman (2019)
408 showed that Salmonella pre-exposed to chloramphenicol resisted the killing by bactericidal
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438 ribosomes. In this new model, called PRDP and proposed by Song and colleagues in 2020,
439 there is evidence of a direct role of the magic spot in the persistent phenotype (83).
440 An interesting issue is the individual variability within a population of cells regarding their
441 tolerance to antibiotics. Whether this heterogeneity is regulated or, on the contrary, is an
442 unavoidable consequence of stochastic fluctuations, remains unknown. In 2004, Balaban and
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473 agents as polymyxins can destabilize the outer membrane in Gram negatives facilitating the
474 entrance of therapeutic molecules, e. g., relacin, or endolysins (108). The absence of known
475 (p)ppGpp synthetases in mammalian cells and the specificity of these inhibitors for the Rel
476 protein, make this protein a good candidate as an antibacterial agent.
477 The second strategy of inhibition of (p)ppGpp accumulation, supported by the 1018 and 1037
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508 Legends
509 Figure 1. Physiological pathways regulated by (p)ppGpp and the stringent response in E. coli.
510 Some of the most common human opportunistic pathogens are represented, such as
511 biofilm-forming P. aeruginosa, intestinal E. coli and skin-borne opportunistic pathogen
512 S. aureus. Focusing on E. coli: 1) increased levels of (p)ppGpp induce transcription of
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541 Acknowledgements
542 This study was funded by grant PI16/01163 and PI19/00878 awarded to M. Tomás within the
543 State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological
544 Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General
545 Directorate for Evaluation and Promotion of Research - European Regional Development Fund
554 O.P., L.B., I.B., L.F-G., A.A., M.L., wrote the manuscript; G.B., R.C., R.G-C., T.K.W., M.T.,
555 supervised and revised the manuscript.
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Year Journal Author Model References
2014 Cell Maisonneuve E. and Gerdes (p)ppGpp induces persistence by activating TA loci via (42, 71-73)*
K. PolyP and Lon protease in E. coli K-12 strain MG1655.
(3, 65, 109)
2016 Scientific Reports Chowdhury N., Kwan B.W, The formation of persister cells in E. coli K-12 strain (75)
and Wood T.K. MG1655 is attributed to production of any toxic protein
(e.g., MazF, RelB and YafO) and (p)ppGpp is not
essential but increases persistence by 1000X.
2019 Science Signalling Pontes M.H. and Groismann Low cytoplasmatic Mg2+ induces S. typhimurium (49)
E.A. tolerance to antibiotic independently of (p)ppGpp and
TA modules. However, (p)ppGpp reduces antibiotic
tolerance under certain conditions.
2020 Biochemical and Song S. and Wood T.K. (p)ppGpp generates persister cells directly by (83)
Biophysical Research inactivation of ribosomes via Rmf and Hpf.
Communication
Table 1.
Rmf: ribosome modulation factor, Hpf: hibernation promoting factor. *Some of these publications have been retracted due to the contamination with the
Φ80 bacteriophage causing artefacts in the results
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Year Author Strategy Mechanism of action References
2012 Wexselblatt and (p)ppGpp analogs inhibit Rel Relacin produced death of B. subtilis with an IC50=200 (91)
colleagues protein: relacin μM, prevention of sporulation and biofilm formation
and induction of a prolonged exponential phase.
2012 De la Fuente-Núñez and Inhibition of (p)ppGpp 1037 reduced expression of flagella-associated genes (95)
colleagues accumulation: 1037 peptide that favorize biofilm establishment in P. aeruginosa and
B. cenocepacia. It also reduced the swarming motility.
2014 De la Fuente-Núñez and Inhibition of (p)ppGpp 1018 marked (p)ppGpp for degradation*: broad anti- (94)
colleagues accumulation: 1018 peptide biofilm activity against Gram positive and negative
bacteria and lack of effect for planktonic cultures.
2017 Syal and colleagues (p)ppGpp analog to inhibit Impairing of biofilm formation by M. smegmatis and (92)
Rel protein: modification of arising of elongated cells. Lack of toxicity, good
relacin permeability to human lung epithelial cells.
2019 Dutta and colleagues (p)ppGpp analog to inhibit Highest inhibitory activity against Rel protein: IC50 of (93)
Rel protein: compound X9 ∼15 μM against purified Rel of M. tuberculosis.
Enhancement of susceptibility against isoniazid.
862
863 Table 2.
864 *Andresen and colleagues rejected this hypothesis two years later, questioning its specificity for (p)ppGpp and for biofilm-forming cells (96).
865
866
867
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Downloaded from http://aac.asm.org/ on July 28, 2020 at EAST CAROLINA UNIV