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RNA antitoxin SprF1 binds ribosomes to attenuate translation and promote persister cell formation in Staphylococcus aureus

Abstract

Persister cells are a subpopulation of transiently antibiotic-tolerant bacteria associated with chronic infection and antibiotic treatment failure. Toxin–antitoxin systems have been linked to persister cell formation but the molecular mechanisms leading to bacterial persistence are mostly unknown. Here, we show that SprF1, a type I antitoxin, associates with translating ribosomes from the major human pathogen Staphylococcus aureus to reduce the pathogen’s overall protein synthesis during growth. Under hyperosmotic stress, SprF1 levels increase due to enhanced stability, accumulate on polysomes and attenuate protein synthesis. Using an internal 6-nucleotide sequence on its 5′-end, SprF1 binds ribosomes and interferes with initiator transfer RNA binding, thus reducing translation initiation. An excess of messenger RNA displaces the ribosome-bound antitoxin, freeing the ribosomes for new translation cycles; however, this RNA antitoxin can also displace ribosome-bound mRNA. This translation attenuation mechanism, mediated by an RNA antitoxin, promotes antibiotic persister cell formation. The untranslated SprF1 is a dual-function RNA antitoxin that represses toxin expression by its 3′-end and fine-tunes overall bacterial translation via its 5′-end. These findings demonstrate a general function for a bacterial RNA antitoxin beyond protection from toxicity. They also highlight an RNA-guided molecular process that influences antibiotic persister cell formation.

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Fig. 1: The SprF1 antitoxin attenuates global translation and associates with polysomes and 70S ribosomes in S. aureus.
Fig. 2: Osmotic-induced SprF1 accumulation attenuates S. aureus protein synthesis.
Fig. 3: The SprF1 antitoxin solution structure.
Fig. 4: A purine-rich sequence at the SprF1 5′-end interacts with S. aureus ribosomes and attenuates translation initiation.
Fig. 5: The SprF1 antitoxin increases the levels of antibiotic-tolerant persisters in S. aureus.
Fig. 6: The dual-function RNA antitoxin SprF1 represses toxin expression via its 3′-end and modulates translation via its 5′-end during S. aureus growth.

Data availability

Full-length blots or gels are provided as Supplementary Information. Additional details on the datasets and protocols that support the findings of this study will be made available by the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Gebetsberger and M. Żywicki for their contributions to the initial phase of the project and J. Berland for help with editing. We also thank C. Gonzalez and M. Hallier for the extracellular protein extracts from the S. aureus strain overexpressing δ-hemolysin. This work was funded by the Agence Nationale pour la Recherche (grant no. ANR-15-CE12-0003-01 ‘sRNA-Fit’ to B.F.), the Fondation pour la Recherche Médicale (grant no. DBF20160635724 ‘Bactéries et champignons face aux antibiotiques et antifongiques’ to B.F.), the universities of Rennes and Sherbrooke and by the Institut National de la Santé et de la Recherche Médicale and the School of Pharmacy and Medical Sciences at Rennes University. Additional funding was provided by the Swiss National Science Foundation (grant nos. 31003A_143388/1 and 31003A_166527 to N.P.)

Author information

Authors and Affiliations

Authors

Contributions

M.-L.P.-M. supervised the project, contributed to the design of the experiments, conducted most of the experiments and their analysis and wrote the manuscript. R.B. designed and conducted the polysome enrichment and persistence experiments, analysed their results and wrote the manuscript. C.R. performed the RT–qPCR analysis shown in Fig. 2a,b, the northern blot analysis shown in Fig. 4a, construction of the pALCΩsprF1-FLAG plasmid, the persistence experiments and interpreted the data. N.G.-A. performed the half-life determination assays, toeprint assays, polysome enrichment, the persistence experiments and contributed to the initial phase of this project. N.P. contributed to the initial phase of this project. B.F. supervised the project, contributed to the design of the experiments, analysed the data and wrote the manuscript.

Corresponding authors

Correspondence to Marie-Laure Pinel-Marie or Brice Felden.

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Competing interests

The authors have filed a patent application (European Patent application no. EP20306315.1) based in part on results presented in this manuscript.

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Extended data

Extended Data Fig. 1 In S. aureus, SprF1 inhibits global translation in vivo but not transcription.

The S. aureus strains N315_pCN35 (WT), N315ΔsprG1/ΔsprF1_pCN35 (ΔΔ), N315ΔsprG1/ΔsprF1_pCN35ΩsprF1 (ΔΔ + SprF1), N315ΔsprG1/ΔsprF1_pCN35ΩsprG1/sprF1 (ΔΔ + SprF1 + SprG1) were cultivated in diluted TSB medium until the exponential growth phase. a, After RNA extraction, northern blot analysis was done on SprG1 and SprF1 expression, with 5 S rRNA used as the loading control (n = 3). b, Growth kinetics of the four S. aureus strains. Error bars show the means and standard deviations of three independent experiments (n = 3). c, The S. aureus strain N315_pCN35 (WT) was cultivated 1 h at 37 °C and incubated with [3H]-uridine in the absence or presence of 20 µg/mL rifampicin or streptomycin over 1 h at 37 °C. The incorporation of the [3H]-uridine into the transcriptome was determined after TCA precipitation and filtration of S. aureus lysates. The incorporation was quantified and is shown compared to the radiolabelled RNAs of the untreated cells (Control), arbitrarily set to 1 (n = 3). d, The strains were incubated for 3 h at 37 °C with [35S]-methionine, with 10 µg/mL chloramphenicol (CHL) used as the positive control. Shown are the results of a representative SDS-PAGE of the newly synthesized radiolabeled proteins, with Coomassie blue staining as the loading control (n = 3). Graph bars represent the mean ± SD and black dots represent individual data points for three independent experiments. Statistical significance was calculated with a two-tailed unpaired t-test and p value are indicated: ***, p < 0.001. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 2 SprF1 does not affect in vitro transcription, but it slows down in vitro translation.

a, Effect of SprF1 on in vitro transcription of sarA mRNA. E. coli RNA polymerase was incubated for 30 min at 37 °C with 10 fmol sarA PCR product, 50 fmol SprF1, 20 µg/ml rifampicin (Rif) used as the positive control, or 20 µg/ml streptomycin (Str) used as the negative control. Shown is a representative urea-PAGE of the radiolabelled sarA mRNA in the absence (-) or presence of SprF1, rifampicin, or streptomycin (n = 3). Quantification of sarA mRNA transcription is expressed as mean fold changes relative to the sarA mRNA transcription found for the control (-), arbitrarily set to 1. b, Effects of SprF1 on in vitro translation of SarA in E. coli cell-free extracts. Shown is a representative SDS PAGE of the radiolabelled SarA in the absence (-) or presence of 4 μM SprF1, with Coomassie blue staining as a loading control (n = 3). Quantification of SarA translation is expressed as mean fold changes relative to the SarA translation found in absence of SprF1, arbitrarily set to 1. a-b, Graph bars represent the mean ± SD and black dots represent individual data points for three independent experiments. Statistical significance was calculated with a two-tailed unpaired t-test and p value are indicated: *, p < 0.05; ****, p < 0.0001. c, Dose-response relationship of SprF1 effects on in vitro translations of luciferase and β-lactamase in E. coli cell-free extracts using the pBESTluc vector. Shown is a representative SDS-PAGE of the radiolabelled luciferase and β-lactamase proteins in the absence (-) or presence of 1-5 μM SprF1, 4 µM SprB (negative control), or 10 µM chloramphenicol (CHL, the positive control) (n = 3). Source data are provided as a Source Data file.

Source data

Extended Data Fig. 3 SprG1 does not affect translation.

a-b, Dose-response relationship of SprG1 effects on in vitro translations of luciferase and β-lactamase in E. coli cell-free extracts using the pBESTluc vector. a, Representative SDS-PAGE of the radiolabelled luciferase and β-lactamase proteins in the absence (-) or presence of 1-5 μM SprG1 (n = 2). b, The amounts of radiolabelled proteins in the presence of 1-5 μM SprG1 are shown as percentages of the control, radiolabelled proteins without SprG1, arbitrarily set to 1. The results are expressed as the mean of two independent experiments (n = 2). Source data are provided as a Source Data file.

Source data

Extended Data Fig. 4 SprF1 associates with the S. aureus polysomes and ribosomes.

a, FPLC profile of ribosomal fraction extracts from S. aureus N315 grown in diluted TSB medium until the exponential growth phase (OD600 = 1). Sucrose gradient separation was done to analyse cell extracts on 18-40% linear sucrose density gradients, and the absorption profiles were measured at 254 nm. The graph corresponds to one of three ribosomal fractions performed (n = 3). Each fraction indicated in the profile was pooled, rinsed, concentrated, and then analysed by agarose gel electrophoresis stained with ethidium bromide (inset). There, 23 S rRNA represents 50 S subunit enrichment, and 16 S rRNA represents 30 S subunit enrichment. b, Northern blot analysis to monitor the presence of SprF1, SprG1 and 5 S rRNA in the ribosomal fractions purified from N315 (n = 3). c, S. aureus strains N315_pCN35 (WT), N315ΔsprG1/ΔsprF1_pCN35 (ΔΔ), N315ΔsprG1/ΔsprF1_pCN35ΩsprF1 (ΔΔ + SprF1), were cultivated in diluted TSB medium until the exponential growth phase. Sucrose gradient separation was done to analyse cell extracts on 18-40% linear sucrose density gradients, and the absorption profiles were measured at 254 nm. The area of each ribosomal fraction from the polysome profiles were quantified (n = 3). d, Filter-binding assays of radiolabelled synthetic RNAs (SprF1, SprG1, SprB, canonical mRNA, or RNAIII) on purified S. aureus 70 S ribosomes. Signals measured in the absence of ribosomal particles (-) were subtracted from the experimental values (n = 3). e, In vitro association of SprF1 with purified S. aureus 70 S, 50 S, and 30 S ribosomal particles. Binding quantification is shown compared to SprF1 association with 70 S, arbitrarily normalized to 1. Signals measured without ribosomal particles were subtracted from the experimental values (n = 3). f, Scatchard analysis of the binding of SprF1 to S. aureus 70 S ribosomes using filter-binding assays, with determination of the dissociation constants (Kd). Error bars show the means and SD of three independent experiments (n = 3). Graph bars represent the mean ± SD and black dots represent individual data points for three independent experiments. Statistical significance was calculated with a two-tailed unpaired t-test and p value are indicated: **, p < 0.01. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 5 SprF1 is a non-coding RNA.

a, Growth kinetics of S. aureus N315 strains carrying pALC, pALCΩsprF1-Flag, or pALCΩsprG1-Flag in presence (right panel) or absence (left panel) of 1 µM anhydrotetracyline (aTc). Error bars show the mean and standard deviation of three independent experiments (n = 3). b, Northern blot analysis of SprF1- and SprG1-flagged RNAs expression levels in S. aureus N315 strains carrying pALC, pALCΩsprF1-Flag, or pALCΩsprG1-Flag after induction by 1 µM anhydrotetracyline (aTc). 5 S rRNA is the internal loading control (n = 3). Source data are provided as a Source Data file.

Source data

Extended Data Fig. 6 SprF1 inhibits in vivo global translation under osmotic stress in S. aureus.

a, FPLC profile of ribosomal fractions from cell extracts during exponential growth phase of S. aureus N315_pCN35 (WT) with 1 M NaCl, analysed by sucrose gradient separation of cell extracts on 18 to 40% linear sucrose density gradients. The absorption profile was recorded at 254 nm. The graph corresponds to one of three ribosomal fractions performed (n = 3). Each fraction was pooled, rinsed, concentrated, and analysed by agarose gel electrophoresis stained with ethidium bromide. 23 S rRNA refers to the 50 S subunit enrichment, and 16 S rRNA to the 30 S subunit enrichment. b, RT-qPCR analysis showing the presence of SprG1 RNA in ribosomal fractions in S. aureus cultivated in the absence (Control) or in the presence of 1 M NaCl. Data were expressed as percentages and compared to its presence in the total fraction, arbitrarily set to 100% and were the mean ± SD of three independent experiments (n = 3). Using the comparative cycle threshold (ΔΔCt) method, the 50 S ribosomal subunit was normalized to 5 S rRNA, and the 30 S was normalized to 16 S rRNA. c, Growth kinetics of S. aureus strains N315_pCN35 (WT), N315ΔsprG1/ΔsprF1_pCN35 (ΔΔ), N315ΔsprG1/ΔsprF1_pCN35ΩsprF1 (ΔΔ + SprF1), N315ΔsprG1/ΔsprF1 _pCN35ΩsprG1/sprF1 (ΔΔ + SprF1 + SprG1), in diluted TSB medium in the presence of 1 M NaCl. Error bars show the mean and standard deviation of three independent experiments (n = 3). d, The N315_pCN35 (WT) strain was incubated in the absence (Control, CTR) or presence of 1 M NaCl during 3 h at 37 °C with [35S]-methionine. A representative SDS-PAGE of the newly synthesized radiolabelled proteins is shown (n = 4). Coomassie blue staining is used as loading control. Graph bars represent the mean ± SD and black dots represent individual data points for three independent experiments. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 7 Secondary structure predictions of the 5’- and 3’-ends of the SprF1 antitoxin and the SprF1 mutants by RNAfold49.

a, Secondary structure prediction of the 5’-end of SprF1. b, Secondary structure prediction of the 3’-end of SprF1. c, Secondary structure prediction of the SprF1∆23 RNA. d, Secondary structure prediction of the SprF1∆6 RNA.

Extended Data Fig. 8 Toeprint assays of SprF1 antitoxin.

a, Toeprint assays of 0.25 pmol alsS mRNA or SprF1 in the absence (-) or presence (+) of 2 pmol of purified E. coli 70 S ribosomes and 12.5 pmol of uncharged tRNAfMet. The A, C, G and U lanes represent the alsS mRNA or SprF1 sequencing lanes. The arrows represent the toeprint position when observed. The gel illustrates one representative experiment among three independent experiments (n = 3). b, Toeprint assays of 0.25 pmol SprF1 or SprF1∆6 mutant in the absence (-) or presence (+) of 2 pmol of purified E. coli 70 S ribosomes and 12.5 pmol of uncharged tRNAfMet. The A, C, G and U lanes represent the SprF1 sequencing lanes. The arrow represents the toeprint position when observed. The gel illustrates one representative experiment among three independent experiments (n = 3). Source data are provided as a Source Data file.

Source data

Extended Data Fig. 9 Conservation of the SprF1 5′-ribosome binding sequence among the S. aureus phylogeny.

Alignments of 14 sprf1 gene sequences extracted from various S. aureus clonal complexes are shown, with emphasis on sequence conservations and variations (light blue). The 5′ sequence involved in ribosomal binding is bold, and contains a 6-nts purine-rich sequence (yellow) that is required for ribosomal loading. Note that this ribosomal-binding site is missing in ED133, an S. aureus ovine mastitis isolate.

Extended Data Fig. 10 Effect of the expression of the SprF1 RNA deleted for the purin-rich sequence on the S. aureus growth and quantification of SprF1 RNA abundance in S. aureus.

a, The strains N315 ΔsprG1/sprF1ΩpCN35sprF1 (ΔΔ + SprF1), and ΔsprG1/sprF1ΩpCN35sprF1∆6 (ΔΔ + SprF1∆6) were cultivated in diluted TSB medium until the exponential (E) and stationary (S) growth phases. After RNA extraction, northern blot analysis was done on SprF1 and SprF1∆6 RNA expression, with 5 S rRNA used as the loading control. b, S. aureus strains N315_pCN35 (WT), N315ΔsprG1/ΔsprF1_pCN35 (ΔΔ), N315ΔsprG1/ΔsprF1_pCN35ΩsprF1 (ΔΔ + SprF1), N315ΔsprG1/ΔsprF1 _pCN35ΩsprG1/sprF1 (ΔΔ + SprF1 + SprG1), and N315ΔsprG1/ΔsprF1_pCN35ΩsprF1∆6 (ΔΔ + SprF1∆6) were grown in diluted TSB medium. At each time point, samples were taken and plated in order to enumerate cfu (colony forming unit). The results are shown as time-kill curves and expressed as the means of two independent experiments (n = 2). c, S. aureus was cultivated to exponential (E) and stationary (S) growth phases. After RNA extraction, 5 µg total RNA were loaded to a denaturing polyacrylamide gel with 0.001-0.01 pmol SprF1 and 0.00625-0.1 pmol tmRNA transcribed in vitro. SprF1 and tmRNA expressions were analysed by northern blot, with 5 S rRNA expression as the loading control. The signals were quantified to determine the amount of SprF1 per 5 µg total RNA. The mean values from three independent experiments were ~2.109 copies per 5 µg total SprF1 RNA at the exponential growth phase, and ~4.109 at the stationary growth phase. For tmRNA, the mean values were ~3.1010 and ~6.1010 (n = 3). Source data are provided as a Source Data file.

Source data

Supplementary information

Supplementary Information

Supplementary Information reference list.

Reporting Summary

Supplementary Tables 1–4

Supplementary Table 1: Strains and plasmids used in this study. Supplementary Table 2: DNA primers used in this study. Supplementary Table 3: Ciprofloxacin and vancomycin minimal inhibitory concentrations for S. aureus N315 strains. Supplementary Table 4: Determination of the minimum duration for killing (MDK99 and MDK99.99) parameters for ciprofloxacin and vancomycin against S. aureus N315 strains.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 1

Unprocessed western blots and gels.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed northern blots.

Source Data Fig. 3

Unprocessed structural probes gels.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 4

Unprocessed toeprint gels.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 5

Unprocessed northern blots.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed northern blots and gels.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 2

Unprocessed gels.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed gels.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed polysome profile, northern blots, dot blots and gels.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed northern blots.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed polysome profile and gels.

Source Data Extended Data Fig. 8

Unprocessed toeprint gels.

Source Data Extended Data Fig. 10

Unprocessed northern blots.

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Pinel-Marie, ML., Brielle, R., Riffaud, C. et al. RNA antitoxin SprF1 binds ribosomes to attenuate translation and promote persister cell formation in Staphylococcus aureus. Nat Microbiol 6, 209–220 (2021). https://doi.org/10.1038/s41564-020-00819-2

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