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N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes

Nature Microbiology volume 2, Article number: 17005 (2017) Cite this article

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An Author Correction to this article was published on 25 June 2018

Abstract

To adapt to changing environments, bacteria have evolved numerous pathways that activate stress response genes. In Gram-positive bacteria, the stressosome, a cytoplasmic complex, relays external cues and activates the sigma B regulon. The stressosome is structurally well-characterized in Bacillus, but how it senses stress remains elusive. Here, we report a genome-wide N-terminomic approach in Listeria that strikingly led to the discovery of 19 internal translation initiation sites and 6 miniproteins, among which one, Prli42, is conserved in Firmicutes. Prli42 is membrane-anchored and interacts with orthologues of Bacillus stressosome components. We reconstituted the Listeria stressosome in vitro and visualized its supramolecular structure by electron microscopy. Analysis of a series of Prli42 mutants demonstrated that Prli42 is important for sigma B activation, bacterial growth following oxidative stress and for survival in macrophages. Taken together, our N-terminonic approach unveiled Prli42 as a long-sought link between stress and the stressosome.

Listeria monocytogenes is a human pathogen that has emerged as a model organism in infection biology1. The genome of L. monocytogenes strain EGD-e was sequenced in 2001 and predicted to encode 2846 open reading frames (ORFs)2. Tiling array-based transcriptomic analyses then explored bacterial transcription3,4, and RNA-sequencing was used to generate genome-wide transcription start (TSS) and termination site (TTS) maps5,6, making this model bacterium ideally suited for further exploration of the regulation of gene expression, at the level of translation. Ribosome profiling in Escherichia coli and Bacillus subtilis recently revealed a wealth of information about translational regulation, including translational pausing at Shine–Dalgarno (SD)-like sequences7. While pause sites can be differentiated from internal translation initiation sites (TISs) in mammalian cells using pulse-chase experiments8, it has been challenging to do so with precision in bacteria9,10. In contrast, N-terminal proteomics-based approaches can unambiguously detect bacterial TISs on a genome-wide scale. N-terminal COFRADIC (combined fractional diagonal chromatography) is one such method that isolates N-terminal peptides via two sequential chromatographic separations11–14. Interestingly, in prokaryotes, N-terminal peptides are formylated and this modification can be exploited to identify TISs (ref. 15).

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Figure 1: A proteogenomic approach to map translation initiation sites (TISs).
Figure 2: An integrated map of Listeria TISs and deviations from the current genome annotation.
Figure 3: Miniprotein Prli42 is highly conserved and interacts with Listeria orthologues of the stressosome.
Figure 4: Membrane miniprotein Prli42 partially tethers RsbR to the bacterial membrane and is essential for the sigma B signalling following oxidative stress and survival in macrophages.

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Acknowledgements

This work was supported by grants to P.C. (European Research Council (ERC) Advanced Grant BacCellEpi (670823), ANR BACNET (BACNET 10-BINF-02-01), ANR Investissement d'Avenir Programme (10-LABX-62-IBEID), Human Frontier Science Program (HFSP; RGP001/2013), ERANET Infect-ERA PROANTILIS (ANR-13-IFEC-0004-02) and the Fondation le Roch les Mousquetaires) and by a grant to M.G.P from the Spanish Ministry of Economy and Competitiveness (BIO2014-55238-R). The authors thank E. Gouin and L. Maranghi for essential technical support, C. O'Byrne and J. Johansson for discussions, C. Thireau for technical support, the Pasteur Ultrapole and C. Rapisarda for help with the EM. The authors thank T. Msadek for providing the L. monocytogenes LO28 ΔclpB strain and the Pasteur Proteomics platform, in particular M. Matondo-Bouzanda and T. Chaze. F.I. received financial support from a Pasteur-Roux Fellowship. L.R. was supported by an HFSP long-term fellowship. A.H.W. was supported by an EMBO long-term fellowship (ALTF 732-2010) and an Institut Carnot–Pasteur Maladies Infectieuses fellowship. P.C. is a Senior International Research Scholar of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Francis Impens

    Present address: †Present address: Medical Biotechnology Center, VIB, Ghent University, 9000 Ghent, Belgium,

  2. Francis Impens, Nathalie Rolhion and Lilliana Radoshevich: These authors contributed equally to this work.

Authors and Affiliations

  1. Département de Biologie Cellulaire et Infection, Institut Pasteur, Unité des Interactions Bactéries-Cellules, F-75015 Paris, France

    Francis Impens, Nathalie Rolhion, Lilliana Radoshevich, Christophe Bécavin, Mélodie Duval, Jeffrey Mellin & Pascale Cossart

  2. Inserm, U604, F-75015 Paris, France

    Francis Impens, Nathalie Rolhion, Lilliana Radoshevich, Christophe Bécavin, Mélodie Duval, Jeffrey Mellin & Pascale Cossart

  3. INRA, Unité sous-contrat 2020, F-75015 Paris, France

    Francis Impens, Nathalie Rolhion, Lilliana Radoshevich, Christophe Bécavin, Mélodie Duval, Jeffrey Mellin & Pascale Cossart

  4. Institut Pasteur, Bioinformatics and Biostatistics Hub, C3BI, USR 3756 IP CNRS, Paris, France

    Christophe Bécavin

  5. Centro Nacional de Biotecnología–Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain

    Francisco García del Portillo & M. Graciela Pucciarelli

  6. Departamento de Biología Molecular, Universidad Autónoma de Madrid, Centro de Biología Molecular ‘Severo Ochoa’ (CBMSO-CSIC), Madrid, Spain

    M. Graciela Pucciarelli

  7. Département de Microbiologie, Institut Pasteur, Unité des Biologie et génétique de la paroi bactérienne, F-75015 Paris, France

    Allison H. Williams

  8. INSERM, Groupe Avenir, F-75015 Paris, France

    Allison H. Williams

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Contributions

P.C. initiated, conceived and supervised the project. F.I. initiated the project and performed the proteomics analysis and validation of the proteomics work and docking model. N.R. identified the oxidative stress phenotype, constructed nearly all the bacterial strains and performed the analysis of sigma B signalling. L.R. performed the macrophage experiments, the fractionation experiments and the virulence experiments. C.B. made the proteogenomics pipeline and is responsible for the bioinformatic analysis of the paper. M.D. performed the northern blots of Sigma B signalling. J.M. constructed the initial bacterial strains for validation. F.G.d.P. and M.G.P. contributed essential reagents. A.H.W. reconstituted the stressosome and imaged it using EM, and performed the docking model and all of the structural biology. L.R. and P.C. wrote the paper, with editing help and discussions from N.R., M.D., F.I. and A.H.W.

Corresponding author

Correspondence to Pascale Cossart.

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Supplementary information

Supplementary Information

Supplementary Figures 1-8, Legends for Supplementary Tables 1–13. (PDF 15010 kb)

Supplementary Table 1

List of predicted undetectable aTIS in L. monocytogenes EGD-e. (XLSX 457 kb)

Supplementary Table 2

List of 1322 L. monocytogenes EGD-e proteins with detected aTIS. (XLSX 199 kb)

Supplementary Table 3

List of 72 L. monocytogenes EGD-e proteins with leaderless mRNAs. (XLSX 14 kb)

Supplementary Table 4

List of 27 L. monocytogenes EGD-e proteins with multiple TIS. (XLSX 84 kb)

Supplementary Table 5

List of 25 L. monocytogenes EGD-e proteins with a corrected TIS. (XLSX 69 kb)

Supplementary Table 6

List of 2 mis-annotated and 2 missing L. monocytogenes EGD-e proteins. (XLSX 59 kb)

Supplementary Table 7

List of 19 L. monocytogenes EGD-e proteins with detected internal TIS. (XLSX 90 kb)

Supplementary Table 8

List of 6 newly identified miniproteins in L. monocytogenes EGD-e. (XLSX 62 kb)

Supplementary Table 9

Results of the homologue search of Rli42 and the stressosome. (XLSX 533 kb)

Supplementary Table 10

List of L. monocytogenes EGD-e proteins identified and quantified by LCMS-MS after co-immunoprecipitation of Prli42-flag or Prli42-R8A-flag. (XLSX 140 kb)

Supplementary Table 11

Results of the RAST re-annotation of the L. monocytogenes EGD-e genome and comparison with the original annotation by Glaser et al. (referred to as NCBI). (XLSX 410 kb)

Supplementary Table 12

Strains, plasmids and primers used in this study. (XLSX 12 kb)

Supplementary Table 13

Spectral counts and peptide numbers for the different datasets. (XLSX 10 kb)

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Impens, F., Rolhion, N., Radoshevich, L. et al. N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes. Nat Microbiol 2, 17005 (2017). https://doi.org/10.1038/nmicrobiol.2017.5

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