Article

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

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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.

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Author information

Author notes

    • Francis Impens

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

    • Francis Impens
    • , Nathalie Rolhion
    •  & Lilliana Radoshevich

    These authors contributed equally to this work.

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Pascale Cossart.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1-8, Legends for Supplementary Tables 1–13.

Excel files

  1. 1.

    Supplementary Table 1

    List of predicted undetectable aTIS in L. monocytogenes EGD-e.

  2. 2.

    Supplementary Table 2

    List of 1322 L. monocytogenes EGD-e proteins with detected aTIS.

  3. 3.

    Supplementary Table 3

    List of 72 L. monocytogenes EGD-e proteins with leaderless mRNAs.

  4. 4.

    Supplementary Table 4

    List of 27 L. monocytogenes EGD-e proteins with multiple TIS.

  5. 5.

    Supplementary Table 5

    List of 25 L. monocytogenes EGD-e proteins with a corrected TIS.

  6. 6.

    Supplementary Table 6

    List of 2 mis-annotated and 2 missing L. monocytogenes EGD-e proteins.

  7. 7.

    Supplementary Table 7

    List of 19 L. monocytogenes EGD-e proteins with detected internal TIS.

  8. 8.

    Supplementary Table 8

    List of 6 newly identified miniproteins in L. monocytogenes EGD-e.

  9. 9.

    Supplementary Table 9

    Results of the homologue search of Rli42 and the stressosome.

  10. 10.

    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.

  11. 11.

    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).

  12. 12.

    Supplementary Table 12

    Strains, plasmids and primers used in this study.

  13. 13.

    Supplementary Table 13

    Spectral counts and peptide numbers for the different datasets.