Detection of prokaryotic mRNA signifies microbial viability and promotes immunity

  • A Corrigendum to this article was published on 21 September 2011

Abstract

Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts1,2,3,4,5,6. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts7. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system8,9, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Sensing bacterial viability induces IFN-β and activates the NLRP3 inflammasome in the absence of virulence factors.
Figure 2: The TLR signalling adaptor TRIF controls ‘viability-induced’ responses.
Figure 3: Bacterial RNA is a vita-PAMP that accesses cytosolic receptors during phagocytosis and in the absence of virulence factors.
Figure 4: Bacterial mRNA constitutes an active vita-PAMP.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Affymetrix Microarray data have been deposited with the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE27960.

References

  1. 1

    Brockstedt, D. G. et al. Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity. Nature Med. 11, 853–860 (2005)

    CAS  Article  Google Scholar 

  2. 2

    Cheers, C. & Zhan, Y. How do macrophages distinguish the living from the dead? Trends Microbiol. 4, 453–455 (1996)

    CAS  Article  Google Scholar 

  3. 3

    Detmer, A. & Glenting, J. Live bacterial vaccines—a review and identification of potential hazards. Microb. Cell Fact. 5, 23 (2006)

    Article  Google Scholar 

  4. 4

    Kawamura, I. et al. Antigen provoking gamma interferon production in response to Mycobacterium bovis BCG and functional difference in T-cell responses to this antigen between viable and killed BCG-immunized mice. Infect. Immun. 62, 4396–4403 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Lauvau, G. et al. Priming of memory but not effector CD8 T cells by a killed bacterial vaccine. Science 294, 1735–1739 (2001)

    CAS  Article  ADS  Google Scholar 

  6. 6

    von Koenig, C. H., Finger, H. & Hof, H. Failure of killed Listeria monocytogenes vaccine to produce protective immunity. Nature 297, 233–234 (1982)

    CAS  Article  ADS  Google Scholar 

  7. 7

    Vance, R. E., Isberg, R. R. & Portnoy, D. A. Patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system. Cell Host Microbe 6, 10–21 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Medzhitov, R. Approaching the asymptote: 20 years later. Immunity 30, 766–775 (2009)

    CAS  Article  Google Scholar 

  9. 9

    Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805–820 (2010)

    CAS  Article  Google Scholar 

  10. 10

    Mariathasan, S. & Monack, D. M. Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation. Nature Rev. Immunol. 7, 31–40 (2007)

    CAS  Article  Google Scholar 

  11. 11

    Schroder, K. & Tschopp, J. The inflammasomes. Cell 140, 821–832 (2010)

    CAS  Article  Google Scholar 

  12. 12

    Wing, H. J., Yan, A. W., Goldman, S. R. & Goldberg, M. B. Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB. J. Bacteriol. 186, 699–705 (2004)

    CAS  Article  Google Scholar 

  13. 13

    Zhou, R., Yazdi, A. S., Menu, P. & Tschopp, J. A role for mitochondria in NLRP3 inflammasome activation. Nature 469, 221–225 (2011)

    CAS  Article  ADS  Google Scholar 

  14. 14

    Pang, I. K. & Iwasaki, A. Inflammasomes as mediators of immunity against influenza virus. Trends Immunol. 32, 34–41 (2011)

    CAS  Article  Google Scholar 

  15. 15

    Kanneganti, T. D. et al. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440, 233–236 (2006)

    CAS  Article  ADS  Google Scholar 

  16. 16

    Davis, M. J. & Swanson, J. A. Technical advance: Caspase-1 activation and IL-1β release correlate with the degree of lysosome damage, as illustrated by a novel imaging method to quantify phagolysosome damage. J. Leukoc. Biol. 88, 813–822 (2010)

    CAS  Article  Google Scholar 

  17. 17

    Hornung, V. et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nature Immunol. 9, 847–856 (2008)

    CAS  Article  Google Scholar 

  18. 18

    Herskovits, A. A., Auerbuch, V. & Portnoy, D. A. Bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system. PLoS Pathog. 3, e51 (2007)

    Article  Google Scholar 

  19. 19

    Shimada, T. et al. Staphylococcus aureus evades lysozyme-based peptidoglycan digestion that links phagocytosis, inflammasome activation, and IL-1β secretion. Cell Host Microbe 7, 38–49 (2010)

    CAS  Article  Google Scholar 

  20. 20

    Piekna-Przybylska, D., Decatur, W. A. & Fournier, M. J. The 3D rRNA modification maps database: with interactive tools for ribosome analysis. Nucleic Acids Res. 36, D178–D183 (2008)

    CAS  Article  Google Scholar 

  21. 21

    Buchmeier, N. A. & Heffron, F. Induction of Salmonella stress proteins upon infection of macrophages. Science 248, 730–732 (1990)

    CAS  Article  ADS  Google Scholar 

  22. 22

    Belasco, J. G. All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay. Nature Rev. Mol. Cell Biol. 11, 467–478 (2010)

    CAS  Article  Google Scholar 

  23. 23

    Rehwinkel, J. & Reis e Sousa, C. RIGorous detection: exposing virus through RNA sensing. Science 327, 284–286 (2010)

    CAS  Article  ADS  Google Scholar 

  24. 24

    Monroe, K. M., McWhirter, S. M. & Vance, R. E. Identification of host cytosolic sensors and bacterial factors regulating the type I interferon response to Legionella pneumophila. PLoS Pathog. 5, e1000665 (2009)

    Article  Google Scholar 

  25. 25

    Nallagatla, S. R., Toroney, R. & Bevilacqua, P. C. A brilliant disguise for self RNA: 5′-end and internal modifications of primary transcripts suppress elements of innate immunity. RNA Biol. 5, 140–144 (2008)

    CAS  Article  Google Scholar 

  26. 26

    Anderson, B. R. et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38, 5884–5892 (2010)

    CAS  Article  Google Scholar 

  27. 27

    Kariko, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Woodward, J. J., Iavarone, A. T. & Portnoy, D. A. c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science 328, 1703–1705 (2010)

    CAS  Article  ADS  Google Scholar 

  29. 29

    Gripenland, J. et al. RNAs: regulators of bacterial virulence. Nature Rev. Microbiol. 8, 857–866 (2010)

    CAS  Article  Google Scholar 

  30. 30

    Raskin, D. M., Seshadri, R., Pukatzki, S. U. & Mekalanos, J. J. Bacterial genomics and pathogen evolution. Cell 124, 703–714 (2006)

    CAS  Article  Google Scholar 

  31. 31

    Torchinsky, M. B., Garaude, J., Martin, A. P. & Blander, J. M. Innate immune recognition of infected apoptotic cells directs TH17 cell differentiation. Nature 458, 78–82 (2009)

    CAS  Article  ADS  Google Scholar 

  32. 32

    Blander, J. M. & Medzhitov, R. Regulation of phagosome maturation by signals from toll-like receptors. Science 304, 1014–1018 (2004)

    CAS  Article  ADS  Google Scholar 

  33. 33

    Sutterwala, F. S. et al. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24, 317–327 (2006)

    CAS  Article  Google Scholar 

  34. 34

    Kuida, K. et al. Altered cytokine export and apoptosis in mice deficient in interleukin-1β converting enzyme. Science 267, 2000–2003 (1995)

    CAS  Article  ADS  Google Scholar 

  35. 35

    Sato, M. et al. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-α/β gene induction. Immunity 13, 539–548 (2000)

    CAS  Article  Google Scholar 

  36. 36

    Maurelli, A. T., Baudry, B., d’Hauteville, H., Hale, T. L. & Sansonetti, P. J. Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. Infect. Immun. 49, 164–171 (1985)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Haraga, A., Ohlson, M. B. & Miller, S. I. Salmonellae interplay with host cells. Nature Rev. Microbiol. 6, 53–66 (2008)

    CAS  Article  Google Scholar 

  38. 38

    Schnupf, P. & Portnoy, D. A. Listeriolysin O: a phagosome-specific lysin. Microbes Infect. 9, 1176–1187 (2007)

    CAS  Article  Google Scholar 

  39. 39

    Gentleman, R. C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004)

    Article  Google Scholar 

  40. 40

    Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193 (2003)

    CAS  Article  Google Scholar 

  41. 41

    Irizarry, R. A. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31, e15 (2003)

    Article  Google Scholar 

  42. 42

    Dai, M. et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 33, e175 (2005)

    Article  Google Scholar 

  43. 43

    Saeed, A. I. et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374–378 (2003)

    CAS  Article  Google Scholar 

  44. 44

    Saeed, A. I. et al. TM4 microarray software suite. Methods Enzymol. 411, 134–193 (2006)

    CAS  Article  Google Scholar 

  45. 45

    Sartor, M. A. et al. Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments. BMC Bioinformatics 7, 538 (2006)

    Article  Google Scholar 

  46. 46

    Storey, J. D. & Tibshirani, R. Statistical significance for genomewide studies. Proc. Natl Acad. Sci. USA 100, 9440–9445 (2003)

    MathSciNet  CAS  Article  ADS  Google Scholar 

  47. 47

    Samarajiwa, S. A., Forster, S., Auchettl, K. & Hertzog, P. J. INTERFEROME: the database of interferon regulated genes. Nucleic Acids Res. 37, D852–D857 (2009)

    CAS  Article  Google Scholar 

  48. 48

    Pahl, H. L. Activators and target genes of Rel/NF-κB transcription factors. Oncogene 18, 6853–6866 (1999)

    CAS  Article  Google Scholar 

  49. 49

    Coll, R. C. & O’Neill, L. A. New insights into the regulation of signalling by toll-like receptors and nod-like receptors. J. Innate Immun. 2, 406–421 (2010)

    CAS  Article  Google Scholar 

  50. 50

    Lim, S. Y., Bauermeister, A., Kjonaas, R. A. & Ghosh, S. K. Phytol-based novel adjuvants in vaccine formulation: 2. Assessment of efficacy in the induction of protective immune responses to lethal bacterial infections in mice. J. Immune Based Ther. Vaccines 4, 5 (2006)

    Article  Google Scholar 

  51. 51

    Hornung, V. et al. 5′-Triphosphate RNA is the ligand for RIG-I. Science 314, 994–997 (2006)

    Article  ADS  Google Scholar 

Download references

Acknowledgements

We are grateful to R. Medzhitov and J. C. Kagan for critical reading of the manuscript; C. B. Lopez for Irf3−/− mice; D. M. Monack for Salmonella ΔSpi1ΔSpi2; M. B. Goldberg for Shigella BS103; and D. A. Portnoy for Listeria ΔHlyΔfliC. We thank M. Rivieccio, I. Brodsky, M. Blander, S. J. Blander, J. Sander and Blander laboratory members for insightful discussions, help and support. L.E.S. was supported by Deutsche Forschungsgemeinschaft grant SA-1940/1-1, D.A. by fellowships from the Academic Medical Center and the Landsteiner Foundation for Blood Research, and M.V.B. and M.M. by the Netherlands Nutrigenomics Centre. This work was supported by NIH grant AI080959A and the Kinship Foundation Searle Scholar award to J.M.B.

Author information

Affiliations

Authors

Contributions

L.E.S. and J.M.B. designed experiments and directed the study. L.E.S. performed all experiments. M.J.D. and L.E.S. performed experiments measuring lysosomal leakage. J.A.S. helped with the design and analysis of the lysosomal leakage experiments. M.V.B. performed gene microarray analysis. M.V.B. and M.M. analysed the gene microarray data and helped with data interpretation. D.A. and J.M.B. performed experiments during the development phase of the project, and C.C.D. helped with the design of RNA-related experiments. B.R. provided bone marrow progenitor cells from Nlrp3−/−, Asc−/− and Casp1−/− mice. L.E.S., D.A. and J.M.B. wrote the manuscript. J.M.B. conceived of the study.

Corresponding author

Correspondence to J. Magarian Blander.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-22 with legends. (PDF 15060 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sander, L., Davis, M., Boekschoten, M. et al. Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. Nature 474, 385–389 (2011). https://doi.org/10.1038/nature10072

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing