Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Glutathione activates virulence gene expression of an intracellular pathogen


Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Forward genetic selection to identify factors required for virulence gene activation during infection.
Figure 2: Listeria monocytogenes ΔgshF is attenuated in vivo.
Figure 3: PrfA* bypasses the requirement for glutathione during infection.
Figure 4: Glutathione-dependent PrfA activation is mediated by allosteric binding, not glutathionylation.


  1. 1

    Xayarath, B. & Freitag, N. E. Optimizing the balance between host and environmental survival skills: lessons learned from Listeria monocytogenes . Future Microbiol. 7, 839–852 (2012)

    CAS  Article  Google Scholar 

  2. 2

    Freitag, N. E., Port, G. C. & Miner, M. D. Listeria monocytogenes—from saprophyte to intracellular pathogen. Nature Rev. Microbiol. 7, 623–628 (2009)

    CAS  Article  Google Scholar 

  3. 3

    Chakraborty, T. et al. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J. Bacteriol. 174, 568–574 (1992)

    CAS  Article  Google Scholar 

  4. 4

    de las Heras, A., Cain, R. J., Bielecka, M. K. & Vázquez-Boland, J. A. Regulation of Listeria virulence: PrfA master and commander. Curr. Opin. Microbiol. 14, 118–127 (2011)

    CAS  Article  Google Scholar 

  5. 5

    Moors, M. A., Levitt, B., Youngman, P. & Portnoy, D. A. Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes . Infect. Immun. 67, 131–139 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Shetron-Rama, L. M., Marquis, H., Bouwer, H. G. A. & Freitag, N. E. Intracellular induction of Listeria monocytogenes actA expression. Infect. Immun. 70, 1087–1096 (2002)

    CAS  Article  Google Scholar 

  7. 7

    Gopal, S. et al. A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis. J. Bacteriol. 187, 3839–3847 (2005)

    CAS  Article  Google Scholar 

  8. 8

    Masip, L., Veeravalli, K. & Georgiou, G. The many faces of glutathione in bacteria. Antioxid. Redox Signal. 8, 753–762 (2006)

    CAS  Article  Google Scholar 

  9. 9

    Newton, G. L. et al. Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J. Bacteriol. 178, 1990–1995 (1996)

    CAS  Article  Google Scholar 

  10. 10

    Newton, G. L. et al. Bacillithiol is an antioxidant thiol produced in Bacilli. Nature Chem. Biol. 5, 625–627 (2009)

    CAS  Article  Google Scholar 

  11. 11

    Meister, A. & Anderson, M. E. Glutathione. Annu. Rev. Biochem. 52, 711–760 (1983)

    CAS  Article  Google Scholar 

  12. 12

    Rouzer, C. A., Scott, W. A., Griffith, O. W., Hamill, A. L. & Cohn, Z. A. Depletion of glutathione selectively inhibits synthesis of leukotriene C by macrophages. Proc. Natl Acad. Sci. USA 78, 2532–2536 (1981)

    CAS  Article  ADS  Google Scholar 

  13. 13

    Zemansky, J. et al. Development of a mariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype. J. Bacteriol. 191, 3950–3964 (2009)

    CAS  Article  Google Scholar 

  14. 14

    Ripio, M. T., Domínguez-Bernal, G., Lara, M., Suárez, M. & Vázquez-Boland, J. A. A. Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes . J. Bacteriol. 179, 1533–1540 (1997)

    CAS  Article  Google Scholar 

  15. 15

    Eiting, M., Hagelüken, G., Schubert, W.-D. & Heinz, D. W. The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif. Mol. Microbiol. 56, 433–446 (2005)

    CAS  Article  Google Scholar 

  16. 16

    Miner, M. D., Port, G. C. & Freitag, N. E. Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA. Microbiology 154, 3579–3589 (2008)

    CAS  Article  Google Scholar 

  17. 17

    Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D. & Milzani, A. Protein S-glutathionylation: a regulatory device from bacteria to humans. Trends Biochem. Sci. 34, 85–96 (2009)

    CAS  Article  Google Scholar 

  18. 18

    Mengaud, J. et al. Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol. Microbiol. 5, 2273–2283 (1991)

    CAS  Article  Google Scholar 

  19. 19

    Kolb, A., Busby, S., Buc, H., Garges, S. & Adhya, S. Transcriptional regulation by cAMP and its receptor protein. Annu. Rev. Biochem. 62, 749–797 (1993)

    CAS  Article  Google Scholar 

  20. 20

    Valladares, A., Flores, E. & Herrero, A. Transcription activation by NtcA and 2-oxoglutarate of three genes involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 190, 6126–6133 (2008)

    CAS  Article  Google Scholar 

  21. 21

    Körner, H., Sofia, H. J. & Zumft, W. G. Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs. FEMS Microbiol. Rev. 27, 559–592 (2003)

    Article  Google Scholar 

  22. 22

    Alkhuder, K., Meibom, K. L., Dubail, I., Dupuis, M. & Charbit, A. Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis . PLoS Pathog. 5, e1000284 (2009)

    Article  Google Scholar 

  23. 23

    Smith, K. & Youngman, P. Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. Biochimie 74, 705–711 (1992)

    CAS  Article  Google Scholar 

  24. 24

    Camilli, A., Tilney, L. G. & Portnoy, D. A. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol. Microbiol. 8, 143–157 (1993)

    CAS  Article  Google Scholar 

  25. 25

    Lauer, P., Chow, M. Y. N., Loessner, M. J., Portnoy, D. A. & Calendar, R. Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J. Bacteriol. 184, 4177–4186 (2002)

    CAS  Article  Google Scholar 

  26. 26

    Sauer, J.-D. et al. The N-ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides. Infect. Immun. 79, 688–694 (2011)

    CAS  Article  Google Scholar 

  27. 27

    Portnoy, D. A., Jacks, P. S. & Hinrichs, D. J. Role of hemolysin for the intracellular growth of Listeria monocytogenes . J. Exp. Med. 167, 1459–1471 (1988)

    CAS  Article  Google Scholar 

  28. 28

    Sun, A. N., Camilli, A. & Portnoy, D. A. Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect. Immun. 58, 3770–3778 (1990)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671–675 (2012)

    CAS  Article  Google Scholar 

  30. 30

    Lauer, P. et al. Constitutive activation of the PrfA regulon enhances the potency of vaccines based on live-attenuated and killed but metabolically active Listeria monocytogenes strains. Infect. Immun. 76, 3742–3753 (2008)

    CAS  Article  Google Scholar 

  31. 31

    Köhler, S., Bubert, A., Vogel, M. & Goebel, W. Expression of the iap gene coding for protein p60 of Listeria monocytogenes is controlled on the posttranscriptional level. J. Bacteriol. 173, 4668–4674 (1991)

    Article  Google Scholar 

  32. 32

    Sauer, J.-D. et al. Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol. Cell Host Microbe 7, 412–419 (2010)

    CAS  Article  Google Scholar 

  33. 33

    Melton-Witt, J. A., McKay, S. L. & Portnoy, D. A. Development of a single-gene, signature-tag-based approach in combination with alanine mutagenesis to identify listeriolysin O residues critical for the in vivo survival of Listeria monocytogenes . Infect. Immun. 80, 2221–2230 (2012)

    CAS  Article  Google Scholar 

  34. 34

    Böckmann, R., Dickneite, C., Middendorf, B., Goebel, W. & Sokolovic, Z. Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron. Mol. Microbiol. 22, 643–653 (1996)

    Article  Google Scholar 

  35. 35

    Bishop, D. K. & Hinrichs, D. J. Adoptive transfer of immunity to Listeria monocytogenes. The influence of in vitro stimulation on lymphocyte subset requirements. J. Immunol. 139, 2005–2009 (1987)

    CAS  PubMed  Google Scholar 

  36. 36

    Skoble, J., Portnoy, D. A. & Welch, M. D. Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility. J. Cell Biol. 150, 527–538 (2000)

    CAS  Article  Google Scholar 

  37. 37

    Cheng, L. W. & Portnoy, D. A. Drosophila S2 cells: an alternative infection model for Listeria monocytogenes . Cell. Microbiol. 5, 875–885 (2003)

    CAS  Article  Google Scholar 

Download references


We thank N. Freitag for providing strains and P. Hwang (UCSF Biosensor Core Facility) for technical support and advice regarding bio-layer interferometry. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 Instrumentation grants S10RR029668 and S10RR027303 and the UCSF Funding Shared Equipment Award. This work was supported by National Institutes of Health grants 1PO1 AI63302 and 1R01 AI27655 to D.A.P.; M.L.R. is supported by F32AI104247; A.T.W. is supported by the NSF GRFP DGE 1106400; K.L.H. is supported by F32GM008487.

Author information




M.L.R., A.T.W., K.L.H. and S.M.J. performed the experiments; P.L. engineered the ‘suicide’ strain; M.L.R., A.T.W., R.G.B. and D.A.P. designed the study; M.L.R. and D.A.P. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Daniel A. Portnoy.

Ethics declarations

Competing interests

D.A.P. has a consulting relationship with and a financial interest in Aduro BioTech, Inc., and both he and the company stand to benefit from the commercialization of the results of this research. P.L. is an employee of Aduro BioTech, Inc.

Extended data figures and tables

Extended Data Figure 1 Listeria monocytogenes ΔgshF is sensitive to hydrogen peroxide.

Bacteria were grown overnight in TSB and then inoculated into top agar and spread on tryptic soy agar plates. Sterile disks soaked in 10 μl of 15% H2O2 (Thermo Fisher Scientific) were placed on the agar and incubated overnight. Plates were then scanned and the area of inhibition was measured (in arbitrary units) using ImageJ software ( The mean ± s.e.m. of four independent experiments is shown. P values were calculated using Student’s t-test; **P < 0.01. a.u., arbitrary units.

Extended Data Figure 2 BSO does not affect L. monocytogenes growth.

BMDM growth curve in which cells were untreated or treated with 2 mM BSO for 16 h before infection and throughout the infection. The mean ± s.e.m. of three independent experiments is shown.

Extended Data Figure 3 The effect of ΔgshF is not specific to actA regulation.

Quantitative RT–PCR of hly (a) or hpt (b) transcript levels. Bacteria were harvested from TSB at mid-log (grey bars) or 4 h post-infection of BMDMs (black bars). Mean ± s.e.m. of three independent experiments is shown. P values were calculated using Student’s t-test. *P < 0.05; ***P < 0.001.

Extended Data Figure 4 Fluorescence polarization binding isotherms.

a, Representative binding isotherms of wild-type PrfA plus DTT (circles), wild-type PrfA plus TCEP (squares), PrfA* (diamonds), and PrfA(C/A)4 (crosses), to the PrfA box of Phly. b, Representative binding isotherms of wild-type PrfA plus DTT (circles), wild-type PrfA plus TCEP (squares), PrfA* (diamonds), PrfA(C/A)4 (crosses), and oxidized wild-type PrfA (plus symbols), to the PrfA box of Phly. This plot underscores the very poor binding of oxidized wild-type PrfA to the PrfA box. In both panels the units of millipolarization (mP, y axis) have been normalized to allow the presentation of all binding isotherms on one graph. The protein concentration is shown in terms of protomer on the x axis.

Extended Data Figure 5 PrfA(C/A)4 expression in L. monocytogenes grown in broth.

Immunoblot of PrfA in L. monocytogenes lysates harvested at early exponential phase in BHI. Mean ± s.e.m. of four independent experiments is shown.

Extended Data Figure 6 The PrfA(C/A)4 gshF::Tn mutant exhibits a significant intracellular growth defect.

The mean ± s.e.m. of four independent experiments is shown. P values were calculated using Student’s t-test.;*P < 0.05; **P < 0.01; ***P < 0.001.

Extended Data Figure 7 Model of glutathione-dependent PrfA activation.

The process of infection or intercellular spread requires that L. monocytogenes inhabit an oxidizing vacuole, which may contain both reactive oxygen and nitrogen species. Upon oxidation, glutathione dimerizes to GSSG, which we have demonstrated does not bind PrfA. In addition, PrfA thiols may be reversibly oxidized, temporarily inactivating the protein by inhibiting DNA binding and leading to a downregulation of PrfA-regulated genes (PRG). L. monocytogenes could then enter the host cytosol, as PrfA activation is dispensable for vacuolar escape in vivo. The host cytosol is a highly reducing environment and upon entry into this compartment, all thiols are expected to be in the reduced form. In the absence of glutathione, it is likely that coenzyme A maintains redox homeostasis in the bacterium, as it is the most abundant LMW thiol in L. monocytogenes. Reduced glutathione could then bind PrfA and activate transcription of PRG. This two-step activation requirement may explain why the mechanism of PrfA activation has been a mystery for over two decades; the redox changes occurring during transit through a vacuole followed by replication in the highly reducing cytosol have yet to be recapitulated in vitro.

Extended Data Table 1 Strains and primers

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Reniere, M., Whiteley, A., Hamilton, K. et al. Glutathione activates virulence gene expression of an intracellular pathogen. Nature 517, 170–173 (2015).

Download citation

Further reading


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.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing