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Glutathione activates virulence gene expression of an intracellular pathogen

Abstract

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.

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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.

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Acknowledgements

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.

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Authors

Contributions

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.

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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 (http://rsbweb.nih.gov/ij). 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

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Reniere, M., Whiteley, A., Hamilton, K. et al. Glutathione activates virulence gene expression of an intracellular pathogen. Nature 517, 170–173 (2015). https://doi.org/10.1038/nature14029

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