Intracellular bacteria engage a STING–TBK1–MVB12b pathway to enable paracrine cGAS–STING signalling


The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS)–stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.

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Fig. 1: Supernatants from cells infected with intracellular bacteria contain IFN-inducing potential.
Fig. 2: Foreign intracellular DNA stimulates IFN-β expression in bystander cells through EVs.
Fig. 3: Listeria infection activates EV-dependent stimulation of type I IFN expression in bystander cells.
Fig. 4: EVs from Listeria-infected cells augment apoptosis in T lymphocytes.
Fig. 5: The sorting of foreign DNA into EVs requires STING and TBK1.
Fig. 6: The sorting of foreign DNA into EVs requires TBK1-mediated phosphorylation of MVB12b.

Data availability

The full next-generation sequencing dataset is available at the European Nucleotide Archive with the identifier ‘ena-STUDY-AARHUS UNIVERSITY 12-12-2018-17:12:31:528-124’, under accession number PRJEB30324. The full mass spectrometry dataset is available at Original immunoblots are shown in Supplementary Fig. 7.


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The technical assistance of K. Stadel Petersen, the next-generation sequencing facility at the Department of Molecular Medicine, Aarhus University Hospital, and the FACS Core facility, Aarhus University is greatly appreciated. This work was funded by The Danish Medical Research Council (12-124330 to S.R.P.), Novo Nordisk Foundation (NNF18OC0030274 to S.R.P.), Lundbeck Foundation (R198-2015-171 to S.R.P.), European Research Council (786602 to S.R.P.), EU FP7 MOBILEX programme (DFF – 5053-00011 to R.N.), BMBF (JPI-AMR - FKZ 01Kl1702) and DFG (SFB/TR-84 TP C01) (both to B.S.), and Austrian Science Fund through grant P 25186-B22 (to T.D.).

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R.N. and S.R.P. conceived the idea and designed the experiments. R.N., R.T., F.M., T.P., A.K., B.Z, S.A., A.M., W.B., A.A., T.K. and M.K.T. performed the experiments. F.M. designed, performed and analysed the phosphoproteomics experiment. E.F. analysed the next-generation sequencing data. C.K.H., B.S., K.A.H., T.H., K.V.G., T.D. and S.R.P. supervised the experiments. R.N. and S.R.P. wrote the manuscript.

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Correspondence to Søren R. Paludan.

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Supplementary Information

Supplementary Figures 1–7 and Supplementary Table 1.

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Supplementary Table 2

Full dataset for phosphoproteome analysis of dsDNA-stimulated cells.

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Nandakumar, R., Tschismarov, R., Meissner, F. et al. Intracellular bacteria engage a STING–TBK1–MVB12b pathway to enable paracrine cGAS–STING signalling. Nat Microbiol 4, 701–713 (2019).

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