Skip to main content

Thank you for visiting nature.com. 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.

  • Letter
  • Published:

Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread

Subjects

Abstract

Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity1. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes, can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells by using the pore-forming toxin listeriolysin O (LLO) and two phospholipase C enzymes2. Expression of the cell surface protein ActA allows L. monocytogenes to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. Here we show that protrusion formation is associated with plasma membrane damage due to LLO’s pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 (encoded by the Timd4 gene) contributes to efficient cell-to-cell spread by L. monocytogenes in macrophages in vitro and growth of these bacteria is impaired in Timd4−/− mice. Thus, L. monocytogenes promotes its dissemination in a host by exploiting efferocytosis. Our results indicate that PS-targeted therapeutics may be useful in the fight against infections by L. monocytogenes and other bacteria that use similar strategies of cell-to-cell spread during infection.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Actin-based motility promotes LLO-mediated membrane damage.
Figure 2: Formation of PS+ structures during L. monocytogenes infection.
Figure 3: Protrusions give rise to PS+ vesicles containing L. monocytogenes.
Figure 4: TIM-4 promotes L. monocytogenes cell-to-cell spread in macrophages and growth in mice.

Similar content being viewed by others

References

  1. Ravichandran, K. S. Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways. Immunity 35, 445–455 (2011)

    Article  CAS  Google Scholar 

  2. Mostowy, S. & Cossart, P. Virulence factors that modulate the cell biology of Listeria infection and the host response. Adv. Immunol. 113, 19–32 (2012)

    Article  CAS  Google Scholar 

  3. Robbins, J. R. et al. Listeria monocytogenes exploits normal host cell processes to spread from cell to cell. J. Cell Biol. 146, 1333–1350 (1999)

    Article  CAS  Google Scholar 

  4. Alberti-Segui, C., Goeden, K. R. & Higgins, D. E. Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread. Cell. Microbiol. 9, 179–195 (2007)

    Article  CAS  Google Scholar 

  5. Gedde, M. M., Higgins, D. E., Tilney, L. G. & Portnoy, D. A. Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes . Infect. Immun. 68, 999–1003 (2000)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Glomski, I. J., Gedde, M. M., Tsang, A. W., Swanson, J. A. & Portnoy, D. A. The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells. J. Cell Biol. 156, 1029–1038 (2002)

    Article  CAS  Google Scholar 

  8. Nomura, T. et al. Irreversible loss of membrane-binding activity of Listeria-derived cytolysins in non-acidic conditions: a distinct difference from allied cytolysins produced by other Gram-positive bacteria. Microbiology 153, 2250–2258 (2007)

    Article  CAS  Google Scholar 

  9. Schnupf, P., Portnoy, D. A. & Decatur, A. L. Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence. Cell. Microbiol. 8, 353–364 (2006)

    Article  CAS  Google Scholar 

  10. Hamon, M. A., Ribet, D., Stavru, F. & Cossart, P. Listeriolysin O: the Swiss army knife of Listeria . Trends Microbiol. 20, 360–368 (2012)

    Article  CAS  Google Scholar 

  11. Cassidy, S. K. et al. Membrane damage during Listeria monocytogenes infection triggers a caspase-7 dependent cytoprotective response. PLoS Pathog. 8, e1002628 (2012)

    Article  CAS  Google Scholar 

  12. Idone, V., Tam, C. & Andrews, N. W. Two-way traffic on the road to plasma membrane repair. Trends Cell Biol. 18, 552–559 (2008)

    Article  CAS  Google Scholar 

  13. Gründling, A., Gonzalez, M. D. & Higgins, D. E. Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells. J. Bacteriol. 185, 6295–6307 (2003)

    Article  Google Scholar 

  14. Draeger, A., Monastyrskaya, K. & Babiychuk, E. B. Plasma membrane repair and cellular damage control: the annexin survival kit. Biochem. Pharmacol. 81, 703–712 (2011)

    Article  CAS  Google Scholar 

  15. Fadeel, B. & Xue, D. The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease. Crit. Rev. Biochem. Mol. Biol. 44, 264–277 (2009)

    Article  CAS  Google Scholar 

  16. Waite, J. C. et al. Dynamic imaging of the effector immune response to Listeria infection in vivo . PLoS Pathog. 7, e1001326 (2011)

    Article  CAS  Google Scholar 

  17. Pust, S., Morrison, H., Wehland, J., Sechi, A. S. & Herrlich, P. Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell. EMBO J. 24, 1287–1300 (2005)

    Article  CAS  Google Scholar 

  18. Babiychuk, E. B., Monastyrskaya, K., Potez, S. & Draeger, A. Blebbing confers resistance against cell lysis. Cell Death Differ. 18, 80–89 (2011)

    Article  CAS  Google Scholar 

  19. Keyel, P. A. et al. Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane. J. Cell Sci. 124, 2414–2423 (2011)

    Article  CAS  Google Scholar 

  20. Feng, D. et al. Cellular internalization of exosomes occurs through phagocytosis. Traffic 11, 675–687 (2010)

    Article  CAS  Google Scholar 

  21. Miyanishi, M. et al. Identification of Tim4 as a phosphatidylserine receptor. Nature 450, 435–439 (2007)

    Article  ADS  CAS  Google Scholar 

  22. Rodriguez-Manzanet, R. et al. T and B cell hyperactivity and autoimmunity associated with niche-specific defects in apoptotic body clearance in TIM-4-deficient mice. Proc. Natl Acad. Sci. USA 107, 8706–8711 (2010)

    Article  ADS  CAS  Google Scholar 

  23. Martin, C. J. et al. Efferocytosis is an innate antibacterial mechanism. Cell Host Microbe 12, 289–300 (2012)

    Article  CAS  Google Scholar 

  24. Appelberg, R. & Leal, I. S. Mutants of Listeria monocytogenes defective in in vitro invasion and cell-to-cell spreading still invade and proliferate in hepatocytes of neutropenic mice. Infect. Immun. 68, 912–914 (2000)

    Article  CAS  Google Scholar 

  25. Drevets, D. A. Dissemination of Listeria monocytogenes by infected phagocytes. Infect. Immun. 67, 3512–3517 (1999)

    Article  CAS  Google Scholar 

  26. Friedrich, N., Hagedorn, M., Soldati-Favre, D. & Soldati, T. Prison break: pathogens’ strategies to egress from host cells. Microbiol. Mol. Biol. Rev. 76, 707–720 (2012)

    Article  CAS  Google Scholar 

  27. Meertens, L. et al. The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry. Cell Host Microbe 12, 544–557 (2012)

    Article  CAS  Google Scholar 

  28. Mercer, J. & Helenius, A. Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 320, 531–535 (2008)

    Article  ADS  CAS  Google Scholar 

  29. Hagedorn, M., Rohde, K. H., Russell, D. G. & Soldati, T. Infection by tubercular mycobacteria is spread by nonlytic ejection from their amoeba hosts. Science 323, 1729–1733 (2009)

    Article  ADS  CAS  Google Scholar 

  30. Hybiske, K. & Stephens, R. S. Mechanisms of host cell exit by the intracellular bacterium Chlamydia . Proc. Natl Acad. Sci. USA 104, 11430–11435 (2007)

    Article  ADS  CAS  Google Scholar 

  31. 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 

  32. Jones, S. & Portnoy, D. A. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect. Immun. 62, 5608–5613 (1994)

    Article  CAS  Google Scholar 

  33. Lauer, P., Chow, M. Y., 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)

    Article  CAS  Google Scholar 

  34. 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)

    Article  CAS  Google Scholar 

  35. Smith, G. A. et al. The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect. Immun. 63, 4231–4237 (1995)

    Article  CAS  Google Scholar 

  36. Shen, A. & Higgins, D. E. The 5′ untranslated region-mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity. Mol. Microbiol. 57, 1460–1473 (2005)

    Article  CAS  Google Scholar 

  37. Rodriguez-Manzanet, R. et al. TIM-4 expressed on APCs induces T cell expansion and survival. J. Immunol. 180, 4706–4713 (2008)

    Article  CAS  Google Scholar 

  38. Munsie, L. N., Caron, N., Desmond, C. R. & Truant, R. Lifeact cannot visualize some forms of stress-induced twisted F-actin. Nature Methods 6, 317 (2009)

    Article  CAS  Google Scholar 

  39. Brumell, J. H., Rosenberger, C. M., Gotto, G. T., Marcus, S. L. & Finlay, B. B. SifA permits survival and replication of Salmonella typhimurium in murine macrophages. Cell. Microbiol. 3, 75–84 (2001)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to S. Gray-Owen, S. Grinstein and D. Portnoy for providing reagents and advice and to D. Holmyard for help with electron microscopy. J.H.B. holds the Pitblado Chair in Cell Biology. Infrastructure for the Brumell laboratory was provided by a Leader’s Opportunity Fund grant from the Canadian Foundation for Innovation and the Ontario Innovation Trust. R.F. was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research in partnership with the Canadian Association of Gastroenterology and the Crohn’s and Colitis Foundation of Canada. S.O. was supported by a postdoctoral fellowship from the Research Training Committee at the Hospital for Sick Children. This work was supported by an operating grant from The Arthritis Society of Canada (#RG11/013) to J.H.B. and a US Public Health Service grant (AI053669) from the National Institutes of Health to D.E.H.

Author information

Authors and Affiliations

Authors

Contributions

J.H.B., M.A.C., S.O. and D.E.H. designed the experiments and wrote the paper. M.A.C., R.F., J.M.v.R., V.C. and S.O. performed the experiments. A.M.M. and V.K.K. contributed reagents and consultations.

Corresponding author

Correspondence to John H. Brumell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Annexins promote membrane repair during L. monocytogenes infection.

a, HeLa cells were treated with the indicated siRNA for 48 h and then infected with wild-type L. monocytogenes at an MOI of 100. At 6 h post-infection, medium was switched to Tyrodes buffer containing 0.5 mg ml−1 PI with or without calcium. Cells were fixed at 60 min after PI addition and then stained for bacteria and DNA (DAPI). The percentage of 100 random infected cells that were PI+ cells were enumerated by microscopic analysis. Averages ± s.d. for three independent experiments are shown. P values were calculated using one-way ANOVA. *P < 0.05. b, Knockdown of gene expression by siRNA was confirmed by western blotting. Images are representative of two independent experiments. c, Recruitment of annexin A2 to PS+ structures containing bacteria. Boxes in low-magnification image indicate areas enlarged in bottom panels. Arrows indicate PS+ structures that co-localize with annexin A2. Images are representative of three independent experiments. Scale bars, 10 µm for low-magnification images, 2 µm for enlarged regions of interest.

Extended Data Figure 2 Actin-based motility promotes LLO-mediated membrane damage during L. monocytogenes infection.

a, HeLa cells were infected with the indicated L. monocytogenes strain. At 6 h post-infection, medium was switched to Tyrodes buffer containing 0.5 mg ml−1 PI with or without calcium. Cells were fixed at 60 min after PI addition and then stained for bacteria and DNA (DAPI). Confocal images representative of three independent experiments are shown (n = 100). PI+ cells were enumerated and results are shown in Fig. 1d. Where indicated, uninfected cells were treated with saponin to permeabilize membranes and allow PI entry, serving as a positive control. Scale bars, 10 µm. b, HeLa cells were infected with wild-type L. monocytogenes and subjected to membrane damage assay as in a in the presence of either DMSO or the actin cytoskeleton inhibitors latrunculin B or cytochalasin D. Averages ± s.d. for three independent experiments are shown (n = 100). P values were calculated using two-tailed Student’s t-test. *P < 0.05.

Extended Data Figure 3 Annexin A5–Alexa 488 as a probe to label PS.

Live HeLa cells were cooled on ice and stained with a fluorescent probe (annexin A5–Alexa 488) for 10 min to label exofacial PS. Cells were then fixed and stained with phalloidin Alexa 568 to visualize F-actin. In uninfected control experiments, low amounts of exofacial PS was detected in the membranes of cells, due to asymmetry of PS distribution in the plasma membrane. By contrast, treatment of cells with the pore-forming surfactant saponin led to robust staining of cells with annexin A5–Alexa 488. Images representative of three independent experiments. Scale bars, 10 µm.

Extended Data Figure 4 Formation of PS+ structures during L. monocytogenes infection.

Low-magnification images used to generate images shown in Fig. 2a. HeLa cells were infected with wild-type L. monocytogenes expressing RFP for 6 h and then cooled on ice and stained with a fluorescent probe (annexin A5–Alexa 488) for 10 min to label exofacial PS. Cells were then fixed and analysed by fluorescence microscopy to identify PS+ structures and bacteria. SEM of the same cell revealed that PS+ structures were associated with the dorsal surface of infected cells. Differential interference contrast (DIC) microscopy of cells was also performed to help identify cells for correlative imaging analysis. Images are representative of two independent experiments. Scale bars, 20 µm.

Extended Data Figure 5 PS+ bacteria are present with a host-derived membrane structure.

a, HeLa cells were infected with wild-type L. monocytogenes expressing RFP for 8 h and then labelled with a probe for exofacial PS (annexin A5–Alexa 488). Cells were then rapidly stained with anti-Listeria antibodies (5 min) to label extracellular bacteria. Cells were then fixed and analysed by fluorescence microscopy to identify PS+ structures and bacteria. Bacteria that co-localize with exofacial PS but are not labelled with anti-Listeria antibodies in the extracellular medium are indicated with arrows. Extracellular bacteria do not label with annexin A5–Alexa 488, indicating that this probe does not bind non-specifically to bacteria. Box in low-magnification image indicates area enlarged in bottom panels. Images are representative of three independent experiments. Scale bars, 10 µm for low magnification, 2 µm for high magnification. b, Cells were infected and stained as in a and analysed by fluorescence microscopy. Bacteria that co-localize with exofacial PS were scored for their accessibility to anti-Listeria antibodies present in the extracellular medium. Data show that the majority of PS+ bacteria are not accessible to anti-Listeria antibodies. Averages ± s.d. for two independent experiments are shown (n = 100).

Extended Data Figure 6 Formation of PS+ structures during L. monocytogenes infection of epithelial cells and macrophages.

a, Henle-407 human intestinal epithelial cells were infected with wild-type L. monocytogenes for 6 h and then incubated with a probe for exofacial PS (annexin A5–Alexa 488; green). Cells were then fixed and stained with phalloidin to visualize F-actin (red) and bacteria (blue). Cells were analysed by fluorescence microscopy to identify PS+ structures and bacteria. Images are representative of three independent experiments. b, Mouse BMDMs from C57BL/6 mice were infected and stained as in a. Scale bars, 10 µm. Images are representative of three independent experiments.

Extended Data Figure 7 Release of PS+ structures containing L. monocytogenes from infected cells.

a, HeLa cells were infected with wild-type L. monocytogenes for 6 h and then incubated with a probe for exofacial PS (annexin A5–Alexa 488; green). Cells were then fixed and stained with phalloidin to visualize F-actin (red) and bacteria (blue). Cells were analysed by fluorescence microscopy to identify PS+ structures and bacteria. Inset shows PS+ bacteria that are not cell associated. Images are representative of three independent experiments. b, HeLa cells were infected with ΔplcAΔplcB mutant bacteria for 6 h. The supernatant from the infected cultures was then removed and centrifuged onto poly-l-lysine-coated coverslips. Bacteria associated with coverslips were then stained with a probe for exofacial PS (annexin A5–Alexa 488; green). Cells were then fixed and stained for bacteria (blue). Coverslips were analysed by fluorescence microscopy to identify PS+ bacteria. Inset shows PS+ bacteria. Scale bars, 10 µm for low-magnification images, 2 µm for insets. Images are representative of three independent experiments.

Extended Data Figure 8 Growth of L. monocytogenes in the cytosol of Timd4−/− macrophages is not impaired.

Gentamicin protection assay to measure intracellular bacterial growth. BMDMs were harvested from C57BL/6 or Timd4−/− mice and seeded at a density of 3 × 105 cells per well. Cells were then infected with wild-type L. monocytogenes in the presence of extracellular gentamicin. At the indicated times, cell lysates were plated and intracellular bacterial numbers (c.f.u.) were determined. Averages ± s.d. for two independent experiments are shown.

Extended Data Figure 9 Cytokine measurements.

a, Measurement of cytokines after in vitro infection of BMDMs from C57BL/6 or Timd4−/− mice with wild-type L. monocytogenes for the indicated time. Data from one of two independent experiments are shown. b, Measurement of basal cytokines in tissues of C57BL/6 or Timd4−/− mice without infection. Averages ± s.d. for three independent experiments are shown. P values were calculated using one-way ANOVA.

Extended Data Figure 10 L. monocytogenes exploits efferocytosis to promote cell-to-cell spread during infection.

Model shows the steps that promote cell-to-cell spread by L. monocytogenes. 1. Protrusion formation via actin-based motility. 2. LLO-mediated damage to the plasma membrane leads to loss of membrane asymmetry and exofacial PS on protrusions. The exofacial exposure of PS promotes protrusion association with neighbouring cells (right). 3. Loss of membrane asymmetry and PS exposure extends along length of protrusions. 4. Calcium entry activates membrane repair pathways that promote scission of the protrusion. Bacteria are released from the cell in PS+ vesicles. 5. Macrophages mediate uptake of PS+ vesicles containing bacteria via the PS-binding receptor TIM-4. PS+ vesicles may be engulfed by neighbouring cells either near the infected cell surface (left side) or within enclosed spaces that form as a result of protrusion penetration into the neighbouring cell (right side). TIM-4 may also promote L. monocytogenes infection indirectly, through its ability to suppress basal levels of pro-inflammatory cytokines as part of its homeostatic function in the immune system.

Supplementary information

Lm protrusion formation leads to exofacial PS exposure on membrane vesicles

Cells were transfected with LifeAct-RFP (red) and then infected with wild type Lm expressing GFP at an MOI of 100 for 6h. Live infected cells were then analyzed by spinning disk confocal microscopy with Annexin V-Alexa 647 in the medium to label exofacial PS (blue). Frames from this video were cropped and are presented in Figure 3B. Images are representative of 3 independent experiments. (MOV 113 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Czuczman, M., Fattouh, R., van Rijn, J. et al. Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread. Nature 509, 230–234 (2014). https://doi.org/10.1038/nature13168

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13168

This article is cited by

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

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