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.

BioID screen of Salmonella type 3 secreted effectors reveals host factors involved in vacuole positioning and stability during infection

Abstract

Many bacterial pathogens express virulence proteins that are translocated into host cells (herein referred to as effectors), where they can interact with target proteins to manipulate host cell processes. These effector–host protein interactions are often dynamic and transient in nature, making them difficult to identify using traditional interaction-based methods. Here, we performed a systematic comparison between proximity-dependent biotin labelling (BioID) and immunoprecipitation coupled with mass spectrometry to investigate a series of Salmonella type 3 secreted effectors that manipulate host intracellular trafficking (SifA, PipB2, SseF, SseG and SopD2). Using BioID, we identified 632 candidate interactions with 381 unique human proteins, collectively enriched for roles in vesicular trafficking, cytoskeleton components and transport activities. From the subset of proteins exclusively identified by BioID, we report that SifA interacts with BLOC-2, a protein complex that regulates dynein motor activity. We demonstrate that the BLOC-2 complex is necessary for SifA-mediated positioning of Salmonella-containing vacuoles, and affects stability of the vacuoles during infection. Our study provides insight into the coordinated activities of Salmonella type 3 secreted effectors and demonstrates the utility of BioID as a powerful, complementary tool to characterize effector–host protein interactions.

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

Fig. 1: BioID, a proximity-based strategy to identify the host targets of Salmonella type 3 secreted effectors.
Fig. 2: The BLOC-2 complex, a candidate target of the effector SifA.
Fig. 3: The BLOC-2 complex interacts with SifA and is involved with SifA-mediated trafficking.
Fig. 4: SifA targets BLOC-2-dependent trafficking in epithelial cells.
Fig. 5: SifA targets BLOC-2 function in Salmonella-infected epithelial cells.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

References

  1. Mead, P. S. et al. Food-related illness and death in the united states. Emerg. Infect. Dis. 5, 607–625 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Figueira, R. & Holden, D. W. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiology 158, 1147–1161 (2012).

    CAS  PubMed  Google Scholar 

  3. Brumell, J. H. & Grinstein, S. Salmonella redirects phagosomal maturation. Curr. Opin. Microbiol. 7, 78–84 (2004).

    CAS  PubMed  Google Scholar 

  4. Stein, M. A., Leung, K. Y., Zwick, M., Garcia-del Portillo, F. & Finlay, B. B. Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells. Mol. Microbiol. 20, 151–164 (1996).

    CAS  PubMed  Google Scholar 

  5. Jiang, X. et al. The related effector proteins SopD and SopD2 from Salmonella enterica serovar Typhimurium contribute to virulence during systemic infection of mice. Mol. Microbiol. 54, 1186–1198 (2004).

    CAS  PubMed  Google Scholar 

  6. Knodler, L. A. & Steele-Mortimer, O. The Salmonella effector PipB2 affects late endosome/lysosome distribution to mediate Sif extension. Mol. Biol. Cell 16, 4108–4123 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Guy, R. L., Gonias, L. A. & Stein, M. A. Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms-aroE intragenic region. Mol. Microbiol. 37, 1417–1435 (2000).

    CAS  PubMed  Google Scholar 

  8. LaRock, D. L., Chaudhary, A. & Miller, S. I. Salmonellae interactions with host processes. Nat. Rev. Microbiol. 13, 191–205 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Liss, V. et al. Salmonella enterica remodels the host cell endosomal system for efficient intravacuolar nutrition. Cell Host Microbe 21, 390–402 (2017).

    CAS  PubMed  Google Scholar 

  10. Szeto, J., Namolovan, A., Osborne, S. E., Coombes, B. K. & Brumell, J. H. Salmonella-containing vacuoles display centrifugal movement associated with cell-to-cell transfer in epithelial cells. Infect. Immun. 77, 996–1007 (2009).

    CAS  PubMed  Google Scholar 

  11. Brumell, J. H. et al. SopD2 is a novel type III secreted effector of Salmonella typhimurium that targets late endocytic compartments upon delivery into host cells. Traffic 4, 36–48 (2003).

    CAS  PubMed  Google Scholar 

  12. Knodler, L. A. et al. Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes. Mol. Microbiol. 49, 685–704 (2003).

    CAS  PubMed  Google Scholar 

  13. Hensel, M. et al. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Mol. Microbiol. 30, 163–174 (1998).

    CAS  PubMed  Google Scholar 

  14. Halici, S., Zenk, S. F., Jantsch, J. & Hensel, M. Functional analysis of the Salmonella pathogenicity island 2-mediated inhibition of antigen presentation in dendritic cells. Infect. Immun. 76, 4924–4933 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Salcedo, S. P. & Holden, D. W. SseG, a virulence protein that targets Salmonella to the golgi network. EMBO J. 22, 5003–5014 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Abrahams, G. L., Muller, P. & Hensel, M. Functional dissection of SseF, a type III effector protein involved in positioning the Salmonella-containing vacuole. Traffic 7, 950–965 (2006).

    CAS  PubMed  Google Scholar 

  17. Boucrot, E., Henry, T., Borg, J. P., Gorvel, J. P. & Meresse, S. The intracellular fate of Salmonella depends on the recruitment of kinesin. Science 308, 1174–1178 (2005).

    CAS  PubMed  Google Scholar 

  18. Beuzon, C. R. et al. Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235–3249 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Choi-Rhee, E., Schulman, H. & Cronan, J. E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci. 13, 3043–3050 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Mojica, S. A. et al. SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors. Mol. Biol. Cell 26, 1918–1934 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Galan, J. E. Common themes in the design and function of bacterial effectors. Cell Host Microbe 5, 571–579 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Gupta, G. D. et al. A dynamic protein interaction landscape of the human centrosome–cilium interface. Cell 163, 1484–1499 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. D’Costa, V. M. et al. Salmonella disrupts host endocytic trafficking by SopD2-mediated inhibition of Rab7. Cell Rep. 12, 1508–1518 (2015).

    PubMed  Google Scholar 

  25. Spano, S., Gao, X., Hannemann, S., Lara-Tejero, M. & Galan, J. E. A bacterial pathogen targets a host Rab-family GTPase defense pathway with a GAP. Cell Host Microbe 19, 216–226 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Harrison, R. E. et al. Salmonella impairs RILP recruitment to Rab7 during maturation of invasion vacuoles. Mol. Biol. Cell 15, 3146–3154 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Henry, T. et al. The Salmonella effector protein PipB2 is a linker for kinesin-1. Proc. Natl Acad. Sci. USA 103, 13497–13502 (2006).

    CAS  PubMed  Google Scholar 

  28. Yu, X. J., Liu, M. & Holden, D. W. Salmonella effectors SseF and SseG interact with mammalian protein ACBD3 (GCP60) to anchor Salmonella-containing vacuoles at the Golgi network. mBio 7, e00474-16 (2016).

  29. Weigele, B. A., Orchard, R. C., Jimenez, A., Cox, G. W. & Alto, N. M. A systematic exploration of the interactions between bacterial effector proteins and host cell membranes. Nat. Commun. 8, 532 (2017).

    PubMed  PubMed Central  Google Scholar 

  30. Kim, D. I. et al. Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc. Natl Acad. Sci. USA 111, E2453–E2461 (2014).

    CAS  PubMed  Google Scholar 

  31. Buchmeier, N. A. & Heffron, F. Inhibition of macrophage phagosome–lysosome fusion by Salmonella typhimurium. Infect. Immun. 59, 2232–2238 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Garcia-del Portillo, F., Zwick, M. B., Leung, K. Y. & Finlay, B. B. Salmonella induces the formation of filamentous structures containing lysosomal membrane glycoproteins in epithelial cells. Proc. Natl Acad. Sci. USA 90, 10544–10548 (1993).

    CAS  PubMed  Google Scholar 

  33. Guignot, J. et al. Microtubule motors control membrane dynamics of Salmonella-containing vacuoles. J. Cell Sci. 117, 1033–1045 (2004).

    CAS  PubMed  Google Scholar 

  34. 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).

    CAS  PubMed  Google Scholar 

  35. Brown, N. F. et al. Mutational analysis of Salmonella translocated effector members SifA and SopD2 reveals domains implicated in translocation, subcellular localization and function. Microbiology 152, 2323–2343 (2006).

    CAS  PubMed  Google Scholar 

  36. McEwan, D. G. et al. PLEKHM1 regulates Salmonella-containing vacuole biogenesis and infection. Cell Host Microbe 17, 58–71 (2015).

    CAS  PubMed  Google Scholar 

  37. Gautam, R. et al. The Hermansky–Pudlak syndrome 3 (cocoa) protein is a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2). J. Biol. Chem. 279, 12935–12942 (2004).

    CAS  PubMed  Google Scholar 

  38. Di Pietro, S. M., Falcon-Perez, J. M. & Dell’Angelica, E. C. Characterization of BLOC-2, a complex containing the Hermansky–Pudlak syndrome proteins HPS3, HPS5 and HPS6. Traffic 5, 276–283 (2004).

    PubMed  Google Scholar 

  39. Li, K. et al. HPS6 interacts with dynactin p150Glued to mediate retrograde trafficking and maturation of lysosomes. J. Cell Sci. 127, 4574–4588 (2014).

    PubMed  Google Scholar 

  40. Ohlson, M. B. et al. Structure and function of Salmonella SifA indicate that its interactions with SKIP, SseJ, and RhoA family GTPases induce endosomal tubulation. Cell Host Microbe 4, 434–446 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhao, W. et al. The Salmonella effector protein SifA plays a dual role in virulence. Sci. Rep. 5, 12979 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Diacovich, L. et al. Interaction between the SifA virulence factor and its host target SKIP is essential for Salmonella pathogenesis. J. Biol. Chem. 284, 33151–33160 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Henry, T., Garcia-Del Portillo, F. & Gorvel, J. P. Identification of Salmonella functions critical for bacterial cell division within eukaryotic cells. Mol. Microbiol. 56, 252–267 (2005).

    CAS  PubMed  Google Scholar 

  44. Aachoui, Y. et al. Caspase-11 protects against bacteria that escape the vacuole. Science 339, 975–978 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Ostrowski, P. P., Fairn, G. D., Grinstein, S. & Johnson, D. E. Cresyl violet: a superior fluorescent lysosomal marker. Traffic 17, 1313–1321 (2016).

    CAS  PubMed  Google Scholar 

  46. Beuzon, C. R., Salcedo, S. P. & Holden, D. W. Growth and killing of a Salmonella enterica serovar Typhimurium sifA mutant strain in the cytosol of different host cell lines. Microbiology 148, 2705–2715 (2002).

    CAS  PubMed  Google Scholar 

  47. Brumell, J. H., Tang, P., Zaharik, M. L. & Finlay, B. B. Disruption of the Salmonella-containing vacuole leads to increased replication of Salmonella enterica serovar Typhimurium in the cytosol of epithelial cells. Infect. Immun. 70, 3264–3270 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Spano, S. & Galan, J. E. A Rab32-dependent pathway contributes to Salmonella typhi host restriction. Science 338, 960–963 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Bultema, J. J., Ambrosio, A. L., Burek, C. L. & Di Pietro, S. M. BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles. J. Biol. Chem. 287, 19550–19563 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Gingras, A. C., Abe, K. T. & Raught, B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Curr. Opin. Chem. Biol. 48, 44–54 (2018).

    PubMed  Google Scholar 

  51. Comartin, D. et al. CEP120 and SPICE1 cooperate with CPAP in centriole elongation. Curr. Biol. 23, 1360–1366 (2013).

    CAS  PubMed  Google Scholar 

  52. Erster, O. & Liscovitch, M. A modified inverse PCR procedure for insertion, deletion, or replacement of a DNA fragment in a target sequence and its application in the ligand interaction scan method for generation of ligand-regulated proteins. Methods Mol. Biol. 634, 157–174 (2010).

    CAS  PubMed  Google Scholar 

  53. Brumell, J. H., Goosney, D. L. & Finlay, B. B. SifA, a type III secreted effector of Salmonella typhimurium, directs Salmonella-induced filament (Sif) formation along microtubules. Traffic 3, 407–415 (2002).

    CAS  PubMed  Google Scholar 

  54. Kessner, D., Chambers, M., Burke, R., Agus, D. & Mallick, P. Proteowizard: open source software for rapid proteomics tools development. Bioinformatics 24, 2534–2536 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Shteynberg, D. et al. iProphet: multi-level integrative analysis of shotgun proteomic data improves peptide and protein identification rates and error estimates. Mol. Cell. Proteomics 10, M111 007690 (2011).

    PubMed  PubMed Central  Google Scholar 

  56. Pedrioli, P. G. Trans-proteomic pipeline: a pipeline for proteomic analysis. Methods Mol. Biol. 604, 213–238 (2010).

    CAS  PubMed  Google Scholar 

  57. Liu, G. et al. ProHits: integrated software for mass spectrometry-based interaction proteomics. Nat. Biotechnol. 28, 1015–1017 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Teo, G. et al. SAINTexpress: improvements and additional features in significance analysis of INTeractome software. J. Proteom. 100, 37–43 (2014).

    CAS  Google Scholar 

  59. Miao, E. A. et al. Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems. Mol. Microbiol. 34, 850–864 (1999).

    CAS  PubMed  Google Scholar 

  60. Freeman, J. A., Ohl, M. E. & Miller, S. I. The Salmonella enterica serovar Typhimurium translocated effectors SseJ and SifB are targeted to the salmonella-containing vacuole. Infect. Immun. 71, 418–427 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

J.H.B. holds the Pitblado Chair in Cell Biology. Infrastructure for the Brumell Laboratory was provided by a John Evans Leadership Fund grant from the Canadian Foundation for Innovation and the Ontario Innovation Trust. V.M.D. was supported by fellowships from the Canadian Institutes of Health Research and the Hospital for Sick Children Research Training Centre, and is a recipient of the L’Oreal-UNESCO Women in Science Award. K.C.B. was supported by scholarships from the Natural Sciences and Engineering Research Council of Canada and the Ontario Graduate Scholarship. We thank Paul Paroutis for help with confocal microscopy. This work was supported by operating grants from the Canadian Institutes of Health Research (grant no. FDN#154329 to J.H.B., grant no. FDN#143202 to S.G. and grant no. MOP#119289 to B.R.). B.R. holds a Canada Research Chair in Proteomics and Molecular Medicine.

Author information

Authors and Affiliations

Authors

Contributions

V.M.D., J.H.B., E.C. and B.R. designed the experiments. V.M.D. and K.C.B. generated the BioID constructs, and V.M.D. generated all other clones and gene mutants. V.M.D. created cell lines and performed experiments for BioID analysis. E.C., E.M.N.L. and J.S. performed peptide isolation and mass spectrometry, and E.C., V.M.D. and K.C.B. analysed subsequent datasets. Downstream cell biological experiments were performed by V.M.D. and T.L. S.G. contributed reagents and technical expertise.

Corresponding authors

Correspondence to Vanessa M. D’Costa or John H. Brumell.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–11, original source images, Supplementary Table legends and Supplementary References.

Reporting Summary

Supplementary Table 1

IP–MS profiling of S. Typhimurium effectors.

Supplementary Table 2

BioID profiling of S. Typhimurium effectors.

Supplementary Table 3

BioID and IP–MS datasets enrichment analysis.

Supplementary Table 4

BioID and IP–MS individual baits individual and comparative enrichment analysis.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

D’Costa, V.M., Coyaud, E., Boddy, K.C. et al. BioID screen of Salmonella type 3 secreted effectors reveals host factors involved in vacuole positioning and stability during infection. Nat Microbiol 4, 2511–2522 (2019). https://doi.org/10.1038/s41564-019-0580-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-019-0580-9

This article is cited by

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