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Peptidoglycan editing by a specific ld-transpeptidase controls the muramidase-dependent secretion of typhoid toxin

Abstract

Protein secretion mechanisms are essential for the virulence of most bacterial pathogens. Typhoid toxin is an essential virulence factor for Salmonella Typhi, the cause of typhoid fever in humans. This toxin is unique in that it is only produced within mammalian cells, and it must be trafficked to the extracellular space before intoxicating target cells. An essential and poorly understood aspect of this transport pathway is the secretion of typhoid toxin from the bacterium into the S. Typhi-containing vacuole. We show here that typhoid toxin secretion requires its translocation to the trans side of the peptidoglycan layer at the bacterial poles for subsequent release through the outer membrane. This translocation process depends on a specialized muramidase, the activity of which requires the localized editing of peptidoglycan by a specific ld-transpeptidase. These studies describe a protein export mechanism that is probably conserved in other bacterial species.

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Fig. 1: TtsA-dependent translocation and agonist-mediated release of typhoid toxin in vitro.
Fig. 2: TtsA mediates the transport of typhoid toxin to the trans side of the PG layer.
Fig. 3: Typhoid toxin and TtsA localize to the bacterial poles.
Fig. 4: TtsA exerts its activity at the bacterial poles.
Fig. 5: YcbB-dependent PG editing is required for typhoid toxin translocation across the bacterial envelope.
Fig. 6: TtsA activity requires YcbB-mediated PG editing.

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All data generated or analysed during this study are included in this published article and its Supplementary Information.

References

  1. Galán, J. & Waksman, G. Protein-injection machines in bacteria. Cell 172, 1306–1318 (2018).

    Article  Google Scholar 

  2. Costa, T. et al. Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat. Rev. Microbiol. 13, 343–359 (2015).

    Article  CAS  Google Scholar 

  3. Green, E. & Mecsas, J. in Virulence Mechanisms of Bacterial Pathogens 5th edn (eds Kudva, I. et al.) 215–239 (ASM Press, Washington, DC, 2016).

  4. Koster, M., Bitter, W. & Tommassen, J. Protein secretion mechanisms in Gram-negative bacteria. Int. J. Med. Microbiol. 290, 325–331 (2000).

    Article  CAS  Google Scholar 

  5. Parry, C., Hien, T. T., Dougan, G., White, N. & Farrar, J. Typhoid fever. N. Engl. J. Med. 347, 1770–1782 (2002).

    Article  CAS  Google Scholar 

  6. Crump, J. & Mintz, E. Global trends in typhoid and paratyphoid fever. Clin. Infect. Dis. 50, 241–246 (2010).

    Article  Google Scholar 

  7. Raffatellu, M., Wilson, R., Winter, S. & Bäumler, A. Clinical pathogenesis of typhoid fever. J. Infect. Dev. Ctries 2, 260–266 (2008).

    PubMed  Google Scholar 

  8. Wain, J., Hendriksen, R., Mikoleit, M., Keddy, K. & Ochiai, R. Typhoid fever. Lancet 385, 1136–1145 (2015).

    Article  Google Scholar 

  9. Dougan, G. & Baker, S. Salmonella enterica serovar Typhi and the pathogenesis of typhoid fever. Annu. Rev. Microbiol. 68, 317–336 (2014).

    Article  CAS  Google Scholar 

  10. Spano, S., Ugalde, J. E. & Galan, J. E. Delivery of a Salmonella Typhi exotoxin from a host intracellular compartment. Cell Host Microbe 3, 30–38 (2008).

    Article  CAS  Google Scholar 

  11. Song, J., Gao, X. & Galan, J. E. Structure and function of the Salmonella Typhi chimaeric A2B5 typhoid toxin. Nature 499, 350–354 (2013).

    Article  CAS  Google Scholar 

  12. Galan, J. E. Typhoid toxin provides a window into typhoid fever and the biology of Salmonella Typhi. Proc. Natl Acad. Sci. USA 113, 6338–6344 (2016).

    Article  CAS  Google Scholar 

  13. Haghjoo, E. & Galan, J. E. Salmonella Typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway. Proc. Natl Acad. Sci. USA 101, 4614–4619 (2004).

    Article  CAS  Google Scholar 

  14. Fowler, C. & Galán, J. Decoding a Salmonella Typhi regulatory network that controls typhoid toxin expression within human cells. Cell Host Microbe 23, 65–76 (2018).

    Article  CAS  Google Scholar 

  15. Chang, S., Song, J. & Galán, J. Receptor-mediated sorting of typhoid toxin during its export from Salmonella Typhi-infected cells. Cell Host Microbe 20, 682–689 (2016).

    Article  CAS  Google Scholar 

  16. Hodak, H. & Galán, J. Salmonella Typhi homolog of bacteriophage muramidases controls typhoid toxin secretion. EMBO Rep. 14, 95–102 (2013).

    Article  CAS  Google Scholar 

  17. Beuzón, C., Banks, G., Deiwick, J., Hensel, M. & Holden, D. pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium. Mol. Microbiol. 33, 806–816 (1999).

    Article  Google Scholar 

  18. Spanò, S. & Galán, J. A Rab32-dependent pathway contributes to Salmonella Typhi host restriction. Science 338, 960–963 (2012).

    Article  Google Scholar 

  19. Darmoise, A., Maschmeyer, P. & Winau, F. The immunological functions of saposins. Adv. Immunol. 105, 25–62 (2010).

    Article  CAS  Google Scholar 

  20. McCormack, R. et al. Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria. eLife 4, e06508 (2015).

    Article  Google Scholar 

  21. Prost, L., Sanowar, S. & Miller, S. Salmonella sensing of anti-microbial mechanisms to promote survival within macrophages. Immunol. Rev. 219, 55–65 (2007).

    Article  CAS  Google Scholar 

  22. Di Domenico, E., Cavallo, I., Pontone, M., Toma, L. & Ensoli, F. Biofilm producing Salmonella Typhi: chronic colonization and development of gallbladder cancer. Int. J. Mol. Sci. 18, E1887 (2017).

    Article  Google Scholar 

  23. Gunn, J. et al. Salmonella chronic carriage: epidemiology, diagnosis, and gallbladder persistence. Trends Microbiol. 22, 648–655 (2014).

    Article  CAS  Google Scholar 

  24. Porat, A., Cho, S. & Beckwith, J. The unusual transmembrane electron transporter DsbD and its homologues: a bacterial family of disulfide reductases. Res. Microbiol. 155, 617–622 (2004).

    Article  CAS  Google Scholar 

  25. Siegrist, M. et al. d-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol. 8, 500–505 (2013).

    Article  CAS  Google Scholar 

  26. Turner, R., Vollmer, W. & Foster, S. Different walls for rods and balls: the diversity of peptidoglycan. Mol. Microbiol. 91, 862–874 (2014).

    Article  CAS  Google Scholar 

  27. Egan, A., Biboy, J., van’t Veer, I., Breukink, E. & Vollmer, W. Activities and regulation of peptidoglycan synthases. Philos. Trans. R. Soc. Lond. B 370, 20150031 (2015).

    Article  Google Scholar 

  28. Vollmer, W., Blanot, D. & de Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).

    Article  CAS  Google Scholar 

  29. Cameron, T., Anderson-Furgeson, J., Zupan, J., Zik, J. J. & Zambryski, P. Peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division. mBio 5, 14 (2014).

    Google Scholar 

  30. Kuru, E., Tekkam, S., Hall, E., Brun, Y. & Van Nieuwenhze, M. Synthesis of fluorescent d-amino acids and their use for probing peptidoglycan synthesis and bacterial growth in situ. Nat. Protoc. 10, 33–52 (2015).

    Article  CAS  Google Scholar 

  31. Glauner, B., Holtje, J. V. & Schwarz, U. The composition of the murein of Escherichia coli. J. Biol. Chem. 263, 10088–10095 (1988).

    CAS  PubMed  Google Scholar 

  32. Holtje, J. V. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 62, 181–203 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Quintela, J., de Pedro, M., Zöllner, P., Allmaier, G. & Garcia-del Portillo, F. Peptidoglycan structure of Salmonella typhimurium growing within cultured mammalian cells. Mol. Microbiol. 23, 693–704 (1997).

    Article  CAS  Google Scholar 

  34. Magnet, S. et al. Identification of the l,d-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan. J. Bacteriol. 189, 3927–3931 (2007).

    Article  CAS  Google Scholar 

  35. Magnet, S., Dubost, L., Marie, A., Arthur, M. & Gutmann, L. Identification of the l,d-transpeptidases for peptidoglycan cross-linking in Escherichia coli. J. Bacteriol. 190, 4782–4785 (2008).

    Article  CAS  Google Scholar 

  36. Alcorlo, M., Martinez-Caballero, S., Molina, R. & Hermoso, J. A. Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases. Curr. Opin. Struct. Biol. 44, 87–100 (2017).

    Article  CAS  Google Scholar 

  37. Kondo, Y. et al. Cloning and characterization of a pair of genes that stimulate the production and secretion of Zymomonas mobilis extracellular levansucrase and invertase. Biosci. Biotechnol. Biochem. 58, 526–530 (1994).

    Article  CAS  Google Scholar 

  38. Oda, Y., Yanase, H., Kato, N. & Tonomura, K. Liberation of sucrose-hydrolyzing enzymes from cells by the zliS gene product that mediates protein secretion in Zymomonas mobilis. J. Ferment. Bioeng. 77, 419–422 (1994).

    Article  CAS  Google Scholar 

  39. Takeda, K. et al. The effect of amphiphilic compounds on the secretion of levansucrase by Zymomonas mobilis. Process Biochem. 40, 3723–3731 (2005).

    Article  Google Scholar 

  40. Rico-Pérez, G. et al. A novel peptidoglycan d,l-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence. Mol. Microbiol. 99, 546–556 (2016).

    Article  Google Scholar 

  41. Takeda, K., Guerrero-Mandujano, A., Hernández-Cortez, C., Ibarra, J. & Castro-Escarpulli, G. The outer membrane vesicles: secretion system type zero. Traffic 18, 425–432 (2017).

    Article  Google Scholar 

  42. Schwechheimer, C. & Kuehn, M. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat. Rev. Microbiol. 13, 605–619 (2015).

    Article  CAS  Google Scholar 

  43. Wang, I., Smith, D. & Young, R. Holins: the protein clocks of bacteriophage infections. Annu. Rev. Microbiol. 54, 799–825 (2000).

    Article  CAS  Google Scholar 

  44. Hamilton, J. et al. A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens. J. Cell Biol. 207, 615–626 (2014).

    Article  CAS  Google Scholar 

  45. Galan, J. E. & Curtiss, R. III Distribution of the invA, -B, -C, and -D genes of Salmonella typhimurium among other Salmonella serovars: invA mutants of Salmonella Typhi are deficient for entry into mammalian cells. Infect. Immun. 59, 2901–2908 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Demarre, G. et al. A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains. Res. Microbiol. 156, 245–255 (2005).

    Article  CAS  Google Scholar 

  47. Kaniga, K., Bossio, J. C. & Galan, J. E. The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins. Mol. Microbiol. 13, 555–568 (1994).

    Article  CAS  Google Scholar 

  48. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    Article  CAS  Google Scholar 

  49. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  50. Galán, J. E. & Curtiss, R. III Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect. Immun. 58, 1879–1885 (1990).

    PubMed  PubMed Central  Google Scholar 

  51. Liu, X., Gao, B., Novik, V. & Galan, J. E. Quantitative proteomics of intracellular Campylobacter jejuni reveals metabolic reprogramming. PLoS Pathog. 8, e1002562 (2012).

    Article  CAS  Google Scholar 

  52. Ducret, A., Quardokus, E. M. & Brun, Y. V. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat. Microbiol. 1, 16077 (2016).

    Article  CAS  Google Scholar 

  53. Seemann, J., Pypaert, M., Taguchi, T., Malsam, J. & Warren, G. Partitioning of the matrix fraction of the Golgi apparatus during mitosis in animal cells. Science 295, 848–851 (2002).

    Article  CAS  Google Scholar 

  54. Heidrich, C. et al. Involvement of N-acetylmuramyl-l-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli. Mol. Microbiol. 41, 167–178 (2001).

    Article  CAS  Google Scholar 

  55. Glauner, B. Separation and quantification of muropeptides with high-performance liquid chromatography. Anal. Biochem. 172, 451–464 (1988).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank H. Rego for useful discussions, and members of the Galán laboratory for critical review of the manuscript. T.G. was supported in part by a Postdoctoral Fellowship (GE 2653/1-1) from the Deutsche Forschungsgemeinschaft (German Research Foundation). This work was supported by the National Institute of Allergy and Infectious Diseases under grant AI079022 (to J.E.G.) and the UK Medical Research Council under grant MR/N002679/1 (to W.V.).

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T.G. was involved in the design and interpretation of experiments and conducted all experiments shown except the biochemical characterization of the PG structure, which was conducted by M.P. with the supervision of W.V., and the LC–MS/MS analysis of culture supernatants, which was carried out by M.L.-T. J.E.G was involved in the design, interpretation and supervision of this study. T.G. and J.E.G. wrote the paper with comments from all authors.

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Correspondence to Jorge E. Galán.

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Supplementary information

Supplementary Information

Supplementary Figures 1–14, Supplementary Tables 1 and 2.

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Supplementary Data Set 1

Raw data for Fig. 1e.

Supplementary Data Set 2

Raw data for Fig. 2b–e.

Supplementary Data Set 3

Raw data for Fig. 3a,b.

Supplementary Data Set 4

Raw data for Fig. 4a–d.

Supplementary Data Set 5

Raw data for Fig. 5a–f.

Supplementary Data Set 6

Data of the LC–MS/MS analysis of bacterial cell supernatants after treatment with various compounds.

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Geiger, T., Pazos, M., Lara-Tejero, M. et al. Peptidoglycan editing by a specific ld-transpeptidase controls the muramidase-dependent secretion of typhoid toxin. Nat Microbiol 3, 1243–1254 (2018). https://doi.org/10.1038/s41564-018-0248-x

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