In vivo tropism of Salmonella Typhi toxin to cells expressing a multiantennal glycan receptor

An Author Correction to this article was published on 13 June 2019

This article has been updated

Abstract

Typhoid fever is a life-threatening disease, but little is known about the molecular bases for its unique clinical presentation. Typhoid toxin, a unique virulence factor of Salmonella Typhi (the cause of typhoid fever), recapitulates in an animal model many symptoms of typhoid fever. Typhoid toxin binding to its glycan receptor Neu5Ac is central, but, due to the ubiquity of Neu5Ac, how typhoid toxin causes specific symptoms remains elusive. Here we show that typhoid toxin displays in vivo tropism to cells expressing multiantennal glycoprotein receptors, particularly on endothelial cells of arterioles in the brain and immune cells, which is in line with typhoid symptoms. Neu5Ac displayed by multiantennal N-glycans, rather than a single Neu5Ac, appears to serve as the high-affinity receptor, as typhoid toxin possesses five identical binding pockets per toxin. Human counterparts also express the multiantennal Neu5Ac receptor. Here we also show that mice immunized with inactive typhoid toxins and challenged with wild-type typhoid toxin presented neither the characteristic in vivo tropism nor symptoms. These mice were protected against a lethal-dose toxin challenge, but Ty21a-vaccinated mice were not. Cumulatively, these results reveal remarkable features describing how a bacterial exotoxin induces virulence exclusively in specific cells at the organismal level.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Typhoid toxin causes neurological complications associated with motor function deficits where both PltB and CdtB subunits play critical roles.
Fig. 2: Typhoid toxin has in vivo tropism to endothelial cells of arterioles in the brain and to immune cells.
Fig. 3: Typhoid toxin binds preferentially to Neu5Ac displayed in the context of multiantennal N-glycans over linear N-glycans.
Fig. 4: Vaccination of naive mice with genetically engineered inactive typhoid toxin or PltB subunit alone completely protects the mice against a lethal-dose toxin challenge.

Change history

  • 13 June 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 1.

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

    CAS  Article  Google Scholar 

  2. 2.

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

    Article  Google Scholar 

  3. 3.

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

    Article  Google Scholar 

  4. 4.

    Crump, J. A., Sjolund-Karlsson, M., Gordon, M. A. & Parry, C. M. Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial resistance, and antimicrobial management of invasive salmonella infections. Clin. Microbiol. Rev. 28, 901–937 (2015).

    CAS  Article  Google Scholar 

  5. 5.

    Ali, G., Rashid, S., Kamli, M. A., Shah, P. A. & Allaqaband, G. Q. Spectrum of neuropsychiatric complications in 791 cases of typhoid fever. Trop. Med. Int. Health 2, 314–318 (1997).

    CAS  Article  Google Scholar 

  6. 6.

    Lutterloh, E. et al. Multidrug-resistant typhoid fever with neurologic findings on the Malawi–Mozambique border. Clin. Infect. Dis. 54, 1100–1106 (2012).

    Article  Google Scholar 

  7. 7.

    Sejvar, J. et al. Neurologic manifestations associated with an outbreak of typhoid fever, Malawi–Mozambique, 2009: an epidemiologic investigation. PLoS ONE 7, e46099 (2012).

    CAS  Article  Google Scholar 

  8. 8.

    Szabo, I. et al. Neurologic complications and sequellae of infectious diseases in Uganda and Kenya: analysis of 288 cases from two rural hospitals. Neuro. Endocrinol. Lett. 34, 28–31 (2013).

    PubMed  Google Scholar 

  9. 9.

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

    CAS  Article  Google Scholar 

  10. 10.

    Hornick, R. B. et al. Typhoid fever: pathogenesis and immunologic control. New Engl. J. Med. 283, 686–691 (1970).

    CAS  Article  Google Scholar 

  11. 11.

    Fraser, A., Paul, M., Goldberg, E., Acosta, C. J. & Leibovici, L. Typhoid fever vaccines: systematic review and meta-analysis of randomised controlled trials. Vaccine 25, 7848–7857 (2007).

    CAS  Article  Google Scholar 

  12. 12.

    Liang, L. et al. Immune profiling with a Salmonella Typhi antigen microarray identifies new diagnostic biomarkers of human typhoid. Sci. Rep. 3, 1043 (2013).

    Article  Google Scholar 

  13. 13.

    Charles, R. C. et al. Characterization of anti-Salmonella enterica serotype Typhi antibody responses in bacteremic Bangladeshi patients by an immunoaffinity proteomics-based technology. Clin. Vaccine Immunol. 17, 1188–1195 (2010).

    CAS  Article  Google Scholar 

  14. 14.

    Tran Vu Thieu, N. et al. An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever. J. Infect. 75, 104–114 (2017).

    Article  Google Scholar 

  15. 15.

    Deng, L. et al. Host adaptation of a bacterial toxin from the human pathogen Salmonella Typhi. Cell 159, 1290–1299 (2014).

    CAS  Article  Google Scholar 

  16. 16.

    Chou, H. H. et al. Inactivation of CMP-N-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution. Proc. Natl Acad. Sci. USA 99, 11736–11741 (2002).

    CAS  Article  Google Scholar 

  17. 17.

    Song, J. et al. A mouse model for the human pathogen Salmonella Typhi. Cell Host Microbe 8, 369–376 (2010).

    CAS  Article  Google Scholar 

  18. 18.

    Chong, A., Lee, S., Yang, Y. A. & Song, J. The role of typhoid toxin in Salmonella Typhi virulence. Yale J. Biol. Med. 90, 283–290 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    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).

    CAS  Article  Google Scholar 

  20. 20.

    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).

    CAS  Article  Google Scholar 

  21. 21.

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

    CAS  Article  Google Scholar 

  22. 22.

    Hatcher, J. P. et al. Development of SHIRPA to characterise the phenotype of gene-targeted mice. Behav. Brain Res. 125, 43–47 (2001).

    CAS  Article  Google Scholar 

  23. 23.

    Rogers, D. C. et al. SHIRPA, a protocol for behavioral assessment: validation for longitudinal study of neurological dysfunction in mice. Neurosci. Lett. 306, 89–92 (2001).

    CAS  Article  Google Scholar 

  24. 24.

    Rutenber, E., Ready, M. & Robertus, J. D. Structure and evolution of ricin B chain. Nature 326, 624–626 (1987).

    CAS  Article  Google Scholar 

  25. 25.

    Geisler, C. & Jarvis, D. L. Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities. Glycobiology 21, 988–993 (2011).

    CAS  Article  Google Scholar 

  26. 26.

    Peng, W. et al. Recent H3N2 viruses have evolved specificity for extended, branched human-type receptors, conferring potential for increased avidity. Cell Host Microbe 21, 23–34 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Cryz, S. J. Jr et al. Construction and characterization of a Vi-positive variant of the Salmonella Typhi live oral vaccine strain Ty21a. Infect. Immun. 57, 3863–3868 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    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 

  29. 29.

    Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

    CAS  Article  Google Scholar 

  30. 30.

    Jo, S., Song, K. C., Desaire, H., MacKerell, A. D. Jr & Im, W. Glycan Reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins. J. Comput. Chem. 32, 3135–3141 (2011).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank J. E. Galán for his suggestion on the typhoid toxoid vaccination study, which was initiated while J.S. was at Yale, N. Nishimura for her comments on brain images, and X. Gao for plasmid pET21a-pltB-His6. This work was supported by a Cornell University Startup, PCCW Affinito-Stewart Award and the USDA/NIFA Hatch project 1010701 (to J.S.), the NIH, NCI, under contract no. HHSN261200800001E (to R.N.), the NIH, NIDCD intramural research programme (to J.Z.), and NIH R01 AI114730 to J.C.P. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Affiliations

Authors

Contributions

Y.-A.Y. conducted the experiments shown in Figs. 1, 2, 3a and 4d,g–k, Supplementary Figs. 1–6 and 9–11 and Supplementary Videos 1–10. S.L. conducted the experiments in Figs. 4a–c,e,f. J.Z. conducted the analyses and simulations in Figs. 3b–e. R.N. supervised the computational project. L.D. contributed to Fig. 3. A.J.T. conducted the experiments in Supplementary Fig. 8. R.M. conducted experiments in Fig. 3f and Supplementary Fig. 7. B.T. contributed to Supplementary Figs. 7 and 8. J.C.P. supervised the glycan microarray project. J.S. was involved in the design, interpretation and supervision of this study. Y.-A.Y. and J.S. wrote the paper and all the authors commented on the manuscript.

Corresponding author

Correspondence to Jeongmin Song.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

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

Electronic supplementary material

Supplementary Information

Supplementary Figures 1–11, Supplementary Tables 1-4, Supplementary Methods, Supplementary References, Supplementary Data

Life Sciences Reporting Summary

Supplementary Table 5

Related to Fig. 3f and Supplementary Fig. 7. Lists of glycans and glycan microarray results. Data for individual glycans are reported as relative fluorescence intensities from four of six replicates (after removal of highest and lowest values)

Supplementary Video 1

Related to Fig. 1c. A representative video showing ataxia signs typically seen 4.5–5 days after WT toxin administration

Supplementary Video 2

Related to Fig. 1c. A representative video showing clonic seizure signs typically seen 5–6 days after WT toxin administration

Supplementary Video 3

Related to Fig. 1d. A representative balance beam test of C57BL/6 mice administered PBS. Balance beam tests were conducted 5 days after PBS buffer administration

Supplementary Video 4

Related to Fig. 1d. A representative result of balance beam tests of mice administered WT toxin. Balance beam tests were conducted 5 days after WT toxin administration

Supplementary Video 5

Related to Fig. 1d. Another representative result of balance beam tests of mice administered WT toxin. Balance beam tests were conducted 5 days after WT toxin administration

Supplementary Video 6

Related to Fig. 1d. A representative result of balance beam tests of mice administered a PltB catalytic mutant toxin. Balance beam tests were conducted 5 days after indicated toxin administration

Supplementary Video 7

Related to Fig. 1f. A representative result of balance beam tests of mice administered a PltA catalytic mutant toxin. Balance beam tests were conducted 5 days after indicated toxin administration

Supplementary Video 8

Related to Fig. 1f. A representative result of balance beam tests of mice administered a CdtB catalytic mutant toxin. Balance beam tests were conducted 5 days after indicated toxin administration

Supplementary Video 9

Related to Supplementary Fig. 2. A representative result of balance beam tests of mice infected with a lethal dose of S. Typhimurium. A group of C57BL/6 mice were infected orally with 107 S. Typhimurium SL1344. The balance beam test was conducted 5 days after infection

Supplementary Video 10

Related to Fig. 4i. A representative result of balance beam tests of mice immunized by typhoid toxoid and challenged by a lethal dose of typhoid toxin. A group of C57BL/6 mice were immunized by subcutaneous injections of typhoid toxoid via a standard prime-boost regimen. Two weeks after boost, the immunized mice were challenged with 2 μg WT typhoid toxin. Balance beam tests were conducted 5 days after the WT toxin challenge

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yang, YA., Lee, S., Zhao, J. et al. In vivo tropism of Salmonella Typhi toxin to cells expressing a multiantennal glycan receptor. Nat Microbiol 3, 155–163 (2018). https://doi.org/10.1038/s41564-017-0076-4

Download citation

Further reading

Search

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