The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and ‘switching’ to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Å-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact.
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Matthews, K. R., McCulloch, R. & Morrison, L. J. The within-host dynamics of African trypanosome infections. Phil. Trans. R. Soc. B https://doi.org/10.1098/rstb.2014.0288 (2015).
Hsia, R., Beals, T. & Boothroyd, J. C. Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins. Mol. Microbiol. 19, 53–63 (1996).
Schwede, A., Macleod, O. J., MacGregor, P. & Carrington, M. How does the VSG coat of bloodstream form African trypanosomes interact with external proteins? PLoS Pathog. 11, e1005259 (2015).
Metcalf, P., Blum, M., Freymann, D., Turner, M. & Wiley, D. C. Two variant surface glycoproteins of Trypanosoma brucei of different sequence classes have similar 6 Å resolution X-ray structures. Nature 325, 84–86 (1987).
Blum, M. L. et al. A structural motif in the variant surface glycoproteins of Trypanosoma brucei. Nature 362, 603–609 (1993).
Carrington, M. et al. Variant specific glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues. J. Mol. Biol. 221, 823–835 (1991).
Bartossek, T. et al. Structural basis for the shielding function of the dynamic trypanosome variant surface glycoprotein coat. Nat. Microbiol. 2, 1523–1532 (2017).
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).
Mendonca-Previato, L., Todeschini, A. R., Heise, N. & Previato, J. O. Protozoan parasite-specific carbohydrate structures. Curr. Opin. Struct. Biol. 15, 499–505 (2005).
Takeuchi, H., Kantharia, J., Sethi, M. K., Bakker, H. & Haltiwanger, R. S. Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats. J. Biol. Chem. 287, 33934–33944 (2012).
Cross, G. A., Kim, H. S. & Wickstead, B. Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol. Biochem. Parasitol. 195, 59–73 (2014).
Black, S. J. et al. Regulation of parasitaemia in mice infected with Trypanosoma brucei. Curr. Top. Microbiol. Immunol. 117, 93–118 (1985).
Pinger, J., Chowdhury, S. & Papavasiliou, F. N. Variant surface glycoprotein density defines an immune evasion threshold for African trypanosomes undergoing antigenic variation. Nat. Commun. 8, 828 (2017).
Lisowska, E. The role of glycosylation in protein antigenic properties. Cell Mol. Life Sci. 59, 445–455 (2002).
Rangappa, S. et al. Effects of the multiple O-glycosylation states on antibody recognition of the immunodominant motif in MUC1 extracellular tandem repeats. Med. Chem. Commun. 7, 1102–1122 (2016).
Hirumi, H. & Hirumi, K. Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J. Parasitol. 75, 985–989 (1989).
Cross, G. A. Release and purification of Trypanosoma brucei variant surface glycoprotein. J. Cell. Biochem. 24, 79–90 (1984).
Kabsch, W. XDS. Acta Crystallogr D 66, 125–132 (2010).
Evans, P. Scaling and assessment of data quality. Acta Crystallogr D 62, 72–82 (2006).
Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D 67, 282–292 (2011).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr D 69, 1204–1214 (2013).
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50, 760–763 (1994).
Sheldrick, G. M. A short history of SHELX. Acta Crystallogr A 64, 112–122 (2008).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66, 213–221 (2010).
Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D 67, 235–242 (2011).
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53, 240–255 (1997).
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D 67, 355–367 (2011).
Ali, L. et al. The O-glycomap of lubricin, a novel mucin responsible for joint lubrication, identified by site-specific glycopeptide analysis. Mol. Cell Proteom. 13, 3396–3409 (2014).
Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).
Böhme, U. & Cross, G. A. M. Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence in Trypanosoma brucei. J. Cell Sci. 115, 805–816 (2002).
Burkard, G., Fragoso, C. M. & Roditi, I. Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei. Mol. Biochem. Parasitol. 153, 220–223 (2007).
Wirtz, E., Leal, S., Ochatt, C. & Cross, G. A. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89–101 (1999).
Leal, S. et al. Virulence of Trypanosoma brucei strain 427 is not affected by the absence of glycosylphosphatidylinositol phospholipase C. Mol. Biochem. Parasitol. 114, 245–247 (2001).
We thank G. Cross (Rockefeller University) and H. Wardemann (DKFZ) for critical reading of the manuscript and for general advice, M. Sanches-Vaz and L. Figueiredo (IMM, Lisbon) for help with mouse experiments and M. Chandra (DKFZ) for providing us with purified VSG615. We also thank the staff at Argonne National Laboratories (NE-CAT) for beamline support, and D. Oren at the Structural Biology and the High-Throughput Sequencing and Spectroscopy Resource Centers at Rockefeller University. NE-CAT is funded by an NIH/NIGMS grant (P41 GM103403) and the Pilatus 6M detector on 24-ID-C beam line is funded by an NIH-ORIP HEI grant (S10 RR029205). The Advanced Photon Source, within which NE-CAT is located, is a US Department of Energy (DOE) User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This work was also supported by funds to C.E.S. and F.N.P. from the German Cancer Research Center (DKFZ, Heidelberg) and Rockefeller University, by NIH/NIAID (AI085973) to F.N.P. and by a Wellcome Trust Senior Investigator Award (101842) to M.A.J.F. The University of Dundee MS facility is supported by Wellcome Trust grant 097045.
The authors declare no competing interests.
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Pinger, J., Nešić, D., Ali, L. et al. African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat. Nat Microbiol 3, 932–938 (2018). https://doi.org/10.1038/s41564-018-0187-6
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