Genetic determinants of genus—Level glycan diversity in a bacterial protein glycosylation system

Autoři: Chris Hadjineophytou aff001;  Jan Haug Anonsen aff001;  Nelson Wang aff001;  Kevin C. Ma aff002;  Raimonda Viburiene aff001;  Åshild Vik aff001;  Odile B. Harrison aff003;  Martin C. J. Maiden aff003;  Yonatan H. Grad aff002;  Michael Koomey aff001
Působiště autorů: Department of Biosciences, Center for Integrative Microbial Evolution, University of Oslo, Oslo, Norway aff001;  Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, United States of America aff002;  Department of Zoology, University of Oxford, Oxford, United Kingdom aff003;  Division of Infectious Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America aff004
Vyšlo v časopise: Genetic determinants of genus—Level glycan diversity in a bacterial protein glycosylation system. PLoS Genet 15(12): e32767. doi:10.1371/journal.pgen.1008532
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008532


The human pathogens N. gonorrhoeae and N. meningitidis display robust intra- and interstrain glycan diversity associated with their O-linked protein glycosylation (pgl) systems. In an effort to better understand the evolution and function of protein glycosylation operating there, we aimed to determine if other human—restricted, Neisseria species similarly glycosylate proteins and if so, to assess the levels of glycoform diversity. Comparative genomics revealed the conservation of a subset of genes minimally required for O-linked protein glycosylation glycan and established those pgl genes as core genome constituents of the genus. In conjunction with mass spectrometric–based glycan phenotyping, we found that extant glycoform repertoires in N. gonorrhoeae, N. meningitidis and the closely related species N. polysaccharea and N. lactamica reflect the functional replacement of a progenitor glycan biosynthetic pathway. This replacement involved loss of pgl gene components of the primordial pathway coincident with the acquisition of two exogenous glycosyltransferase genes. Critical to this discovery was the identification of a ubiquitous but previously unrecognized glycosyltransferase gene (pglP) that has uniquely undergone parallel but independent pseudogenization in N. gonorrhoeae and N. meningitidis. We suggest that the pseudogenization events are driven by processes of compositional epistasis leading to gene decay. Additionally, we documented instances where inter-species recombination influences pgl gene status and creates discordant genetic interactions due ostensibly to the multi-locus nature of pgl gene networks. In summary, these findings provide a novel perspective on the evolution of protein glycosylation systems and identify phylogenetically informative, genetic differences associated with Neisseria species.

Klíčová slova:

Genetic loci – Genomic databases – Glycosylation – Glycosyltransferases – Neisseria – Neisseria meningitidis – Sequence alignment


1. Mostowy RJ, Holt KE. Diversity-Generating Machines: Genetics of Bacterial Sugar-Coating. Trends Microbiol. 2018;26(12):1008–21. doi: 10.1016/j.tim.2018.06.006 30037568; PubMed Central PMCID: PMC6249986.

2. Bentley SD, Aanensen DM, Mavroidi A, Saunders D, Rabbinowitsch E, Collins M, et al. Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes. PLoS Genet. 2006;2(3):e31. doi: 10.1371/journal.pgen.0020031 16532061; PubMed Central PMCID: PMC1391919.

3. Wang L, Wang Q, Reeves PR. The variation of O antigens in gram-negative bacteria. Subcell Biochem. 2010;53:123–52. doi: 10.1007/978-90-481-9078-2_6 20593265.

4. Eichler J, Koomey M. Sweet New Roles for Protein Glycosylation in Prokaryotes. Trends Microbiol. 2017;25(8):662–72. doi: 10.1016/j.tim.2017.03.001 28341406.

5. Borud B, Anonsen JH, Viburiene R, Cohen EH, Samuelsen AB, Koomey M. Extended glycan diversity in a bacterial protein glycosylation system linked to allelic polymorphisms and minimal genetic alterations in a glycosyltransferase gene. Molecular microbiology. 2014;94(3):688–99. Epub 2014/09/13. doi: 10.1111/mmi.12789 25213144.

6. Nothaft H, Scott NE, Vinogradov E, Liu X, Hu R, Beadle B, et al. Diversity in the protein N-glycosylation pathways within the Campylobacter genus. Molecular & cellular proteomics: MCP. 2012;11(11):1203–19. Epub 2012/08/04. doi: 10.1074/mcp.M112.021519 22859570; PubMed Central PMCID: PMC3494190.

7. Scott NE, Kinsella RL, Edwards AV, Larsen MR, Dutta S, Saba J, et al. Diversity within the O-linked protein glycosylation systems of acinetobacter species. Molecular & cellular proteomics: MCP. 2014;13(9):2354–70. doi: 10.1074/mcp.M114.038315 24917611; PubMed Central PMCID: PMC4159654.

8. Coyne MJ, Fletcher CM, Chatzidaki-Livanis M, Posch G, Schaffer C, Comstock LE. Phylum-wide general protein O-glycosylation system of the Bacteroidetes. Molecular microbiology. 2013;88(4):772–83. doi: 10.1111/mmi.12220 23551589; PubMed Central PMCID: PMC3656502.

9. Bennett JS, Jolley KA, Earle SG, Corton C, Bentley SD, Parkhill J, et al. A genomic approach to bacterial taxonomy: an examination and proposed reclassification of species within the genus Neisseria. Microbiology (Reading, England). 2012;158(Pt 6):1570–80. Epub 2012/03/17. doi: 10.1099/mic.0.056077–0 22422752; PubMed Central PMCID: PMC3541776.

10. Tønjum T. Genus I. Neisseria. In: Garrity G. M. BDJ, Krieg N. R, Staley J. R, editor. Bergey’s Manual of Systematic Bacteriology. New York: Springer-Verlag; 2005. p. 777–98.

11. Marri PR, Paniscus M, Weyand NJ, Rendon MA, Calton CM, Hernandez DR, et al. Genome sequencing reveals widespread virulence gene exchange among human Neisseria species. PloS one. 2010;5(7):e11835. Epub 2010/08/03. doi: 10.1371/journal.pone.0011835 20676376; PubMed Central PMCID: PMC2911385.

12. Snyder LA, Saunders NJ. The majority of genes in the pathogenic Neisseria species are present in non-pathogenic Neisseria lactamica, including those designated as 'virulence genes'. BMC Genomics. 2006;7:128. doi: 10.1186/1471-2164-7-128 16734888; PubMed Central PMCID: PMC1538595.

13. Stabler RA, Marsden GL, Witney AA, Li Y, Bentley SD, Tang CM, et al. Identification of pathogen-specific genes through microarray analysis of pathogenic and commensal Neisseria species. Microbiology (Reading, England). 2005;151(Pt 9):2907–22. doi: 10.1099/mic.0.28099–0 16151203.

14. Veyrier FJ, Biais N, Morales P, Belkacem N, Guilhen C, Ranjeva S, et al. Common Cell Shape Evolution of Two Nasopharyngeal Pathogens. PLoS Genet. 2015;11(7):e1005338. doi: 10.1371/journal.pgen.1005338 26162030; PubMed Central PMCID: PMC4498754.

15. Borud B, Viburiene R, Hartley MD, Paulsen BS, Egge-Jacobsen W, Imperiali B, et al. Genetic and molecular analyses reveal an evolutionary trajectory for glycan synthesis in a bacterial protein glycosylation system. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(23):9643–8. Epub 2011/05/25. doi: 10.1073/pnas.1103321108 21606362; PubMed Central PMCID: PMC3111294.

16. Aas FE, Li X, Edwards J, Hongro Solbakken M, Deeudom M, Vik A, et al. Cytochrome c-based domain modularity governs genus-level diversification of electron transfer to dissimilatory nitrite reduction. Environmental microbiology. 2015;17(6):2114–32. Epub 2014/10/21. doi: 10.1111/1462-2920.12661 25330335.

17. Clemence MEA, Maiden MCJ, Harrison OB. Characterization of capsule genes in non-pathogenic Neisseria species. Microbial genomics. 2018;4(9). doi: 10.1099/mgen.0.000208 30074474; PubMed Central PMCID: PMC6202450.

18. Borud B, Barnes GK, Brynildsrud OB, Fritzsonn E, Caugant DA. Genotypic and Phenotypic Characterization of the O-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates. Journal of bacteriology. 2018;200(16). Epub 2018/03/21. doi: 10.1128/jb.00794-17 29555702; PubMed Central PMCID: PMC6060354.

19. Borud B, Aas FE, Vik A, Winther-Larsen HC, Egge-Jacobsen W, Koomey M. Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species. Journal of bacteriology. 2010;192(11):2816–29. Epub 2010/04/07. doi: 10.1128/JB.00101-10 20363948; PubMed Central PMCID: PMC2876500.

20. Hartley MD, Morrison MJ, Aas FE, Borud B, Koomey M, Imperiali B. Biochemical characterization of the O-linked glycosylation pathway in Neisseria gonorrhoeae responsible for biosynthesis of protein glycans containing N,N'-diacetylbacillosamine. Biochemistry. 2011;50(22):4936–48. Epub 2011/05/06. doi: 10.1021/bi2003372 21542610; PubMed Central PMCID: PMC3108506.

21. Aas FE, Vik A, Vedde J, Koomey M, Egge-Jacobsen W. Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure. Molecular microbiology. 2007;65(3):607–24. Epub 2007/07/05. doi: 10.1111/j.1365-2958.2007.05806.x 17608667; PubMed Central PMCID: PMC1976384.

22. Chamot-Rooke J, Rousseau B, Lanternier F, Mikaty G, Mairey E, Malosse C, et al. Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(37):14783–8. Epub 2007/09/07. doi: 10.1073/pnas.0705335104 17804791; PubMed Central PMCID: PMC1976187.

23. Power PM, Roddam LF, Rutter K, Fitzpatrick SZ, Srikhanta YN, Jennings MP. Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis. Molecular microbiology. 2003;49(3):833–47. Epub 2003/07/17. doi: 10.1046/j.1365-2958.2003.03602.x 12864863.

24. Johannessen C, Koomey M, Borud B. Hypomorphic glycosyltransferase alleles and recoding at contingency loci influence glycan microheterogeneity in the protein glycosylation system of Neisseria species. Journal of bacteriology. 2012;194(18):5034–43. Epub 2012/07/17. doi: 10.1128/JB.00950-12 22797763; PubMed Central PMCID: PMC3430319.

25. Anonsen JH, Vik A, Borud B, Viburiene R, Aas FE, Kidd SW, et al. Characterization of a Unique Tetrasaccharide and Distinct Glycoproteome in the O-Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica. Journal of bacteriology. 2016;198(2):256–67. Epub 2015/10/21. doi: 10.1128/JB.00620-15 26483525; PubMed Central PMCID: PMC4751800.

26. Wang N, Anonsen JH, Viburiene R, Lam JS, Vik A, Koomey M. Disrupted Synthesis of a Di-N-acetylated Sugar Perturbs Mature Glycoform Structure and Microheterogeneity in the O-Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica. Journal of bacteriology. 2019;201(1). Epub 2018/10/17. doi: 10.1128/jb.00522-18 30322851; PubMed Central PMCID: PMC6287464.

27. Kahler CM, Martin LE, Tzeng YL, Miller YK, Sharkey K, Stephens DS, et al. Polymorphisms in pilin glycosylation Locus of Neisseria meningitidis expressing class II pili. Infection and immunity. 2001;69(6):3597–604. Epub 2001/05/12. doi: 10.1128/IAI.69.6.3597-3604.2001 11349019; PubMed Central PMCID: PMC98345.

28. Power PM, Roddam LF, Dieckelmann M, Srikhanta YN, Tan YC, Berrington AW, et al. Genetic characterization of pilin glycosylation in Neisseria meningitidis. Microbiology (Reading, England). 2000;146 (Pt 4):967–79. Epub 2000/04/28. doi: 10.1099/00221287-146-4-967 10784055.

29. Vik A, Aas FE, Anonsen JH, Bilsborough S, Schneider A, Egge-Jacobsen W, et al. Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(11):4447–52. Epub 2009/03/03. doi: 10.1073/pnas.0809504106 19251655; PubMed Central PMCID: PMC2648892.

30. Lamelas A, Harris SR, Roltgen K, Dangy JP, Hauser J, Kingsley RA, et al. Emergence of a new epidemic Neisseria meningitidis serogroup A Clone in the African meningitis belt: high-resolution picture of genomic changes that mediate immune evasion. mBio. 2014;5(5):e01974–14. Epub 2014/10/23. doi: 10.1128/mBio.01974-14 25336458; PubMed Central PMCID: PMC4212839.

31. Viburiene R, Vik A, Koomey M, Borud B. Allelic variation in a simple sequence repeat element of neisserial pglB2 and its consequences for protein expression and protein glycosylation. Journal of bacteriology. 2013;195(15):3476–85. Epub 2013/06/05. doi: 10.1128/JB.00276-13 23729645; PubMed Central PMCID: PMC3719539.

32. Gault J, Ferber M, Machata S, Imhaus AF, Malosse C, Charles-Orszag A, et al. Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation. PLoS pathogens. 2015;11(9):e1005162. Epub 2015/09/15. doi: 10.1371/journal.ppat.1005162 26367394; PubMed Central PMCID: PMC4569582.

33. Anonsen JH, Vik A, Egge-Jacobsen W, Koomey M. An extended spectrum of target proteins and modification sites in the general O-linked protein glycosylation system in Neisseria gonorrhoeae. Journal of proteome research. 2012;11(12):5781–93. Epub 2012/10/04. doi: 10.1021/pr300584x 23030644.

34. Ku SC, Schulz BL, Power PM, Jennings MP. The pilin O-glycosylation pathway of pathogenic Neisseria is a general system that glycosylates AniA, an outer membrane nitrite reductase. Biochemical and biophysical research communications. 2009;378(1):84–9. Epub 2008/11/18. doi: 10.1016/j.bbrc.2008.11.025 19013435.

35. Swanson J. Studies on gonococcus infection. IV. Pili: their role in attachment of gonococci to tissue culture cells. J Exp Med. 1973;137(3):571–89. doi: 10.1084/jem.137.3.571 4631989; PubMed Central PMCID: PMC2139381.

36. Virji M, Saunders JR, Sims G, Makepeace K, Maskell D, Ferguson DJ. Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin. Molecular microbiology. 1993;10(5):1013–28. Epub 1993/12/01. doi: 10.1111/j.1365-2958.1993.tb00972.x 7934852.

37. Hobbs MM, Sparling PF, Cohen MS, Shafer WM, Deal CD, Jerse AE. Experimental Gonococcal Infection in Male Volunteers: Cumulative Experience with Neisseria gonorrhoeae Strains FA1090 and MS11mkC. Front Microbiol. 2011;2:123. doi: 10.3389/fmicb.2011.00123 21734909; PubMed Central PMCID: PMC3119411.

38. Jen FE, Warren MJ, Schulz BL, Power PM, Swords WE, Weiser JN, et al. Dual pili post-translational modifications synergize to mediate meningococcal adherence to platelet activating factor receptor on human airway cells. PLoS pathogens. 2013;9(5):e1003377. doi: 10.1371/journal.ppat.1003377 23696740; PubMed Central PMCID: PMC3656113.

39. Jennings MP, Jen FE, Roddam LF, Apicella MA, Edwards JL. Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells. Cellular microbiology. 2011;13(6):885–96. Epub 2011/03/05. doi: 10.1111/j.1462-5822.2011.01586.x 21371235; PubMed Central PMCID: PMC3889163.

40. Vik A, Aspholm M, Anonsen JH, Borud B, Roos N, Koomey M. Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C-terminally tagged pilin: a role for O-linked glycosylation. Molecular microbiology. 2012;85(6):1166–78. Epub 2012/08/14. doi: 10.1111/j.1365-2958.2012.08166.x 22882659.

41. Zhang QY, DeRyckere D, Lauer P, Koomey M. Gene conversion in Neisseria gonorrhoeae: evidence for its role in pilus antigenic variation. Proceedings of the National Academy of Sciences of the United States of America. 1992;89(12):5366–70. doi: 10.1073/pnas.89.12.5366 1351681; PubMed Central PMCID: PMC49292.

42. Power PM, Seib KL, Jennings MP. Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli. Biochemical and biophysical research communications. 2006;347(4):904–8. Epub 2006/07/28. doi: 10.1016/j.bbrc.2006.06.182 16870136.

43. Siddique A, Buisine N, Chalmers R. The transposon-like Correia elements encode numerous strong promoters and provide a potential new mechanism for phase variation in the meningococcus. PLoS Genet. 2011;7(1):e1001277. Epub 2011/02/02. doi: 10.1371/journal.pgen.1001277 21283790; PubMed Central PMCID: PMC3024310.

44. Robertson BD, Frosch M, van Putten JP. The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide. Molecular microbiology. 1993;8(5):891–901. Epub 1993/05/01. doi: 10.1111/j.1365-2958.1993.tb01635.x 8355614.

45. Ison CA, Golparian D, Saunders P, Chisholm S, Unemo M. Evolution of Neisseria gonorrhoeae is a continuing challenge for molecular detection of gonorrhoea: false negative gonococcal porA mutants are spreading internationally. Sexually transmitted infections. 2013;89(3):197–201. Epub 2012/12/18. doi: 10.1136/sextrans-2012-050829 23241969.

46. Kuo CH, Ochman H. The extinction dynamics of bacterial pseudogenes. PLoS Genet. 2010;6(8). Epub 2010/08/12. doi: 10.1371/journal.pgen.1001050 20700439; PubMed Central PMCID: PMC2916853.

47. Heidrich N, Bauriedl S, Barquist L, Li L, Schoen C, Vogel J. The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq. Nucleic Acids Res. 2017;45(10):6147–67. doi: 10.1093/nar/gkx168 28334889; PubMed Central PMCID: PMC5449619.

48. Swartley JS, Marfin AA, Edupuganti S, Liu LJ, Cieslak P, Perkins B, et al. Capsule switching of Neisseria meningitidis. Proceedings of the National Academy of Sciences of the United States of America. 1997;94(1):271–6. Epub 1997/01/07. doi: 10.1073/pnas.94.1.271 8990198; PubMed Central PMCID: PMC19312.

49. Hill DM, Lucidarme J, Gray SJ, Newbold LS, Ure R, Brehony C, et al. Genomic epidemiology of age-associated meningococcal lineages in national surveillance: an observational cohort study. Lancet Infect Dis. 2015;15(12):1420–8. doi: 10.1016/S1473-3099(15)00267-4 26515523; PubMed Central PMCID: PMC4655307.

50. Tonjum T, Freitag NE, Namork E, Koomey M. Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria. Molecular microbiology. 1995;16(3):451–64. Epub 1995/05/01. doi: 10.1111/j.1365-2958.1995.tb02410.x 7565106.

51. Johnston DM, Cannon JG. Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection. Gene. 1999;236(1):179–84. doi: 10.1016/s0378-1119(99)00238-3 10433979.

52. Wattam AR, Davis JJ, Assaf R, Boisvert S, Brettin T, Bun C, et al. Improvements to PATRIC, the all-bacterial Bioinformatics Database and Analysis Resource Center. Nucleic Acids Res. 2017;45(D1):D535–D42. doi: 10.1093/nar/gkw1017 27899627; PubMed Central PMCID: PMC5210524.

53. Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016;44(D1):D457–62. doi: 10.1093/nar/gkv1070 26476454; PubMed Central PMCID: PMC4702792.

54. Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PloS one. 2010;5(6):e11147. doi: 10.1371/journal.pone.0011147 20593022; PubMed Central PMCID: PMC2892488.

55. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution. 2013;30(4):772–80. doi: 10.1093/molbev/mst010 23329690; PubMed Central PMCID: PMC3603318.

56. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–3. doi: 10.1093/bioinformatics/btu033 24451623; PubMed Central PMCID: PMC3998144.

57. Thingholm TE, Larsen MR. Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides. In: vS L., editor. Phospho-Proteomics Methods in Molecular Biology. 1355. New York, NY 2016.

58. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular biology and evolution. 2016;33(7):1870–4. doi: 10.1093/molbev/msw054 27004904.

59. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics (Oxford, England). 2014;30(14):2068–9. Epub 2014/03/20. doi: 10.1093/bioinformatics/btu153 24642063.

60. Price MN, Dehal PS, Arkin AP. FastTree 2—approximately maximum-likelihood trees for large alignments. PloS One. 2010;5(3):e9490. Epub 2010/03/13. doi: 10.1371/journal.pone.0009490 20224823; PubMed Central PMCID: PMC2835736.

61. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MT, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics (Oxford, England). 2015;31(22):3691–3. Epub 2015/07/23. doi: 10.1093/bioinformatics/btv421 26198102; PubMed Central PMCID: PMC4817141.

62. Bovre K, Holten E. Neisseria elongata sp.nov., a rod-shaped member of the genus Neisseria. Re-evaluation of cell shape as a criterion in classification. J Gen Microbiol. 1970;60(1):67–75. doi: 10.1099/00221287-60-1-67 5488467.

63. Muzzi A, Mora M, Pizza M, Rappuoli R, Donati C. Conservation of meningococcal antigens in the genus Neisseria. mBio. 2013;4(3):e00163–13. Epub 2013/06/14. doi: 10.1128/mBio.00163-13 23760461; PubMed Central PMCID: PMC3685207.

Genetika Reprodukční medicína

Článek vyšel v časopise

PLOS Genetics

2019 Číslo 12

Nejčtenější v tomto čísle
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se