Bordetella bronchiseptica Glycosyltransferase Core Mutants Trigger Changes in Lipid A Structure

  • Adriana C. Casabuono
  • Federico Sisti
  • Julieta Fernández
  • Daniela Hozbor
  • Alicia S. CoutoEmail author
Research Article


Bordetella bronchiseptica, known to infect animals and rarely humans, expresses a lipopolysaccharide that plays an essential role in host interactions, being critical for early clearance of the bacteria. On a B. bronchiseptica 9.73 isolate, mutants defective in the expression of genes involved in the biosynthesis of the core region were previously constructed. Herein, a comparative detailed structural analysis of the expressed lipids A by MALDI-TOF mass spectrometry was performed. The Bb3394 LPS defective in a 2-amino-2-deoxy-d-galacturonic acid lateral residue of the core presented a penta-acylated diglucosamine backbone modified with two glucosamine phosphates, similar to the wild-type lipid A. In contrast, BbLP39, resulting in the interruption of the LPS core oligosaccharide synthesis, presented lipid A species consisting in a diglucosamine backbone N-substituted with C14:0(3-O-C12:0) in C-2 and C14:0(3-O-C14:0) in C-2′, O-acylated with C14:0(3-O-C10:0(3-OH) in C-3′ and with a pyrophosphate in C-1. Regarding Bb3398 also presenting a rough LPS, the lipid A is formed by a hexa-acylated diglucosamine backbone carrying one pyrophosphate group in C-1 and one phosphate in C-4′, both substituted with ethanolamine groups. As far as we know, this is the first description of a phosphoethanolamine modification in B. bronchiseptica lipid A. Our results demonstrate that although gene deletions were not directed to the lipid A moiety, each mutant presented different modifications. MALDI-TOF mass spectrometry was an excellent tool to highlight the structural diversity of the lipid A structures biosynthesized during its transit through the periplasm to the final localization in the outer surface of the outer membrane.

Graphical Abstract


B. bronchiseptica Lipopolysaccharide UV-MALDI-TOF MS Lipid A modifications 



This work was supported by CONICET, Grant PIP-11220110100660, and by ANPCyT, (Grant PICT 2013-0736 and 2013-0092) and UBA Grant 20020130100476BA. The Ultraflex II (Bruker) TOF/TOF mass spectrometer was supported by ANPCyT Grant PME 125 (CEQUIBIEM). A.C. and F.S. are members of the Scientific Career of CONICET.

Supplementary material

13361_2019_2233_MOESM1_ESM.doc (210 kb)
ESM 1 (DOC 210 kb)
13361_2019_2233_MOESM2_ESM.doc (78 kb)
ESM 2 (DOC 78 kb)
13361_2019_2233_MOESM3_ESM.docx (85 kb)
ESM 3 (DOCX 85 kb)
13361_2019_2233_MOESM4_ESM.docx (117 kb)
ESM 4 (DOCX 116 kb)


  1. 1.
    Whitfield, C., Trent, M.S.: Biosynthesis and export of bacterial lipopolysaccharides. Annu. Rev. Biochem. 83, 99–128 (2014)CrossRefGoogle Scholar
  2. 2.
    Caroff, M., Karibian, D.: Structure of bacterial lipopolysaccharides. Carbohydr. Res. 338, 2431–2447 (2003)CrossRefGoogle Scholar
  3. 3.
    Trent, M.S., Stead, C.M., Tran, A.X., Hankins, J.V.: Diversity of endotoxin and its impact on pathogenesis. J. Endotoxin Res. 12, 205–223 (2006)Google Scholar
  4. 4.
    Zhou, X., Gao, X., Broglie, P.M., Kebaier, C., Anderson, J.E., Thom, N., Apicella, M.A., Sempowski, G.D., Duncan, J.A.: Hexa-acylated lipid A is required for host inflammatory response to Neisseria gonorrhoeae in experimental gonorrhea. Infect. Immun. 82(1), 184–192 (2014)CrossRefGoogle Scholar
  5. 5.
    Mattoo, S., Cherry, J.D.: Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin. Microbiol. Rev. 18, 326–382 (2005)CrossRefGoogle Scholar
  6. 6.
    Garbal, M., Adaszek, L., Lyp, P., Frymus, J., Winiarczyk, M., Winiarczyk, S.: Occurence of Bordetella bronchiseptica in domestic cats with upper respiratory tract infections. Pol. J. Vet. Sci. 19(2), 353–358 (2016)CrossRefGoogle Scholar
  7. 7.
    Cherry, J.D., Seaton, B.L.: Patterns of Bordetella parapertussis respiratory illnesses: 2008-2010. Clin Infect Dis. 54(4), 534–537 (2012)CrossRefGoogle Scholar
  8. 8.
    Preston, A., Petersen, B.O., Duus, J., Kubler-Kielb, J., Ben-Menachem, G., Li, J., Vinogradov, E.: Complete structures of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharides. J. Biol. Chem. 281, 18135–18144 (2006)CrossRefGoogle Scholar
  9. 9.
    Sisti, F., Fernández, J., Rodríguez, M.E., Lagares, A., Guiso, N., Hozbor, D.F.: In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipo-polysaccharide structure. Infect. Immun. 70, 1791–1798 (2002)CrossRefGoogle Scholar
  10. 10.
    Sisti, F., Fernández, J., Higgins, S.C., Casabuono, A., Couto, A., Mills, K.H.G., Hozbor, D.: A deep rough type structure in Bordetella bronchiseptica lipopolysaccharide modulates host immune responses. Microbiol. Immunol. 55, 847–854 (2011)CrossRefGoogle Scholar
  11. 11.
    Caroff, M., Aussel, L., Zarrouk, H., Martin, A., Richards, J.C., Thérisod, H., Perry, M.B., Karibian, D.: Structural variability and originality of the Bordetella endotoxins. J. Endotoxin. Res. 7, 63–68 (2001)CrossRefGoogle Scholar
  12. 12.
    MacArthur, I., Mann, P.B., Harvill, E.T., Preston, A.: IEIIS meeting minireview: Bordetella evolution: lipid A and toll-like receptor 4. J. Endotoxin Res. 13, 243–247 (2007)CrossRefGoogle Scholar
  13. 13.
    Zhang, X., Rodríguez, M.E., Harvill, E.T.: O antigen allows B. parapertussis to evade B. pertussis vaccine-induced immunity by blocking binding and functions of cross-reactive antibodies. PLoS One. 4, 9 e6989 (2009)CrossRefGoogle Scholar
  14. 14.
    Aussel, L., Therisod, H., Karibian, D., Perry, M.B., Bruneteau, M., Caroff, M.: Novel variation of lipid A structures in strains of different Yersinia species. FEBS Lett. 465, 87–92 (2000)CrossRefGoogle Scholar
  15. 15.
    Zarrouk, H., Karibian, D., Bodie, S., Perry, M.B., Richards, J.C., Caroff, M.: Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution. J. Bacteriol. 179, 3756–3760 (1997)CrossRefGoogle Scholar
  16. 16.
    MacArthur, I., Jones, J.W., Goodlett, D.R., Ernst, R.K., Preston, A.: Role of pagL and lpxO in Bordetella bronchiseptica lipid A biosynthesis. J. Bacteriol. 193, 4726–4735 (2011)CrossRefGoogle Scholar
  17. 17.
    Preston, A., Maxim, E., Toland, E., Pishko, E.J., Harvill, E.T., Caroff, M., Maskell, D.J.: Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract. Mol. Microbiol. 48, 725–736 (2003)CrossRefGoogle Scholar
  18. 18.
    Basheer, S.M., Guiso, N., Tirsoaga, A., Caroff, M., Novikov, A.: Structural modifications occurring in lipid A of Bordetella bronchiseptica clinical isolates as demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 25, 1075–1081 (2011)CrossRefGoogle Scholar
  19. 19.
    Rolin, O., Muse, S.J., Safi, C., Elahi, S., Gerdts, V., Hittle, L.E., Ernst, R.K., Harvill, E.T., Preston, A.: Enzymatic modification of lipid A by ArnT protects Bordetella bronchiseptica against cationic peptides and is required for transmission. Infect. Immun. 82, 491–499 (2014)CrossRefGoogle Scholar
  20. 20.
    Preston, A., Thomas, R., Maskell, D.J.: Mutational analysis of the Bordetella pertussis wlb LPS biosynthesis locus. Microb. Pathog. 33, 91–95 (2002)CrossRefGoogle Scholar
  21. 21.
    Sisti, F., Fernandez, J., Cordero, A., Casabuono, A., Couto, A., Hozbor, D.: Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity. Bioorg. Med. Chem. Lett. 27, 432–436 (2017)CrossRefGoogle Scholar
  22. 22.
    Raetz, C.R., Guan, Z., Ingram, B.O., Six, D.A., Song, F., Wang, X., Zhao, J.: Discovery of new biosynthetic pathways: the lipid A story. J. Lipid Res. 50(Suppl), S103–S108 (2009)CrossRefGoogle Scholar
  23. 23.
    Darveau, R.P., Hancock, R.E.: Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155(2), 831–838 (1983)Google Scholar
  24. 24.
    Lukasiewicz, J., Jachymek, W., Niedziela, T., Kenne, L., Lugowski, C.: Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides. J. Lipid Res. 51, 564–574 (2010)CrossRefGoogle Scholar
  25. 25.
    Domon, B., Costello, C.E.: A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397–409 (1988)CrossRefGoogle Scholar
  26. 26.
    Jones, J.W., Shaffer, S.A., Ernst, R.K., Goodlett, D.R., Turecek, F.: Determination of pyrophosphorylated forms of lipid A in Gram-negative bacteria using a multivaried mass spectrometric approach. Proc. Natl. Acad. Sci. U. S. A. 105, 12742–12747 (2008)CrossRefGoogle Scholar
  27. 27.
    Marr, N., Hajjar, A.M., Shah, N.R., Novikov, A., Yam, C.S., Caroff, M., Fernandez, R.C.: Substitution of the Bordetella pertussis lipid a phosphate groups with glucosamine is required for robust NF-κB activation and release of proinflammatory cytokines in cells expressing human but not murine toll-like receptor 4-MD-2-CD14. Infect Immun. 78, 2060–2069 (2010)CrossRefGoogle Scholar
  28. 28.
    Marr, N., Tirsoaga, A., Blanot, D., Fernandez, R., Caroff, M.: Glucosamine found as a substituent of both phosphate groups in Bordetella lipid A backbones: role of a BvgAS-activated ArnT ortholog. J. Bacteriol. 190(12), 4281–4290 (2008)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Facultad de Ciencias Exactas y Naturales, Departamento de Química Orgánica - Consejo Nacional de Investigaciones Científicas y Técnicas, Centro de Investigación en Hidratos de Carbono (CIHIDECAR)Universidad de Buenos AiresBuenos AiresArgentina
  2. 2.Instituto de Biotecnología y Biología Molecular CCT La Plata CONICETFacultad de Ciencias Exactas UNLPLa PlataArgentina

Personalised recommendations