Advertisement

Purification and Analysis of Mycobacterial Phosphatidylinositol Mannosides, Lipomannan, and Lipoarabinomannan

  • Kathryn C. Rahlwes
  • Julia Puffal
  • Yasu S. MoritaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)

Abstract

Mycobacteria and related bacteria in the Actinobacteria phylum are unusual in that they produce phosphatidylinositol (PI) as a major phospholipid species. PI can be further modified by glycan polymers, leading to the synthesis of PI mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). Small lipids such as PI and PIMs are extracted with a mixture of chloroform, methanol, and water and analyzed by thin layer chromatography. For larger glycolipids, such as LM and LAM, more hydrophilic solvent is needed for the extraction, and SDS-PAGE is better suited for the analysis. For LM, further structural characterization can be performed by MALDI-TOF mass spectrometry. Precise quantification of PIMs, LM, and LAM can be performed by quantification of glycan staining using analytical software. The metabolic radiolabeling protocol is also described.

Key words

Glycolipids Glycan staining Lipomannan Lipoarabinomannan Phosphatidylinositol mannosides 

Notes

Acknowledgments

This work was supported by a Biomedical Research Grant (RG-414805) from the American Lung Association and a Research Grant from the Pittsfield Anti-Tuberculosis Association to YSM.

References

  1. 1.
    Mishra AK, Driessen NN, Appelmelk BJ et al (2011) Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host-pathogen interaction. FEMS Microbiol Rev 35:1126–1157CrossRefGoogle Scholar
  2. 2.
    Oldenburg R, Demangel C (2017) Pathogenic and immunosuppressive properties of mycobacterial phenolic glycolipids. Biochimie 141:3–8CrossRefGoogle Scholar
  3. 3.
    Ishikawa E, Mori D, Yamasaki S (2017) Recognition of mycobacterial lipids by immune receptors. Trends Immunol 38:66–76CrossRefGoogle Scholar
  4. 4.
    Källenius G, Correia-Neves M, Buteme H et al (2016) Lipoarabinomannan, and its related glycolipids, induce divergent and opposing immune responses to Mycobacterium tuberculosis depending on structural diversity and experimental variations. Tuberculosis 96:120–130CrossRefGoogle Scholar
  5. 5.
    Achkar JM, Lawn SD, Moosa MY et al (2011) Adjunctive tests for diagnosis of tuberculosis: serology, ELISPOT for site-specific lymphocytes, urinary lipoarabinomannan, string test, and fine needle aspiration. J Infect Dis 204(Suppl 4):S1130–S1141CrossRefGoogle Scholar
  6. 6.
    Sabur NF, Esmail A, Brar MS et al (2017) Diagnosing tuberculosis in hospitalized HIV-infected individuals who cannot produce sputum: is urine lipoarabinomannan testing the answer? BMC Infect Dis 17:803CrossRefGoogle Scholar
  7. 7.
    LaCourse SM, Cranmer LM, Njuguna IN et al (2018) Urine tuberculosis lipoarabinomannan predicts mortality in hospitalized human immunodeficiency virus-infected children. Clin Infect Dis 66:1798–1801CrossRefGoogle Scholar
  8. 8.
    Shah M, Hanrahan C, Wang ZY et al (2016) Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in HIV-positive adults. Cochrane Database Syst Rev 19:CD011420Google Scholar
  9. 9.
    Ballou CE, Vilkas E, Lederer E (1963) Structural studies on the myo-inositol phospholipids of Mycobacterium tuberculosis (var. bovis, strain BCG). J Biol Chem 238:69–76PubMedGoogle Scholar
  10. 10.
    Lee YC, Ballou CE (1964) Structural studies on the myo-inositol mannodides from the glycolipids of Mycobacterium tuberculosis and Mycobacterium phlei. J Biol Chem 239:1316–1327PubMedGoogle Scholar
  11. 11.
    Morita YS, Fukuda T, Sena CBC et al (2011) Inositol lipid metabolism in mycobacteria: biosynthesis and regulatory mechanisms. Biochim Biophys Acta 1810:630–641CrossRefGoogle Scholar
  12. 12.
    Sancho-Vaello E, Albesa-Jové D, Rodrigo-Unzueta A et al (2017) Structural basis of phosphatidyl-myo-inositol mannosides biosynthesis in mycobacteria. Biochim Biophys Acta 1862:1355–1367CrossRefGoogle Scholar
  13. 13.
    Jankute M, Cox JAG, Harrison J et al (2015) Assembly of the mycobacterial cell wall. Annu Rev Microbiol 69:405–423CrossRefGoogle Scholar
  14. 14.
    Daffé M, Crick DC, Jackson M (2014) Genetics of capsular polysaccharides and cell envelope (glyco)lipids. Microbiol Spectr 2. MGM2–0021–2013Google Scholar
  15. 15.
    Angala SK, Belardinelli JM, Huc-Claustre E et al (2014) The cell envelope glycoconjugates of Mycobacterium tuberculosis. Crit Rev Biochem Mol Biol:1–39Google Scholar
  16. 16.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682CrossRefGoogle Scholar
  17. 17.
    Patterson JH, Waller RF, Jeevarajah D et al (2003) Mannose metabolism is required for mycobacterial growth. Biochem J 372:77–86CrossRefGoogle Scholar
  18. 18.
    Haites RE, Morita YS, McConville MJ et al (2005) Function of phosphatidylinositol in mycobacteria. J Biol Chem 280:10981–10987CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kathryn C. Rahlwes
    • 1
  • Julia Puffal
    • 1
  • Yasu S. Morita
    • 1
    Email author
  1. 1.Department of MicrobiologyUniversity of MassachusettsAmherstUSA

Personalised recommendations