This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Andreu, N.
Right arrow Articles by Sirakova, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Andreu, N.
Right arrow Articles by Sirakova, T.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, March 2004, p. 1379-1380, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1379-1380.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Neutral Red Staining of Cells of a Sulfolipid-Deficient Mycobacterium tuberculosis pks2 Mutant Proves that Sulfolipids Are Not Responsible for This Cytochemical Reaction


arrow
LETTER
 
In 1948 Dubos and Middlebrook reported that the cells of the virulent H37Rv Mycobacterium tuberculosis strain were able to reduce neutral red and become red in color (NR+), whereas the cells of the avirulent H37Ra M. tuberculosis were not (NR-) (3, 7). During the following decade, neutral red staining was performed on M. tuberculosis clinically isolated strains, Mycobacterium bovis, and environmental mycobacteria (1, 4-6). Recently, we have also performed this staining in M. tuberculosis strains (11). The different studies showed a concordance of results; that is, cells of the virulent M. tuberculosis and M. bovis strains stained red, while cells of the majority of environmental mycobacteria remained unstained.

In 1959 Middlebrook and coworkers studied the cellular components responsible for neutral red staining and established a positive correlation between the sulfolipid content and the neutral-red-staining cells (8). Although the results of this single work were not conclusive, the idea that sulfolipids are responsible for the NR+ cells of the M. tuberculosis virulent strains has been widely quoted and accept- ed. However, no direct evidence has confirmed this conclusion.

Recently, a pks2 mutant of M. tuberculosis H37Rv has been generated that is unable to produce hepta- and octamethyl phthioceranic acids, the major acyl constituents of sulfolipids, and is thus unable to produce sulfolipids (10). The availability of this mutant made it possible to test directly whether sulfolipids are responsible for neutral red staining.

The M. tuberculosis strains used in this study were the two reference strains H37Rv (ATCC 27294) and H37Ra (ATCC 25177), the H37Rv pks2 mutant, and the H37Rv msl3 mutant (2). This last mutant is able to produce sulfolipids but is deficient in polyacyl and diacyl trehaloses and has been used as a control together with the H37Rv wild-type and H37Ra strains.

The neutral red staining was performed in a test tube as described previously (11). The strains were tested several times, and the results shown in Fig. 1 were obtained each time. As shown in Fig. 1, cells of the pks2 mutant were NR+, as were cells of the wild-type H37Rv strain and the msl3 mutant. H37Ra cells were NR-. Consequently, we can conclude that sulfolipids are not responsible for the ability of the cells of virulent M. tuberculosis strains to become red stained.



View larger version (101K):
[in this window]
[in a new window]
  		FIG. 1. Neutral red staining in a test tube. From left to right, the M. tuberculosis strains are pks2, msl3, H37Ra, and H37Rv.

In the past a correlation was accepted between neutral red staining and virulence in M. tuberculosis, and as sulfolipids were thought to be responsible for this reaction, they were considered a virulence factor until recently. However, a new study has shown that the M. tuberculosis pks2 sulfolipid-deficient mutant showed the same degree of virulence as the wild type (9). The present finding that sulfolipids are not involved in neutral red staining is consistent with this result. The mycobacterial compound(s) responsible for neutral red staining in M. tuberculosis, with a possible correlation to virulence, remains to be identified.


arrow
ACKNOWLEDGMENTS
 
This work was supported by grant QLK2-CT-1999-01093 from the European Union, grant 2002SGR-00099 from the Generalitat de Catalunya, and grants AI46582 and AI35272 from the U.S. National Institutes of Health.


arrow
REFERENCES
 
    1
  1. Desbordes, J., and E. Fournier. 1954. Action des substances colorantes sur les mycobactéries. I. Colorants basiques. Étude de la cinétique de la réaction. Ann. Inst. Pasteur (Paris) 86:657-660.[Medline]
    2. 2
  2. Dubey, V. S., Sirakova, T. D., and P. E. Kolattukudy. 2002. Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregation. Mol. Microbiol. 45:1451-1459.[CrossRef][Medline]
    3. 3
  3. Dubos, R. J., and G. Middlebrook. 1948. Cytochemical reaction of virulent tubercle bacilli. Am. Rev. Tuberc. 58:698-699.[Medline]
    4. 4
  4. Haudoroy, M. P., and M. Y. Postenak. 1949. Sur une réaction permettant de distinguer les mycobactéries virulentes des mycobactéries avirulentes. C. R. Hebd. Séances Acad. Sci. 228:781.
    5. 5
  5. Haudoroy, P., and M. Cevey. 1953. Importance des réactions cyto-chimiques pour la détermination de la virulence des mycobactéries et intérèt pour la classification. Ann. Inst. Pasteur (Paris) 84:1034-1035.[Medline]
    6. 6
  6. Hughes, D., M. Moss, M. Hood, and M. Henson. 1954. Virulence of Mycobacterium tuberculosis: evaluation of a test using neutral red indicator. Am. J. Clin. Pathol. 24:621-625.[Medline]
    7. 7
  7. Lillie, R. D. 1977. Quinone-imines dyes and azines, p. 376-392. In R. D. Lillie (ed.), H. J. Conn's biological stains, 9th ed. Williams and Wilkins, Baltimore, Md.
    8. 8
  8. Middlebrook, G., C. M. Coleman, and W. B. Schaefer. 1959. Sulfolipid from virulent tubercle bacilli. Proc. Natl. Acad. Sci. USA 45:1801-1804.[Free Full Text]
    9. 9
  9. Rousseau, C., O. C. Turner, E. Rush, Y. Bordat, T. D. Sirakova, P. E. Kolattukudy, I. M. Orme, B. Gicquel, and M. Jackson. 2003. Sulfolipid deficiency does not affect the virulence of Mycobacterium tuberculosis. Infect. Immun. 71:4684-4690.[Abstract/Free Full Text]
    10. 10
  10. Sirakova, T. D., A. K. Thirumala, V. S. Dubey, H. Sprecher, and P. E. Kolattukudy. 2001. The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis. J. Biol. Chem. 276:16833-16839.[Abstract/Free Full Text]
    11. 11
  11. Soto, C. Y., N. Andreu, I. Gibert, and M. Luquin. 2002. Simple and rapid differentiation of Mycobacterium tuberculosis H37Ra from M. tuberculosis clinical isolates through two cytochemical tests using neutral red and nile blue stains. J. Clin. Microbiol. 40:3021-3024.[Abstract/Free Full Text]
Núria Andreu
Isidre Gibert
Institut de Biotecnologia i de BiomedicinaUniversitat Autònoma de Barcelona08193 Bellaterra (Barcelona), Spain

Marina Luquin*
Departament de Genètica i de Microbiologia
Universitat Autònoma de Barcelona
08193 Bellaterra (Barcelona), Spain

Pappachan E. Kolattukudy
Tatiana Sirakova
Neurobiotechnology Center and Departments of Biochemistry  and Molecular and Cellular Biochemistry
The Ohio State University
Columbus, OH 43210

* Phone: 34 935812540,Fax: 34 935812387,E-mail: Marina.Luquin{at}uab.es

Journal of Clinical Microbiology, March 2004, p. 1379-1380, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1379-1380.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Andreu, N.
Right arrow Articles by Sirakova, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Andreu, N.
Right arrow Articles by Sirakova, T.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»