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

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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. ​Fig.11 were obtained each time. As shown in Fig. ​Fig.1,1, 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. 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.