The gross structural features of five families of multiacylated trehalose 2-sulfates elaborated by Mycobacterium tuberculosis strain H37Rv are described. The principal sufatide SL-I is a 2,3,6,6'-tetraacyl-alpha,alphs'-D-trehalose 2'-sulfate, whose component carboxylate substituents (and homolgy) were previously established. In the present study the specific locations of the acyl substituents were assigned. The desulfated glycolipid (SL-I-CF) was methanolyzed on a column of diethylaminoethylcellulose (free base form), affording tri-, di-, and monoacylated trehalose mixtures. The most abundant diacyltrehalose generated was identified as 6,6'-bis-(2,4,6,8,10,12,14,16-octamethyl-17-hydroxydotriaconta-noyl)trehalose (6,6'-bis(C40-hydroxyphthioceranoyl)trehalose), along with lower and higher homologues.A small amount (about 15%) of the unhydroxylated analogue (phthioceranate) was also recognized. From the monoacylated carbohydrate mixture (chiefly 6-(C40-hydroxyphthioceranoyl)trehalose) surviving trehalose monopalmitate(s) were isolated by preparative gas chromatography of the trimethylsilylated products. Trehalose 2-palmitate was identified as the principal component. Small amounts of the 3 isomer may also be present, but no 6-palmitate was detectable. Gentle acidic solvolysis, which minimizes the possibility of acyl migrations, afforded a different diacyltrehalose, identified by mass spectrometry of the permethylated derivative as principally 2-palmitoyl(stearoyl)-3-phthioceranoyltrehalose. A variant in which hydroxyphthioceranate substitutes at the 3 position was also recognized. The results indicate that the biological acylation processes at the trehalose core are not entirely specific, but instead yield an SL-I family, for the chief member of which a logical structural expression is deduced.

No abstract

Lipid compositions of 40 patient isolates of Mycobacterium tuberculosis derived from Madras, Burma, Rangoon, and East Africa were studied, and two major populations of tubercle bacilli were distinguished. Nearly all of the strains previously designated as attenuated for the guinea pig (D. A. Mitchison) are characterized by a specific phenolic phthiocerol diester which is identified with the aglycone moieties of mycosides A and B (and probably G). This lipid (AI) was not seen in any of the more virulent strains. Thus, presence of AI may be taken as definitive for attenuation (P << 0.001). Phthiocerol dimycoserosate (DIM), a companion substance, is ubiquitous for the series of 40 strains. However, a dramatic attenuation found in a DIM-less H37Rv mutant may support a role for this substance in the virulent state. Since mycosides A, G, and B seem to be restricted to certain chromogenic and bovine species, respectively, we speculate that lysogenization or transduction of fully virulent M. tuberculosis may have provided the genetic determinants for attenuation and Al synthesis, and thus led to the two classes of tubercle bacilli.

From examination of some 40 patient strains of Mycobacterium tuberculosis , a statistically very significant correlation (Spearman's rho) can be drawn between the root index of virulence for the guinea pig (D. A. Mitchison) and the ability of the individual strains to elaborate strongly acidic lipids (SAL) in culture. These include both sulfatides (SL) and phospholipids (PL). Since essentially all, if indeed not all, of the virulent and only few attenuated strains are prolific in elaborating SAL, this criterion may be a necessary requirement for the expression of virulence in M. tuberculosis. Tested by chi-square, this premise is seen to be statistically and pragmatically highly significant. We speculate that SL may contribute to the pathogenesis of tuberculosis because of a demonstrable activity directed against host liver mitochondrial membranes (manuscript in preparation) and its synergistic potentiation of the specific toxicity of trehalose dimycolate (cord factor). The activity may also be expressed against phagosomal and lysosomal membranes within macrophages. Because of their strongly anionic character, SL and PL may interact with cationic sites on lysosomal hydrolases with resultant immobilization and/or inactivation of the enzymes. By a similar mechanism, these ionic lipids may alter the activity of bactericidal basic proteins, previously recognized in the lysosomal armamentarium. Since a minor but significant fraction of demonstrably attenuated strains is nevertheless prolific in SL or SAL elaboration, this facility alone is evidently not a sufficient criterion for expression of virulence.

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes ofl-[14C]alanine and [14C]taurine in thin limbs of Henle’s loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle’s loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle’s loop may occur via a paracellular route.