Intracellular parasites (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, and some Chlamydiae) may promote their survival within the host by acting from within phagosomes to prevent phagolysosome formation, thus avoiding exposure to the lysosomal hydrolases. The present studies demonstrate that when sulfatides of M. tuberculosis (anionic trehalose glycolipids largely responsible for the neutral red reactivity of virulent strains) are administered to cultured mouse peritoneal macrophages, they accumulate in the secondary lysosomes, which are rendered incompetent for fusion with phagosomes containing suitable target particles such as viable yeasts. This antifusion effect is also exhibited when small amounts of sulfatide are introduced directly into phagosomes by attachment to the target yeasts prior to their ingestion. The sulfatides evidently exert a selective inhibitory influence on membrane fusion, analogous to what occurs typically when macrophage cultures are infected with tubercle baci li. This effect may be due to ionic interaction between the polyanionic micelles of bacterial sulfatide and organelle membranes, modifying the latter and inducing dysfunction. The sulfatides of Mycobacterium tuberculosis (1) have been characterized as several closely related glycolipids-more specifically, multiacylated trehalose 2-sulfates (2). Because a significant correlation has been established between production of strongly acidic lipids, including sulfatides (SL), and order of "infectivity" (3) or virulence (4) for some 40 strains of M. tuberculosis, it was inferred that these substances might be a factor in the virulence of these organisms.We have previously speculated that in parasitized host cells the glycolipids might influence phagosomal or lysosomal membranes, so as to interfere with their capability to fuse and form phagolysosomes (4). Such interference could underlie a survival mechanism for some intracellular parasites, as exhibited by viable virulent tubercle bacilli during infection of cultured macrophages (5, 6j. A similar failure of lysosomes to fuse with phagocytic vacuoles containing viable Chlamydiae (7) or Toxoplasma gondii (8) has also been established.Our present studies with purified sulfatides from M. tuberculosis strain H37Rv show that they are potent inhibitors of phagolysosome formation in cultured mouse peritoneal macrophages. The inhibition may involve dysfunction in either phagosomal or lysosomal membranes-or both. We present a preliminary report of these investigations. MATERIALS AND METHODSTissue Culture and Related Procedures. Unelicited peritoneal macrophages of normal female mice of the albino P strain were maintained as coverslip monolayers in Chang medium (9) in Leighton tubes as described previously (5, 10). The monolayers were used after about 10 days in culture.Sulfatides of M. tuberculosis. Thirty-day surface cultures of strain H37Rv were harvested and the principal sulfatide (SL-I), a 2,3,6,6'-tetraacyl trehalose 2'-sulfate (molecular weight about 2400), was recovered an...

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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.

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