Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host–pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0 -deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro , despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.
The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope ( 34 SO 4 2؊ ). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.T he modification of primary and secondary metabolites by the addition or removal of sulfate can have a profound influence on their biological properties (1-5). Typically, sulfated molecules are directed outside the cell, where they serve as modulators of cell-cell interactions. As a notable example pertinent to human health, sulfation of the glycans of endothelial CD34 engenders high-affinity binding with the leukocyte adhesion molecule L-selectin, facilitating an interaction that eventually leads to the recruitment of lymphocytes into peripheral lymph nodes (6). Similarly, sulfation of tyrosyl residues found on the chemokine receptor CCR5 is a modification required for binding of HIV gp120 and therefore efficient viral entry (7).The roles of sulfated compounds in prokaryotes and other microbes are less clear. In one well-characterized case, however, sulfation acts as a modulator of cell-cell communication, similar to its role in eukaryotes. The nitrogen-fixing bacterium Sinorhizobium meliloti utilizes a sulfated glycolipid as a secondary messenger to induce root nodulation in its plant host alfalfa (4,8). Mutants lacking the sulfotransferase that installs this sulfate ester are unable to induce root nodulation in alfalfa but gain the ability to colonize the roots of vetch. That sulfation of a single glycolipid plays such a vital role in nitrogen fixation has far-reaching implications for the agricultural community and presents a possible target for chemical or genetic engineering.Sulfation may also be relevant to the process of bacterial pathogenesis (9). Several mycobacteria, including the human pathogens Mycobacterium tuberculosis and Mycobacterium avium, are known to produce sulfated compounds. One example is Sulfatide-1 (SL-1, Fig. 1A), a sulfated glycolipid from M....
Sulfolipid-I (SL-I) is an abundant metabolite found in the cell wall of Mycobacterium tuberculosis that is comprised of a trehalose 2-sulfate core modified with four fatty acyl substituents. The correlation of its abundance with the virulence of clinical isolates suggests a role for SL-I in pathogenesis, although its biological functions remain unknown. Here we describe the synthesis of a SL-I analogue bearing unnatural lipid substituents. A key feature of the synthesis was application of an intramolecular aglycon delivery reaction to join two differentially protected glucose monomers, one prepared with a novel α-selective glycosylation. The route developed for the model compound can be readily extended to the synthesis of native SL-I as well as additional analogues for use in the investigation of SL-I's functions.
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