Mycobacterium tuberculosis synthesizes specific polyketide lipids that interact with the host and are required for virulence. Using a mass spectrometric approach to simultaneously monitor hundreds of lipids, we discovered that the size and abundance of two lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1 (SL-1), are controlled by the availability of a common precursor, methyl malonyl CoA (MMCoA). Consistent with this view, increased levels of MMCoA led to increased abundance and mass of both PDIM and SL-1. Furthermore, perturbation of MMCoA metabolism attenuated pathogen replication in mice. Importantly, we detected increased PDIM synthesis in bacteria growing within host tissues and in bacteria grown in culture on odd-chain fatty acids. Because M. tuberculosis catabolizes host lipids to grow during infection, we propose that growth of M. tuberculosis on fatty acids in vivo leads to increased flux of MMCoA through lipid biosynthetic pathways, resulting in increased virulence lipid synthesis. Our results suggest that the shift to host lipid catabolism during infection allows for increased virulence lipid anabolism by the bacterium.lipid virulence factor ͉ metabolic flux ͉ pathogenesis ͉ PDIM ͉ sulfolipid-1

pathogenesis ͉ biochemistry ͉ glycolipid ͉ sulfation T he thick Mycobacterium tuberculosis (M. tb) cell wall consists of numerous glycolipids that are distinctive to the mycobacterial genus, including phosphatidylinositol mannosides, trehalose mycolates, and lipoarabinomannans (1). These molecules are essential for many of the characteristics that distinguish mycobacterial pathogenesis, such as the inhibition of phagosomal maturation, drug resistance, and alteration of the host immune response (2-6). A family of cell surface sulfated lipids (dubbed sulfatides) were identified in M. tb extracts and correlated to strain virulence (7-9). The most abundant sulfatide, termed Sulfolipid-1 (SL-1), consists of a trehalose core, four fatty acyl groups, and a sulfate ester (Fig. 1A) (10-13). Despite the discovery of SL-1 nearly 50 years ago, the biological function of the molecule is not known. Conflicting reports suggest a role for SL-1 in superoxide (O 2 Ϫ ) release from human neutrophils or monocytes, alteration of trehalose dimycolate toxicity, and inhibition of trehalose dimycolate-induced macrophage recruitment (14-19). The relevance of these studies to the physiological role of SL-1 in M. tb infection is debatable.Although the role of SL-1 remains elusive, advances in genetics and metabolite analysis have sped the discovery of genes, proteins, and intermediates associated with SL-1 biosynthesis (20). Currently, three proteins are known to be involved in SL-1 assembly: Stf0, Pks2, and MmpL8. The sulfotransferase Stf0 sulfates trehalose at the 2-position, forming trehalose-2-sulfate (T2S), thereby initiating SL-1 biosynthesis (21). Meanwhile, the polyketide synthase Pks2 synthesizes the phthioceranoyl and hydroxyphthioceranoyl lipids that occupy the 6-, 6Ј-, and 3Ј-positions of SL-1 (Fig. 1 A) (22). The proteins responsible for transfer of the Pks2 products and the palmitoyl group to the T2S core, and the order in which these lipids are added, have not yet been defined.Insight into the order of lipid addition came from characterization of the putative lipid transporter MmpL8 (23,24). A mutant strain, ⌬mmpL8, lacks SL-1 but accumulates the diacylated intermediate SL 1278 (named for its observed mass) inside the cell (Fig. 1B). This intermediate possesses two of the four SL-1-associated lipids: a hydroxyphthioceranoyl group at the 3Ј-position and a palmitoyl group at the 2Ј-position (24). SL 1278 was recently found to be an immunostimulant in human tuberculosis patients (25). The glycolipid is presented on the surface of M. tb-infected antigen-presenting cells by CD1b, a member of the MHC class I-like CD1 family. Intriguingly, the ⌬mmpL8 mutant, which lacks SL-1 but accumulates SL 1278 , shows attenuated virulence in mice (23,24). By contrast, a ⌬pks2 mutant, which lacks both SL-1 and SL 1278 , is indistinguishable from WT M. tb in mice and guinea pigs (23,26). These observations suggest that SL 1278 , and possibly other SL-1 intermediates, modulate M. tb pathogenesis.In our effort to define the functions of M. tb sulf...

Background: Sulfolipid-1 (SL-1) is a Mycobacterium tuberculosis outer membrane lipid whose biosynthesis is not fully understood.Results: Chp1 catalyzes two acyl transfer reactions to form SL-1. Sap modulates SL-1 levels and transmembrane transport.Conclusion: The activities of Chp1 and Sap complete the SL-1 pathway.Significance: Lipid biosynthesis and transport are coupled at the membrane interface by multiple proteins that may regulate substrate specificity and flux.

Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans, has a complex cell wall that contains a number of unique glycolipids intimately linked to mycobacterial pathogenesis (1, 2). The biosynthesis of many of these virulence factors, including the trehalose mycolates, phenolic glycolipids, and sulfolipid-1 (SL-1), 3 is largely understood (3-5). In contrast, relatively little is known about the biosynthesis of other prominent M. tuberculosis glycolipids, such as di-, tri-, and polyacyltrehaloses. These acyltrehaloses are located in the outer surface of the cell wall and contain di-and tri-methyl branched fatty acids that are only found in pathogenic species of mycobacteria (6, 7). Previous studies suggest a role for these glycolipids in anchoring the bacterial capsule, which impedes phagocytosis by host cells (6). The major polyacyltrehalose (PAT) of M. tuberculosis, also referred to as pentaacyl or polyphthienoyl trehalose, consists of five acyl chains, four mycolipenic (phthienoic) acids and one fully saturated fatty acid, linked to trehalose (Fig. 1A) (8). The mycolipenic acid side chains of PAT are products of the polyketide synthase gene pks3/4 (7). Disruption of pks3/4 (also referred to as msl3 (7)) abolishes PAT biosynthesis and causes cell aggregation. At present, the remaining proteins required for PAT assembly have not been characterized.Interestingly, the PAT biosynthetic gene cluster strongly resembles that of SL-1, which is a structurally similar trehalosebased glycolipid unique to pathogenic mycobacteria (Fig. 1B) (9). Both gene clusters contain polyketide synthase (pks), ...

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.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces unique sulfated metabolites associated with virulence. One such metabolite from M. tuberculosis lipid extracts, S881, has been shown to negatively regulate the virulence of M. tuberculosis in mouse infection studies, and its cell-surface localization suggests a role in modulating host-pathogen interactions. However, a detailed structural analysis of S881 has remained elusive. Here we use high resolution, high mass accuracy, and tandem mass spectrometry to characterize the structure of S881. Exact mass measurements showed that S881 is highly unsaturated, tandem mass spectrometry indicated a polyisoprene-derived structure, and characterization of synthetic structural analogs confirmed that S881 is a previously-undescribed sulfated derivative of dihydromenaquinone-9, the primary quinol electron carrier in M. tuberculosis. To our knowledge, this is the first example of a sulfated menaquinone produced in any prokaryote. Together with previous studies, these findings suggest that this redox cofactor may play a role in mycobacterial pathogenesis.Tuberculosis (TB) affects approximately one third of the world's population and kills approximately two million people a year (1). In order to be an effective pathogen, Mycobacterium tuberculosis, the causative agent of TB, must not only survive the initial onslaught of the host immune response, but also carefully modulate adaptive immunity to allow for bacterial persistence. Sulfated metabolites have been shown to serve as signaling molecules between both symbiotic and pathogenic bacteria and their hosts (2-4), and the sulfate modification is also key to a number of mammalian extracellular signaling events (5). A number of sulfated metabolites have been isolated from the mycobacterial family (6-9), many of which are found in the cell wall (10,11). While the best-characterized of these molecules is the M. tuberculosis-specific metabolite sulfolipid-1 (SL-1) (9,12), another sulfated metabolite identified in M. tuberculosis lipid extracts has also been localized to the outer envelope of the cell (8,10). This previously-uncharacterized metabolite was termed S881 based on its measured mass. Isotopic labeling of S881 with 34 SO 4 2− indicated that it contains only one sulfate moiety (8,10). Despite the identification of this novel metabolite in M. tuberculosis

Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope of M. tuberculosis and negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3 -phosphoadenosine-5 -phosphosulfate and a previously uncharacterized sulfotransferase, stf3. These findings extend the known functions of sulfated molecules as general modulators of cell-cell interactions to include those between a bacterium and a human host.Fourier transform ion cyclotron resonance ͉ hypervirulent ͉ sulfate assimilation ͉ kinase ͉ adenosine-5-phosphosulfate A wide variety of organisms use sulfated molecules to control extracellular events. In mammals, sulfation of tyrosine residues on cell surface proteins is important for the interactions of chemokines with certain chemokine receptors, and for viral binding and entry (1-6). Sulfated glycans modulate processes such as leukocyte homing to lymph nodes, clearance of serum glycoproteins, and blood coagulation (7-9). Members of the glypican family that are modified with sulfated glycosaminoglycans guide organ development in Drosophila by maintaining a morphogen concentration gradient (10).In bacteria, sulfated glycolipids have been shown to serve as extracellular signaling molecules (11). The nitrogen fixing bacterium Sinorhizobium meliloti secretes the nodulation factor NodRm-1, a tetrasaccharide bearing both sulfate and lipid modifications (12). This molecule binds a receptor on the host plant, normally alfalfa, and induces root nodule formation. The sulfate group is critical for the function of NodRm-1, because the unsulfated form fails to induce root nodulation in alfalfa. In the rice blight-causing pathogen Xanthomonas oryzae, several genes involved in the synthesis of sulfated metabolites have been identified as avirulence factors with respect to certain host strains (13,14). These examples suggest that bacterial sulfated metabolites can participate in dialogue with eukaryotic hosts, analogous to their role in mammalian cell-cell communication.Mycobacteria produce an unusually complex array of sulfated structures (11). Sulfolipid-1 (SL-1), an abundant component of the cell envelope of M. tuberculosis, is the best characterized of these molecules. SL-1 has generated much interest because of its elaborate structure and the observation that its abundance correlates with strain virulence (15)(16)(17)(18)(19)(20)(21)(22). Advances in M. tuberculosis genetics and genome sequence data facilitated several contemporary studies that addressed aspects of the biosynthesis and the function of SL-1 in v...

Pathogenic bacteria have developed numerous mechanisms to survive inside a hostile host environment. The human pathogen Mycobacterium tuberculosis (M. tb) is thought to control the human immune response with diverse biomolecules, including a variety of exotic lipids. One prevalent M. tb-specific sulfated metabolite, termed sulfolipid-1 (SL-1), has been correlated with virulence though its specific biological function is not known. Recent advances in our understanding of SL-1 biosynthesis will help elucidate the role of this curious metabolite in M. tb infection. Furthermore, the study of SL-1 has led to questions regarding the significance of sulfation in mycobacteria. Examples of sulfated metabolites as mediators of interactions between bacteria and plants suggest that sulfation is a key modulator of extracellular signaling between prokaryotes and eukaryotes. The discovery of novel sulfated metabolites in M. tb and related mycobacteria strengthens this hypothesis. Finally, mechanistic and structural data from sulfate-assimilation enzymes have revealed how M. tb controls the flux of sulfate in the cell. Mutants with defects in sulfate assimilation indicate that the fate of sulfur in M. tb is a critical survival determinant for the bacteria during infection and suggest novel targets for tuberculosis drug therapy.