Michael W. Schelle

Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling

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.

PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor Sulfolipid-1

Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. A diacylated intermediate in SL-1 biosynthesis, SL1278, has been shown to activate the adaptive immune response in human patients. Although several proteins involved in SL-1 biosynthesis have been identified, the enzymes that acylate the T2S core to form SL1278 and SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL1278 and are essential for SL-1 biosynthesis. In vitro, recombinant PapA2 converts T2S to 2′-palmitoyl T2S, and PapA1 further elaborates this newly identified SL-1 intermediate to an analog of SL1278. Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Finally, the ΔpapA2 and ΔpapA1 mutants were screened for virulence defects in a mouse model of infection. The loss of SL-1 (and SL1278) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection.

Sulfate Metabolism in Mycobacteria

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.

PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis

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.

Conditional glycosylation in eukaryotic cells using a biocompatible chemical inducer of dimerization.

Chemical inducers of dimerization (CIDs) are cell-permeable small molecules capable of dimerizing two protein targets. The most widely used CID, the natural product rapamycin and its relatives, is immunosuppressive due to interactions with endogenous targets and thus has limited utility in vivo. Here we report a new biocompatible CID, Tmp-SLF, which dimerizes E. coli DHFR and FKBP and has no endogenous mammalian targets that would lead to unwanted in vivo side effects. We employed Tmp-SLF to modulate gene expression in a yeast three-hybrid assay. Finally, we engineered the Golgi-resident glycosyltransferase FucT7 for tunable control by Tmp-SLF in mammalian cells.

Structural Characterization of a Novel Sulfated Menaquinone produced by stf3 from Mycobacterium tuberculosis

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.

Elucidation and Chemical Modulation of Sulfolipid-1 Biosynthesis in Mycobacterium tuberculosis

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 unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.

Sulfolipid-1 Biosynthesis Restricts Mycobacterium tuberculosis Growth in Human Macrophages

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 Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging.

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.

Directing flux in glycan biosynthetic pathways with a small molecule switch.

The diverse array of complex glycans displayed on the surface of mammalian cells is synthesized by the coordinate action of Golgi-resident glycosyltransferases. Deciphering glycosyltransferases’ relative contributions in oligosaccharide biosynthesis is complicated by their functional redundancy and the embryonic lethality of gene knock-outs. While the in vitro activities and specificities of certain glycosyltransferases have been delineated, cellular activity of these enzymes is often more limited. Localization of glycosyltransferases to particular subcompartments of the Golgi complex is one mechanism for their restricted cellular activity. Taking advantage of the critical role of Golgi localization, we previously reported a general method for small-molecule control of glycosyltransferase activity in cells. This method (Scheme 1A) does not demand the synthesis of bioavailable active-site inhibitors ; rather, it takes advantage of the modularity of glycosyltransferases’ localization (Loc) and catalytic (Cat) domains. 3] The two domains are expressed as separate polypeptides, each fused to a rapamycinbinding protein, either FKBP or FRB. In the absence of rapamycin, the catalytic domain fails to localize to the Golgi and, consequently, is unable to access its normal substrates. Therefore, no product is observed. The addition of rapamycin induces heterodimerization of the localization and catalytic domains, reconstituting the enzyme and restoring activity. In these experiments, we use the T2098L mutant of FRB, which is susceptible to degradation in the absence of rapamycin. The activity increase that we observe with addition of rapamycin may be due in part to rapamycin-induced stabilization of FRB-containing constructs. Here we apply the small-molecule method to direct flux through the biosynthetic pathways that determine the ultimate fate of glycans terminating in b-linked galactose (Scheme 1B). Fucosyltransferase 1 (FUT1) catalyzes the transfer of fucose to the 2-position of terminal b-linked galactose; this generates Fuca1,2Gal, also known as H antigen. Terminal blinked galactose is also a substrate for a variety of other Golgiresident glycosyltransferases, including a-1,3-galactosyltransferases, a-2,3-sialyltransferases, and a-2,6-sialyltransferases. Modification of galactose by any of these other enzymes precludes the production of H antigen. In any particular glycan, the observed modification of galactose will depend on the set of glycosyltransferases expressed in that cell type and their localization within the Golgi to sites proximal to enzymes responsible for generating terminal galactose substrate. Thus, modulation of FUT1 activity effectively directs glycan biosynthetic traffic toward, or away from, production of H antigen. Competition between FUT1 and a-1,3-galactosyltransferase (GGTA1) for Galb1,4GlcNAc (LacNAc) is of particular interest in the area of xenotransplantation. The action of GGTA1 on galactose generates a structure (Gala1,3Gal) known as aGal [a] Prof. Dr. C. R. Bertozzi Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute University of California and Materials Sciences Division Lawrence Berkeley National Laboratory Berkeley, CA 94720 (USA) E-mail : crb@berkeley.edu [b] Dr. J. J. Kohler, Dr. J. L. Czlapinski, S. T. Laughlin, M. W. Schelle, Dr. C. L. de Graffenried, Prof. Dr. C. R. Bertozzi Department of Chemistry, University of California, Berkeley B84 Hildebrand Hall, Berkeley, CA 94720 (USA) Fax: (+1)510-643-2628 [c] Dr. C. L. de Graffenried Current address: Department of Cell Biology, Yale University School of Medicine 333 Cedar Street, New Haven, Connecticut 06520 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. Directing biosynthetic traffic by small-molecule activation of FUT1. A) Schematic representation of the method by which rapamycin activates FUT1, which results in cell-surface expression of FUT1’s product. B) Terminal b-linked galactose is a substrate for four different families of glycosyltransferases: a-1,2-fucosyltransferases, a-1,3-galactosyltransferases, a-2,3-sialyltransferases, and a-2,6-sialyltransferases. The relative activities and localization of these enzymes determines the biosynthetic flux toward possible final glycans. H antigen (Fuca1,2Gal) can be detected with Ulex europaeus lectin 1 (UEA) while aGal (Gala1,3Gal) is recognized by isolectin B4 from Bandeiraea simplicifolia (IB4).