5′-Adenosinephosphosulfate Lies at a Metabolic Branch Point in Mycobacteria

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...

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“…1A; ref. 30). This observation suggests that PAPS functions exclusively as a sulfate donor for sulfotransferases.…”

Section: Resultsmentioning

confidence: 99%

A sulfated metabolite produced bystf3negatively regulates the virulence ofMycobacteriumtuberculosis

Mougous

Senaratne

Kim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Specifically, the same isotope incorporation experiment was performed in a mutant strain, ⌬cysH, which lacks the ability to reduce adenosine-5Ј-phosphosulfate (APS) to sulfite, the first committed step in the biosynthesis of r-sulfur (17). Thus, when a ⌬cysH strain was grown in 32 S-methionine ( 32 met) ϩ Na 2 34 SO 4 -containing media, only sulfated compounds incorporated the heavy sulfur isotope.…”

Section: Identification Of R-sulfur and Sulfatedmentioning

confidence: 99%

“…2B) was used . APS lies at a branchpoint in the pathway in mycobacteria (17). APS kinase (CysC) phosphorylates APS to form 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS), the sulfate donor for sulfotransferases.…”

Section: Identification Of R-sulfur and Sulfatedmentioning

confidence: 99%

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

Mougous

Leavell

Senaratne

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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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....

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“…Gene expression and proteome analysis of various models of dormant M. tuberculosis also consistently identified genes involved in sulfur metabolism as up-regulated (7)(8)(9). Inhibition of cysteine biosynthesis therefore appears to be a potential target for novel antibacterial agents specifically directed toward this pathogen in its persistent phase (10,11).…”

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Cysteine Synthase (CysM) of Mycobacterium tuberculosis Is an O-Phosphoserine Sulfhydrylase

Ågren

Schnell

Oehlmann

et al. 2008

Journal of Biological Chemistry

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The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-Å resolution.

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5′-Adenosinephosphosulfate Lies at a Metabolic Branch Point in Mycobacteria

Cited by 86 publications

References 38 publications

A sulfated metabolite produced bystf3negatively regulates the virulence ofMycobacteriumtuberculosis

A sulfated metabolite produced bystf3negatively regulates the virulence ofMycobacteriumtuberculosis

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

Cysteine Synthase (CysM) of Mycobacterium tuberculosis Is an O-Phosphoserine Sulfhydrylase

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