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Proc Natl Acad Sci U S A. Mar 14, 2006; 103(11): 4258–4263.
Published online Mar 6, 2006. doi:  10.1073/pnas.0510861103
PMCID: PMC1449680
Microbiology

A sulfated metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis

Abstract

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.

Keywords: 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 (16). Sulfated glycans modulate processes such as leukocyte homing to lymph nodes, clearance of serum glycoproteins, and blood coagulation (79). 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 (1522). 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 vivo (2326). Although these studies have greatly increased our understanding of SL-1, its function in the life cycle of the bacterium remains unknown. Other sulfated molecules identified in mycobacteria include sulfated glycopeptidolipids present in the cell envelopes of M. avium (27) and M. fortuitum (28), and trehalose-2-sulfate (29) in M. smegmatis. The functions of these metabolites are also unknown.

Sulfotransferases are the enzymes responsible for installing sulfate esters on metabolites by transfer of the sulfuryl group from the universal donor molecule, 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Because the sulfate ester is generally a determinant of function, these enzymes are attractive targets for the perturbation and study of sulfated molecules. In previous reports, we used conserved sulfotransferase motifs and mycobacterial genome sequence information to identify four sulfotransferases (stf0-3) in M. tuberculosis (11, 24). The abundance of stf genes suggested that M. tuberculosis has the capacity to synthesize sulfated metabolites in addition to SL-1. Subsequently, mass spectrometric analysis of M. tuberculosis extracts revealed a previously undescribed sulfated compound (m/z 881.56, now termed S881) that is structurally unrelated to SL-1 (29).

In this report, we provide genetic evidence that S881 is a sulfated compound that requires PAPS and a specific M. tuberculosis sulfotransferase (stf3) for its biosynthesis. We further demonstrate that S881 localizes to the outer envelope of M. tuberculosis, a position consistent with a role in cell–cell communication. Finally, we show that deletion of stf3 causes a hastened progression of tuberculosis in mice. These data indicate that S881 is a negative regulator of virulence and implicate sulfated metabolites as modulators of human host–pathogen interactions.

Results

Inactivation of cysC Abrogates Sulfation and S881 Biosynthesis.

S881 was initially identified in a mass spectrometric screen of M. tuberculosis extracts (29). The low abundance of S881 precluded detailed structural investigation; however, the presence of a sulfate ester was suggested by isotopic labeling and fragmentation analysis. This structural insight prompted us to investigate the biosynthetic origin of the sulfate modification.

All sulfotransferases characterized to date require PAPS as a high-energy sulfate donor. We have shown that mycobacteria use an adenosine-5′-phosphosulfate (APS) reductase (CysH) to initiate the reductive branch of their sulfate assimilation pathway (Fig. 1A; ref. 30). This observation suggests that PAPS functions exclusively as a sulfate donor for sulfotransferases. Two enzymes, ATP sulfurylase and APS kinase, are required for the production of PAPS from ATP and sulfate (Fig. 1A). In Escherichia coli, three ORFs, cysC, cysD, and cysN, are required to perform this function (31). ATP sulfurylase is a heteromultimer composed of CysN, a regulatory GTPase, and CysD, the catalytic domain that converts ATP and sulfate to APS. CysC, the APS kinase, then phosphorylates APS to form PAPS. In many bacteria, including M. tuberculosis, cysC is fused in-frame to the 3′ end of cysN, forming cysNC (32, 33). Mycobacterium avium has two highly similar copies of cysN and cysC, one present as cysNC and the other present in unfused form (Fig. 1B). These varied genomic arrangements suggested that the activities of cysNC may be modular and retain activity if separated. We exploited this observation to design a mutant M. tuberculosis strain devoid of all sulfated molecules (Fig. 1B).

Fig. 1.
Sulfate assimilation pathways and design of a global sulfation knockout. (A) Overview of the sulfate assimilation pathway of M. tuberculosis. CysD and CysN comprise ATP sulfurylase, which activates sulfate by forming APS from ATP and inorganic sulfate. ...

The mutation we designed (ΔcysC) removes the APS kinase domain of the bifunctional cysNC gene. To determine whether ΔcysC retained a functional ATP sulfurylase, we tested for production of APS by growth on sulfate as the sole sulfur source (Fig. 2A). The observed sulfate prototrophy of ΔcysC demonstrated that truncation of CysNC after the regulatory domain does not affect the ability of the mutant to produce APS for the reductive branch of the sulfate assimilation pathway. This result is supported further by biochemical and structural analyses of the truncated complex (34).

Fig. 2.
ΔcysC is a global sulfation knockout of M. tuberculosis. (A) Inactivation of cysC does not inhibit the reductive branch of the sulfate assimilation pathway. Growth of WT (□), ΔcysC (○), and ΔcysH ([open triangle]) in ...

Extracts of ΔcysC were next analyzed for the presence of sulfated molecules. To validate the mutant, we analyzed extracts for the presence of sulfated SL-1, the biosynthesis of which has been shown to rely on a PAPS-dependent sulfotransferase (24). Extracts from ΔcysC were devoid of SL-1 as determined by both Fourier transform ion cyclotron resonance (FT-ICR) MS (Fig. 2B) and 14C-propionic acid labeling (Fig. 2C). ΔcysC was successfully complemented (ΔcysC-ctrl) by expressing only the APS kinase domain of CysNC. To confirm that the defect in SL-1 biosynthesis was due to a lack of sulfation, we also analyzed extracts from ΔcysC and WT for the first committed sulfated precursor of SL-1, trehalose-2-sulfate (24). As expected, ΔcysC did not contain detectable levels of trehalose-2-sulfate (Fig. 2D). In addition to these experiments, we used 35SO42− labeling to monitor global sulfation in ΔcysC and found no detectable incorporation into sulfated molecules as observed by TLC (Fig. 5, which is published as supporting information on the PNAS web site). These data confirm that ΔcysC is unable to produce PAPS and, consequently, all sulfated molecules that rely on PAPS-dependent sulfotransferases.

With a global sulfation mutant in hand, we next tested whether the biosynthesis of S881 also relies on a PAPS-dependent sulfotransferase. Using FT-ICR MS with parameters that favor high resolution, S881 can be resolved from a neighboring isobar in crude lipid extracts of M. tuberculosis (Fig. 3A; ref. 29). When extracts of ΔcysC were analyzed in this manner, they were found to specifically lack S881 (Fig. 3A). Moreover, the biosynthesis of S881 was restored in the complemented strain ΔcysC-ctrl, thus providing evidence that S881 is sulfated by a PAPS-dependent sulfotransferase.

Fig. 3.
S881 is a sulfated molecule localized to the outer envelope of M. tuberculosis and its biosynthesis depends on PAPS and stf3. (A) S881 is not synthesized by M. tuberculosis lacking PAPS (ΔcysC) or stf3. Mass spectra of crude extracts from the ...

Stf3 Is Required for S881 Biosynthesis.

Having confirmed that S881 is sulfated, we sought to identify the responsible sulfotransferase. Of the candidate sulfotransferase genes, stf0-3, we ruled out stf0 based on our previous characterization of the enzyme as the trehalose-2-sulfate sulfotransferase required to initiate SL-1 biosynthesis (24). Thus, stfs1-3 were individually disrupted and probed for the ability to synthesize S881. FT-ICR MS analysis of crude extracts from these mutants showed that only Δstf3 lacked S881 (Fig. 3A). This phenotype was not due to a general lack of sulfation, because SL-1 biosynthesis was not disrupted in Δstf3 (Fig. 3B). Moreover, expression of Stf3 in Δstf3stf3-ctrl) restored the biosynthesis of S881 to levels similar to WT (Fig. 3A).

Growth of Δstf3-ctrl in minimal media containing either 32SO42− or 34SO42− as the sole sulfur source showed the expected specific incorporation of the isotope into S881 (Fig. 3C). Tandem MS analysis of these isotopically enriched samples revealed a fragment ion at m/z 97, corresponding to 32HSO4 in the 32SO42−-labeled sample and a peak at m/z 99, corresponding to 34HSO4 in the 34SO42−-labeled sample (Fig. 6, which is published as supporting information on the PNAS web site). This analysis provided direct chemical evidence of the sulfate modification on S881. These data, taken together with our prior sequence analysis of the stf family, provide strong evidence that stf3 encodes the sulfotransferase responsible for sulfating S881.

S881 Is Localized to the Outer Envelope of M. tuberculosis.

Consistent with their functions as modulators of cell–cell interactions, sulfated molecules found in bacteria and higher organisms are often localized outside of the cell (2, 7, 11, 35). Indeed, SL-1 (23) and the two known sulfated glycopeptidolipids produced by M. avium (27) and M. fortuitum (28) are found outside of the cell.

To evaluate whether S881 has the potential to mediate interactions outside of the cell, we determined its localization by using a lipid fractionation procedure described in ref. 23. This method has been shown to effectively separate a cell membrane-associated precursor of SL-1 from the fully elaborated molecule found in the cell envelope (23). As shown in Fig. 3D, S881 and several metabolites with similar m/z were observed in the cell membrane-associated fraction. Within the same limited mass range, however, the cell envelope fraction contained only S881. The identity of the m/z 881 ion observed in the cell envelope fraction was further confirmed by analyzing the same fraction from ΔcysC (Fig. 3D). These results show that S881 was selectively extracted in the cell envelope fraction and suggest that it is actively secreted to the outer envelope of M. tuberculosis.

Stf3 Negatively Regulates the Virulence of M. tuberculosis.

Sulfated molecules produced and secreted by pathogenic and symbiotic bacteria have been implicated in modulating responses in their plant hosts (1214). To investigate whether a secreted sulfated molecule produced by a human pathogen could play a similar role, we performed mouse infection studies with Δstf3. Interestingly, the mean time to death (TTD) for BALB/c mice infected i.v. with Δstf3 (12 weeks) was significantly shorter than mice infected with WT (20 weeks; P < 0.01) (Fig. 4A). To test for potential attenuation of the WT strain, two additional mutants were generated and analyzed in parallel experiments by using identical methods. One of these mutants, a deletion of a nonessential gene, behaved as WT, and the other, a sulfur auxotroph (ΔcysH) (29, 36), was attenuated relative to WT (Fig. 4A). It is also of note that the mean TDD observed for Δstf3 was shorter than a previous report (37) that used a similar infection model with H37Rv, the parental strain used in our study (37). Taken together, these data suggest that disruption of stf3 enhances the virulence of M. tuberculosis.

Fig. 4.
stf3 negatively regulates the virulence of M. tuberculosis. (A) Deletion of stf3 results in shorted TTD in a mouse model of tuberculosis. TTD analysis of BALB/c mice infected i.v. with WT (□), Δstf3 (○), ΔcysH ([open triangle]), ...

With in vitro biochemical data showing that the defect in S881 biosynthesis observed in Δstf3 was corrected in Δstf3-ctrl (Fig. 3A), we performed additional mouse infection studies to test whether the phenotype observed in vivo could also be complemented. In our initial experiment, we used a Δstf3-ctrl strain with an extrachromosomal plasmid containing stf3. Although the Δstf3-infected group began dying in significant numbers by the 7th week, the complemented strain did not show any mortality until the 13th week (Fig. 4B). However, by the 15th week, the two groups had converged and the Δstf3-ctrl group eventually succumbed to the infection at approximately same mean TTD as the Δstf3 group (15 weeks; P > 0.1). We reasoned that selection against bacteria with a functional plasmid in vivo may have caused this result. This phenomenom could also explain the delayed mortality of Δstf3-ctrl-infected mice, because curing the plasmid would likely require numerous generations of bacteria. This hypothesis was confirmed by analyzing for plasmid-encoded antibiotic resistance in bacteria isolated from mice at an advanced stage of infection with Δstf3-ctrl. At 15 weeks after infection, only 4% of the bacteria were resistant to hygromycin, indicating that they retained the plasmid (Fig. 4B Inset). We therefore used a stable integrating complementation plasmid and, in addition, the more resistant C57BL/6 mouse and the aerosol route of infection. Under these more physiologically relevant conditions, Δstf3 again displayed a shortened TTD (21 weeks) relative to WT (41 weeks; P < 0.01) (Fig. 4C). Importantly, the stable complemented strain was now attenuated relative to Δstf3 (P < 0.01) and displayed virulence comparable to WT (P > 0.1).

Colony-forming unit (cfu) measurements taken from the lungs of infected mice suggested that the increased virulence of Δstf3 is due to increased replication during the acute phase of infection. Three weeks after infecting mice by the aerosol route, Δstf3 replicated to levels approaching 10-fold above WT (Fig. 4D). This heightened bacterial load was maintained throughout the infection. Importantly, Δstf3 and the stably complemented Δstf3-ctrl showed no difference in growth rate in vitro (data not shown).

For further characterization, we submitted histological sections of lungs from C57BL/6 mice at 10 weeks after infection with the above strains to an independent pathologist. A summary of the pathology report is provided in Table 1, which is published as supporting information on the PNAS web site. Lungs from mice infected with Δstf3 had a higher degree of inflammation and increased bacterial load compared to those infected with WT or Δstf3-ctrl. At a gross level, a decrease in alveolar space caused by increased immune cell infiltration was clearly observed in lungs infected with Δstf3 (Fig. 4E).

During the course of these experiments, we noted that the WT strain used in our studies had lower levels of the outer envelope lipid phthiocerol dimycocerosate (PDIM) than either Δstf3 or Δstf3-ctrl (data not shown). Spontaneous loss of PDIM has been reported to occur frequently in H37Rv, the WT strain used in our study (26). Because PDIM is an important component of the M. tuberculosis cell envelope (3840), we considered the possibility that altered PDIM levels might underlie the enhanced virulence of Δstf3 relative to WT. Several lines of evidence contradict this interpretation. Perhaps most compelling is the fact that Δstf3-ctrl synthesizes high levels of PDIM but is attenuated relative to Δstf3 and has the same mean TTD as WT. Also, the differential in colony forming units we observed between Δstf3 and WT is in excess of that previously reported between WT M. tuberculosis H37Rv and a PDIM-deficient strain (41).

Discussion

Increasing evidence suggests that bacterial plant pathogens and symbionts use secreted sulfated metabolites to modulate interactions with host cells. Pathogenic mycobacteria have a complex lifecycle, involving several stages of interaction with their host that could be mediated by small molecules. Many lipid-based metabolites have shown to be virulence determinants (23, 40, 4245), but the sulfated components of the mycobacterial metabolome remain essentially uncharacterized. In this study, we provide evidence that the human pathogen M. tuberculosis, secretes a previously uncharacterized sulfated metabolite that negatively regulates virulence. This finding broadens our understanding of the evolutionarily conserved function of sulfated molecules.

Identification of the molecular mechanism by which S881 acts on host cells to regulate virulence is an important direction for future study. For now, recent progress in the legume symbiosis field has offered some interesting precedents for host pathways that could respond to sulfated metabolites such as S881. Receptor kinases containing extracellular LysM lectin domains in Lotus japonicus have been shown to mediate the stringent recognition of sinorhizobial nodulation factors (4648). These receptors appear to act in concert with a separate class of receptor kinases that contain extracellular leucine-rich repeats (LRR), termed symbiosis receptor-like kinase proteins (49, 50). Mammalian LRR-containing proteins of the Toll-like receptor (TLR) and NOD families, which are known to participate in innate recognition of bacteria, are therefore candidate receptors for S881. Indeed, various preparations of mycobacteria and several specific mycobacterial products have already been shown to stimulate signaling through TLR2, TLR4, TLR1, and TLR6 (51). Furthermore, a recent study identified SL1278, a sulfated precursor of SL-1, as a CD1d-restricted antigen that can activate T cells from tuberculosis patients in vitro (52).

Structural elucidation of S881 will be an important milestone toward understanding its mechanism of action in M. tuberculosis pathogenesis. Exact mass measurements performed by FT-ICR MS have allowed us to rule out the possibility that S881 is a close structural relative to SL-1 or any other characterized mycobacterial sulfated metabolite. Based on preliminary MS fragmentation data, its solubility in organic solvents, and its localization in the cell envelope, we believe S881 possesses lipid character. In addition, Stf3 has significant similarity to known carbohydrate sulfotransferases (11), suggesting that S881 has a carbohydrate component as well.

The discovery of stf3, a gene linked to the discrete metabolite S881, as a negative regulator of virulence marks a significant advance in our understanding of factors that control the delicate balance between the replication of this pathogen and the survival of its host. Previous studies have implicated the mce operon (53) and several two-component signaling modules (54) as additional negative regulators of M. tuberculosis virulence. In these studies, however, the pleiotropic nature of the mutants may complicate efforts to define the molecular underpinnings of the phenotype. As noted above, numerous M. tuberculosis cell envelope constituents have been identified as positive contributors to virulence. Given the advantages that a latent phase of infection offers M. tuberculosis, it is not surprising that the organism also has developed pathways that modulate the host response to limit its replicative potential.

Materials and Methods

Generation of M. tuberculosis Mutants.

Genomic sequence data for M. tuberculosis H37Rv was obtained from the TubercuList web site (http://genolist.pasteur.fr/TubercuList). All mutants were constructed by using the method of Parish and Stoker (55). Disrupted alleles were created by amplifying flanking regions of ≈2 kb in length around the targeted gene. After digestion with the appropriate restriction enzymes, these PCR products were subcloned into the p2NIL vector and subsequently interrupted by a hygromycin resistance cassette. Mutant selection and additional vector information have been described in ref. 30 and 55. Mutants were analyzed by PCR and Southern blot.

For design of Δcys C, sequence alignments clearly indicated a nonconserved linker between the ATP sulfurylase (cysN) and APS kinase (cysC) regions of cysNC. The reverse primer used to amplify the flanking region that included cysN contained a stop codon in this nonconserved region (amino acid 422 of cysNC).

Extrachromosomal complementation of the mutants was achieved by using the pMSGS vector described by our laboratory in ref. 24. For Δcys C, amino acids 428–617 of the M. smegmatis cysNC gene were amplified and inserted into the vector. To obtain stable complementation of Δstf3, a cassette containing the entire stf3 ORF under control of the glutamine synthase promoter was excised from the pMSGS vector and ligated into the integrating vector pMV306-kan.

Preparation of M. tuberculosis Lipid Extracts.

For sulfur isotope enrichment, 5.0-ml cultures of M. tuberculosis were grown in modified Saunton media (all sulfate salts replaced with chloride equivalents) containing either 2 mM Na232SO4 or Na234SO4 (ICON Isotopes, Summit, NJ) as the sole sulfur source. Once cells had reached stationary phase, they were pelleted by centrifugation, and the resulting cell pellet was extracted with 1.0 ml of 2:1 chloroform:methanol by vigorous shaking for 2 h at room temperature. The organic phase was clarified by centrifugation and removed for MS analysis. Unlabeled extracts analyzed by MS were prepared in a similar manner, with the exception that the growth medium was 7H9 with ADC supplement. Subcellular lipid fractionation and the preparation of these samples for MS was performed as described in ref. 23. Briefly, 50 ml of M. tuberculosis cultures were grown to OD600 = 0.8–1.0, pelleted by centrifugation, resuspended in 1 ml of hexane, and gently sonicated. Centrifugation and subsequent organic extraction (56) of the supernatant and pellet fractions of this sample produced the envelope and cellular fractions, respectively.

Mass Spectrometry.

The chloroform:methanol extracts were analyzed on an Apex II FT-ICRMS (Bruker Daltonics, Billerica, MA) equipped with a 7 T actively shielded superconducting magnet. The extracts were introduced to the ion source via a syringe pump at a flow rate of 2 μl/min. Ions were generated with an Apollo (Bruker Daltonics) pneumatically assisted electrospray ionization source operating in negative ion mode.

After ionization, the ions were accumulated for 2 s in a rf-only hexapole and subsequently transferred to the ICR cell by a series of ion optics (57). The elemental composition of S881 was determined by accurate mass measurement with internal calibration. To facilitate characterization of S881 a correlated sweep was used to reduce the complexity of the surrounding mass range (58). Sustained off-resonance irradiation collision induced dissociation was applied to S881 to generate structural data (59). A pulse of argon was introduced into the ultrahigh vacuum region of the ICR cell while exciting the ion 1.5 kHz above its resonance frequency. Mass spectra consist of 512,000 data points (average of 24 scans) and were acquired of the FT-ICR data station, operating xmass 6.0.

Mouse Infections.

Female C57BL/6 mice were purchased from The Jackson Laboratory, and female BALB/c mice were purchased from Charles River Laboratories. Seven- to 8-week-old mice were infected either by i.v. or aerosol route. Bacteria were aerosolized by using the Inhalation Exposure System (Glas-col, Terre Haute, IN) to deliver 100 to 200 bacilli per mouse right lung. For i.v. infections, mice were infected by injection of 106 bacteria in 0.2 ml of PBS containing 0.05% Tween 80 (PBST) into the lateral tail vein. In both methods of infection (with the exception of data presented in Fig. 3A), organs (right lung, liver, and spleen for i.v. infection and right lung only for aerosol infection) from three mice were harvested 24 h after infection to determine the number of bacteria seeded. Bacterial numbers were enumerated by plating serial dilutions of organ homogenates from three mice for each group on 7H10-OADC. Colonies were counted after 3–4 weeks.

Loss of weight accompanied by failure to drink and eat, failure to groom (poor coat condition), and lethargy were used to determine the point of premorbid state in addition (in some instances) to the advice obtained from the veterinary staff of North Animal Facility of the University of California, Berkeley. Health of the mice was monitored daily by the above veterinary staff.

Mouse left lungs fixed in 10% neutral (PBS)-buffered formalin were embedded in paraffin, sectioned, and stained for histology with either hematoxylin/eosin (H&E) or by the Ziehl-Neelsen technique. Sectioning and staining was carried out by Histology Consultation Services, Everson, WA. For comparative purposes, sections were obtained from the same regions of all lungs. Three to six sections were obtained from each lung. Sections obtained from the top and the bottom parts of the lung were H&E stained, whereas the section obtained from the middle region was stained by Ziehl-Neelsen technique. Pathology of each lung was assessed with two to four H&E-stained sections, and one to two sections stained with the Ziehl-Neelsen technique. Lung sections from three mice were used per time point per M. tuberculosis strain for pathological analysis. All pathological analysis was performed by an independent veterinary pathologist from the School of Veterinary Medicine, University of California, Davis.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Aimee Shen for critical review of the manuscript and members of the Bertozzi, Cox, Leary, and Riley groups for helpful suggestions. J.D.M. was supported by a Fellowship from the Ford Foundation. This work was supported by a National Institutes of Health Grant AI51622 (to C.R.B.).

Glossary

Abbreviations:

APS
adenosine-5′-phosphosulfate
FT-ICR
Fourier transform ion cyclotron resonance
PAPS
3′-phosphoadenosine-5′-phosphosulfate
PDIM
phthiocerol dimycocerosate
SL-1
sulfolipid-1
TTD
time to death.

Footnotes

Conflict of interest statement: No conflicts declared.

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