Abstract
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.
Keywords: pathogenesis, biochemistry, glycolipid, sulfation
The 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 (O2−) 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.
Fig. 1.
SL-1, SL1278, and genomic organization. (A) Structure of SL-1. (B) Structure of SL1278. (C) The genomic arrangement of the SL-1 biosynthetic cluster. papA1 resides downstream of pks2, whereas papA2 lies further downstream of mmpL8.
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-, 6′-, and 3′-positions of SL-1 (Fig. 1A) (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, ΔmmpL8, lacks SL-1 but accumulates the diacylated intermediate SL1278 (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 3′-position and a palmitoyl group at the 2′-position (24). SL1278 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 ΔmmpL8 mutant, which lacks SL-1 but accumulates SL1278, shows attenuated virulence in mice (23, 24). By contrast, a Δpks2 mutant, which lacks both SL-1 and SL1278, is indistinguishable from WT M. tb in mice and guinea pigs (23, 26). These observations suggest that SL1278, and possibly other SL-1 intermediates, modulate M. tb pathogenesis.
In our effort to define the functions of M. tb sulfolipids, we sought to identify the machinery underlying conversion of T2S to SL1278. As a starting point we turned our attention to other M. tb lipid biosynthetic pathways for which the acyltransferases had been characterized. Recently, an acyltransferase that installs the lipid groups on the M. tb virulence factor phthiocerol dimycocerosate (PDIM) was identified as the polyketide synthase-associated protein PapA5 (27–29). This enzyme transfers a mycocerosyl group from the acyl carrier protein domain of the polyketide synthase Mas, which, like many polyketide synthases, including Pks2, lacks a thioesterase domain that would otherwise release the lipid (6, 30). papA5 belongs to a family comprising four additional members (27, 31). Two of these, papA2 and papA1, reside in proximity to other SL-1 genes in the M. tb genome (Fig. 1C). Given the similarity of the PDIM biosynthetic gene cluster to that of SL-1, we reasoned that the corresponding proteins, PapA2 and PapA1, may be involved in SL-1 biosynthesis.
Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S and are required for the synthesis of SL1278 and SL-1 in vivo. The order of addition of the two lipids found in SL1278 was first determined by using purified enzymes and synthetic substrates. These results were validated by disrupting papA2 and papA1 in M. tb and analyzing the metabolite profile of each mutant using high-resolution MS and radiolabeling. Finally, these mutants were evaluated for virulence defects in a mouse model of tuberculosis.
Results
papA2 and papA1 Reside in an SL-1 Biosynthetic Gene Cluster.
The genes encoding Pks2 and MmpL8 are clustered in the M. tb genome (Fig. 1C). Within the same cluster reside two genes annotated as polyketide synthase-associated proteins, papA2 and papA1 (31). As discussed above, the related gene papA5 encodes an acyltransferase that esterifies phthiocerol with mycocerosic acids in the assembly of PDIM (27–29). papA5 is associated with mas, the polyketide synthase responsible for mycocerosic acid synthesis. Similarly, papA1 is associated with pks2, which encodes the enzyme that synthesizes the phthioceranic acids of SL-1. However, papA2 is distal from pks2 in the M. tb genome and is not directly associated with another polyketide synthase. This arrangement is unique among the M. tb pap genes, suggesting that PapA2 may function independent of a polyketide synthase. Further biochemical analysis of the interactions between the Pap proteins and their associate Pks proteins will be needed to validate these genetic implications.
PapA2 Is an Acyltransferase That Esterifies T2S.
Given the unique genomic arrangement of papA2, we reasoned that PapA2 may be the acyltransferase that transfers the palmitoyl group to the 2′-position of T2S. To test this hypothesis we amplified papA2 from M. tb H37Rv genomic DNA and expressed the protein in Escherichia coli. Purified PapA2 was then incubated with 14C-palmitoyl-CoA (PCoA) and either trehalose or synthetic T2S. The reaction mixtures were separated by TLC and imaged by phosphorescence. Hydrolysis of PCoA was seen in the reactions containing PapA2 in the absence of another substrate. However, a unique product was observed in the reaction containing PapA2, PCoA, and T2S (Fig. 2A). Unsulfated trehalose was not a competent substrate for PapA2. Kinetic analysis of the PapA2 reaction containing T2S and PCoA showed substrate inhibition by PCoA, similar to that reported for PapA5 [supporting information (SI) Fig. 6]. The Km value for T2S was determined to be 2.5 mM whereas the Km for PCoA was 6.0 μM (Table 1). For comparison, PapA5 exhibits a Km of 0.5 mM for 1-octanol and 4 μM for PCoA.
Fig. 2.
PapA2 and PapA1 are acyltransferases that act sequentially on T2S. (A) PapA2 was incubated with radiolabeled PCoA and either trehalose (Tre) or T2S, and the reaction was analyzed by TLC. A product was observed only in the reaction with T2S (arrowhead). (B) FT-ICR MS of the PapA2 reaction. Synthetic trehalose-2-sulfate-2′-palmitate (SL659) exhibits an m/z = 659.30. The same ion was observed in the PapA2 reaction (PapA2 rxn). The control reaction lacking PapA2 showed no product at m/z = 659. (C) PapA1 was incubated with radiolabeled PCoA and the product of the PapA2 reaction (PapA2 rxn). The reaction was analyzed by TLC, and a new product was observed (arrowhead). (D) FT-ICR MS of the PapA1 reaction. An ion with m/z = 897.54 was observed in the PapA1 reaction (PapA1 rxn), whereas the control reaction lacking PapA1 showed no such product.
Table 1.
Kinetic constants for PapA2 and PapA1
Substrate | PapA2 |
PapA1 |
||
---|---|---|---|---|
kcat, min−1 | Km, μM | kcat, min−1 | Km, μM | |
Trehalose | ND | ND | ND | ND |
T2S | 0.40 ± 0.02 | 2,500 ± 331 | ND | ND |
Trehalose-2-palmitate | ND | ND | ND | ND |
SL659 | ND | ND | 0.12 ± 0.005 | 332 ± 60 |
PCoA | 0.19 ± 0.001 | 5.96 ± 0.90 | ND | ND |
Values ± SEM were determined from triplicate measurements. ND, not detectable.
We then characterized the product of the PapA2 reaction by Fourier transform ion cyclotron resonance (FT-ICR) MS and NMR spectroscopy. Analysis of the crude PapA2 reaction by FT-ICR MS yielded a major ion with m/z = 659.30, corresponding to the exact mass of palmitoyl T2S (Fig. 2B). Fragmentation of the ion at m/z = 659.30 by tandem MS yielded peaks that were identical to those derived from chemically synthesized trehalose-2-sulfate-2′-palmitate (SI Fig. 7). NMR analysis of the PapA2 reaction product showed the presence of a palmitoyl group at the 2′-position of T2S (SI Fig. 8). These data demonstrate that PapA2 palmitoylates T2S at the 2′-position to form trehalose-2-sulfate-2′-palmitate (termed SL659 based on its observed mass).
PapA1 Acylates SL659, the Product of PapA2.
We next reasoned that the PapA2 reaction product, SL659, could be a substrate for PapA1. To test this hypothesis we expressed and purified PapA1 and then incubated the protein with PCoA and SL659. A new product appeared only in the presence of all three reaction components (Fig. 2C). The palmitoyl group of SL659 was found to be essential for modification by PapA1, as T2S was not modified. Further kinetic experiments showed substrate inhibition by SL659 but not by PCoA (SI Fig. 9). The Km of PCoA for PapA1 was much higher than that observed for PapA2, suggesting that PCoA is not the physiological substrate of the enzyme (Table 1). The related PapA5 acyltransferase has been shown to transfer mycocerosate directly from the polyketide synthase Mas (29). Thus, it is likely that PapA1's biological function is to transfer (hydroxy)phthioceranoyl groups from Pks2 and that the acylated Pks2 acyl carrier protein domain is its bona fide substrate. The product produced by the PapA1 reaction with SL659 was analyzed by FT-ICR MS. The major ion observed, with m/z = 897.54, was consistent with dipalmitoylated T2S (Fig. 2D). Further analysis of this ion by tandem MS indicated that both palmitoyl groups were on the same glucose residue, consistent with the structure of SL1278, which positions a phthioceranoyl group at the 3′-position. By contrast, PapA2 was not able to further modify SL659, and neither PapA1 nor PapA2 showed activity toward unsulfated trehalose-2′-palmitate.
PapA2 and PapA1 Are Required for SL-1 Biosynthesis.
We used a combined genetic and MS method (32) to show that both PapA2 and PapA1 are essential for SL-1 biosynthesis in M. tb. The genes encoding these proteins were disrupted in the Erdman strain of M. tb, creating the mutant strains ΔpapA2 and ΔpapA1. The production of sulfated metabolites in each strain was monitored by growing the cells in the presence of Na235SO4. The cells were extracted with organic solvents, and the crude lipids were separated by TLC. A compound with an Rf corresponding to SL-1 was readily seen in WT M. tb extracts but was absent from extracts from both mutant strains (Fig. 3A). The absence of SL-1 in the ΔpapA2 and ΔpapA1 cultures was confirmed by FT-ICR MS analysis as previously described (21, 32). Ions corresponding to the lipid envelope of SL-1 were observed in WT M. tb extracts, yet these peaks were not seen in extracts from either mutant strain (Fig. 3B). These observations confirm that both PapA2 and PapA1 are essential for the production of SL-1 in M. tb.
Fig. 3.
papA2 and papA1 are essential for SL-1 biosynthesis in vivo. (A) WT M. tb, ΔpapA2, and ΔpapA1 were labeled with Na235SO4. The total lipids were extracted and analyzed by TLC. All three extracts show T2S. The ΔpapA2 extract lacks a spot corresponding to SL659, the product of the PapA2 reaction in vitro. Both ΔpapA2 and ΔpapA1 extracts lack spots corresponding to SL1278 and SL-1. (B) FT-ICR MS analysis of total lipids from WT, ΔpapA2, and ΔpapA1. WT M. tb extracts display the characteristic SL-1 lipoforms, which are absent from the ΔpapA2 and ΔpapA1 extracts. (C) Identification of a peak corresponding to the exact mass of SL659 in WT M. tb and ΔpapA1 samples. This metabolite is absent from ΔpapA2 extracts.
PapA2 and PapA1 Act Sequentially to Form SL1278 in Vivo.
The experiments detailed above suggest that PapA2 acylates T2S to form SL659 in vitro and that PapA1 acts on SL659 to form SL1278 in vivo, and dipalmitoylated T2S in our in vitro reaction with PCoA. To validate these observation in vivo we probed for the presence of SL-1 biosynthetic intermediates by metabolic labeling of M. tb cultures and FT-ICR MS analysis. TLC separation of Na235SO4-labeled organic extracts from WT M. tb, ΔpapA2, and ΔpapA1 allowed us to visualize T2S, SL659, and SL1278 along with SL-1 (Fig. 3A). Although WT M. tb cells produced each of these metabolites, the ΔpapA2 mutant lacked SL659 and SL1278 while retaining the ability to produce T2S. Similarly, the ΔpapA1 mutant was deficient in SL1278 synthesis but still produced T2S and SL659. These data were corroborated by FT-ICR MS analysis of culture extracts. A peak with m/z = 659.30 was seen in both WT and ΔpapA1 M. tb extracts but was absent from the ΔpapA2 extracts (Fig. 3C). Also, the lipid envelope of SL1278 was observed in WT M. tb extracts but was absent from both the ΔpapA2 and ΔpapA1 extracts (SI Fig. 10). These data support our proposal that, in M. tb, T2S is first elaborated by PapA2 to form SL659, which is then acylated by PapA1 with a (hydroxy)phthioceranoyl group, yielding SL1278. These results thus establish the order of the first two lipid additions in SL-1 biosynthesis.
Mutation of papA2 or papA1 Does Not Affect M. tb Virulence.
Previous studies have addressed the contribution of SL-1 to the virulence of M. tb in animal models. The SL-1-deficient Δpks2 mutant was previously screened in mice for virulence defects (23, 26). However, the mutation did not hinder replication of the bacteria in the lungs, livers, or spleens, indicating that SL-1 is not critical to infection in mice. Curious as to how ΔpapA2 and ΔpapA1 would fare in vivo, we infected BALB/c mice with the two mutants and assessed the ability of the bacteria to replicate in the lungs and traffic to the spleen and liver (Fig. 4). Similar to the Δpks2 mutant, the ΔpapA2 and ΔpapA1 mutants showed no virulence defect when compared with WT M. tb.
Fig. 4.
Disruption of papA2 or papA1 does not alter the virulence of M. tb in mice. BALB/c mice were infected via aerosol with WT (filled squares), ΔpapA2 (open circles), or ΔpapA1 (open triangles) strains at an initial inoculum of ≈250 bacteria per lung. Growth in the lungs (A), spleen (B), and liver (C) was monitored by harvesting organs at 0, 10, 23, and 44 days after infection. Bacillary loads were determined by plating dilutions on solid media. Each data point represents the average of cfu from four to five mice, and error bars indicate the standard deviation from the means.
Discussion
The biochemical and genetic data presented here reveal the order of addition of the first two lipids to the T2S core of SL-1, as well as the acyltransferases responsible for their installation. Based on our current work and other recent studies, we propose the biosynthetic route for SL-1 shown in Fig. 5. The process begins with the installation of a sulfate group at the 2-position of trehalose by Stf0. PapA2 then installs the straight chain lipid found in SL-1 and is specific for T2S, further indicating that sulfation of trehalose is the first step in SL-1 biosynthesis (21). The resulting SL659 is then acylated by PapA1 with a (hydroxy)phthioceranic acid group, the product of Pks2 (22). Despite significant sequence similarity, PapA2 and PapA1 are very specific for T2S and SL659, respectively, and their roles in SL-1 biosynthesis are not interchangeable. There remains uncertainty regarding the physiological acyl donor substrates of the two acyltransferases. Both enzymes are capable of using PCoA as a substrate in vitro, and this may be a physiological substrate for PapA2. However, we cannot rule out the possibility that PapA2's natural substrate is another acyl pantotheine-based cofactor, such as a pantotheinyl acyl carrier protein. Moreover, our genetic evidence points to PapA1 as a mediator of (hydroxy)phthioceranoyl group transfer, but its transfer from Pks2 may be direct or involve a (hydroxy)phthioceranoyl-acyl carrier protein intermediate. Resolution of these remaining questions will require advances in polyketide synthase enzymology, including the development of recombinant expression systems and in vitro biochemical assays.
Fig. 5.
Proposed SL-1 biosynthetic pathway. (A) Trehalose is first sulfated by Stf0 to form T2S. PapA2 then acylates the 2′-position of T2S to form SL659. A (hydroxy)phthioceranic acid is then synthesized by Pks2 and transferred directly by PapA1 onto SL659 to generate SL1278. (B) The diacylated SL1278 is then transferred by MmpL8 to the exterior of the cell where the final acylations occur.
The last characterized step in SL-1 biosynthesis is the transport of SL1278 to the outer leaflet of the cell by MmpL8. The enzymes or processes responsible for the final acylation of SL1278 to SL-1 have yet to be determined. The proposed biosynthetic route indicates that the final lipid modifications are extracellular. This hypothesis is strengthened by the accumulation of SL1278 on the interior of the cell in the ΔmmpL8 mutant (23). However, the (hydroxy)phthioceranoyl groups at the 6- and 6′-positions of SL-1 are putatively synthesized intracellularly by Pks2, suggesting that these lipids are transported to the exterior of the cell and attached to SL1278 by an unknown acyltransferase (22). One proposed candidate for SL1278 elaboration is antigen 85, an extracellular acyltransferase involved in trehalose mycolate synthesis (23, 33). However, because of the dissimilarity between phthioceranic acid and mycolic acid, we feel that it is unlikely that this enzyme participates in SL-1 biosynthesis.
Another possibility is that PapA1 is responsible for all three (hydroxy)phthioceranoyl transfer reactions and that these occur intracellularly before transport by MmpL8. No direct evidence of this reaction has been observed, however. The low solubility of SL1278 (or SL1278 analogs) makes it difficult to address this possibility using in vitro biochemical assays. The polyketide synthases involved in PDIM biosynthesis sequester the lipids they produce, effectively solubilizing the lipid during biosynthesis (29, 34). Also, MmpL7 associates with the phthiocerol polyketide synthase PpsE and is proposed to coordinate PDIM biosynthesis (35). In vivo, MmpL8 may sequester SL1278 and present the 6- and 6′-positions for acylation by PapA1 or act as a scaffold for SL-1 synthesis, similar to MmpL7. In this scheme, MmpL8 would transport fully formed SL-1 to the capsular layer of the bacteria. Further biochemical and genetic experiments are necessary to determine which hypothesis is most likely.
The availability of reliable genetic techniques and the development of high-resolution metabolite analysis tools have sped our progress toward understanding the details of SL-1 biosynthesis. However, the lack of an observable phenotypic difference between SL-1-deficient strains and WT M. tb in the murine tuberculosis model has undermined efforts to assign a function to the metabolite. The activity of SL1278 as a CD1b-restricted T cell antigen is intriguing and may be relevant to human infection. However, mice do not possess a counterpart of CD1b (36, 37). Thus, efforts to elucidate the role of SL-1 and its intermediates in pathogenesis may ultimately be frustrated in this model. Indeed, other bacterial sulfated metabolites are known to have host-specific functions. The alfalfa symbiont Sinorhizobium meliloti and the rice pathogen Xanthomonas oryzae both produce sulfated metabolites that are sensed by their hosts but not required for viability in other environments (38–41). Until tuberculosis models that more closely reflect the human disease are developed, we may not be able to assign a role for many metabolites produced by M. tb, including SL-1.
Materials and Methods
Expression and Purification of PapA2 and PapA1.
DNA sequences encoding M. tb PapA2 and PapA1 were amplified from the H37Rv genomic DNA by PCR, cloned into pET28a (Novagen, Madison, WI), and transformed into the E. coli strain BL21 for protein expression. Cells were grown to an OD600 of 0.6 at 37°C, and protein expression was induced with 0.1 mM isopropyl-β-d-thiogalactoside for 15 h at 22°C. PapA1 was expressed with both N- and C-terminal His6 tags, and PapA2 was expressed as a C-terminal His6-tagged protein. Cells were harvested by centrifugation and resuspended in Ni-NTA followed by anion exchange chromatography using a HiTrap Q column on an AKTA FPLC system. Eluted protein was concentrated and frozen in 100 mM sodium phosphate buffer (pH 7.2; 1 mM DTT and 10% glycerol). Protein concentrations were measured using the Bradford assay. On the basis of the densitometry data, both proteins were determined to be >90% pure.
Reagents and Chemicals.
The synthesis for SL659 is described in SI Text. Trehalose-2-palmitate and T2S were synthesized as previously described (42–45). NMR and MS data were consistent with previous reports. PCoA (55 mCi/mmol) was purchased from ARC Radiochemicals (St. Louis, MO). The plasmid pET28a and Ni-NTA affinity resins were purchased from Novagen and Qiagen (Valencia, CA), respectively. All other chemicals were purchased from Sigma (St. Louis, MO) or Fluka (St. Louis, MO) and used without further purification.
Biochemical Characterization of PapA1 and PapA2.
PapA1 or PapA2 (1 μM) was incubated with 20 μM PCoA and 1 mM trehalose or T2S in reaction buffer [100 mM sodium phosphate (pH 7.5), 1 mM DTT, and 10% glycerol] at 25°C for 60 min. Subsequently, the reactions were quenched by the addition of an equal volume of ethanol. The products were analyzed by TLC, eluting with 35:65 methanol:chloroform, and were quantified by using phosphorimaging followed by densitometry. Kinetic parameters for PapA2 and PapA1 were determined by obtaining the initial velocities of the reactions at varying substrate concentrations.
Tandem Reactions with PapA2 and PapA1.
PapA2 (1 μM) was incubated with 20 μM unlabeled PCoA and 1 mM T2S in reaction buffer at 25°C for 4 h to generate trehalose-2-sulfate-2′-palmitate (SL659) in situ. Subsequently, the enzyme was heat-killed by boiling the reaction mixture at 100°C for 5 min, followed by addition of 1 μM PapA1 and 20 μM PCoA. The reaction products were analyzed by TLC as described above.
FT-ICR MS Characterization of Reaction Products.
PapA2 (1 μM) was incubated with 100 μM unlabeled PCoA and 1 mM T2S in 100 mM ammonium bicarbonate (pH 7.2) for 6 h at room temperature. In a separate reaction, 1 μM PapA1 was incubated with 200 μM synthetic SL659 and 100 μM unlabeled PCoA in 100 mM ammonium bicarbonate (pH 7.2) for 6 h at room temperature. The resulting reaction products and appropriate control reactions were analyzed by FT-ICR MS.
Construction of M. tb Mutants.
M. tb cells (Erdman strain) were cultured in 7H9 medium supplemented with 10% oleic acid albumin dextrose catalase, 0.5% glycerol, and 0.05% Tween 80, or on 7H10 solid agar medium supplemented with 10% oleic acid albumin dextrose catalase and 0.5% glycerol. Hygromycin (50 μg/ml) was used when necessary. The ΔpapA2 and ΔpapA1 mutant strains were created by homologous recombination using specialized transduction phages phmws103 and phmws113, respectively (46). These deletions replaced 1,171 bp of papA2 (amino acids 39–429) and 1,064 bp of papA1 (amino acids 103–457) with a hygromycin resistance cassette.
Biochemical Analysis of Sulfolipids.
M. tb cultures were synchronized and grown to OD600 = 0.8. Aliquots (10 ml) were resuspended in an equal volume of PBS supplemented with 1 mg/ml sodium acetate and labeled with 150 μCi of [35S]sulfate for 18 h at 37°C. Cell pellets were extracted with chloroform:methanol (2:1) and analyzed by TLC, eluting with chloroform:methanol:water (60:12:1) or water:isopropanol:ammonium hydroxide (1:6:1) as the developing solvent. Radioactivity was quantified by using phosphorimaging followed by densitometry.
MS.
All mass spectra were acquired on a Bruker Apex II FT-ICR MS equipped with a 7T superconducting magnet (Bruker Daltonics, Billerica, MA). Extracts of M. tb were ionized by using electrospray ionization (Analytica, Branford, CT) in negative ion mode and were infused at a rate of 2 μl/min in 2:1 chloroform:methanol. The ions were accumulated in an external hexapole for 0.5–2 s (the time was adjusted to maximize ion signal without depleting spectral resolution) before being transferred to the ICR cell for high-resolution analysis. Spectra were acquired by using XMass 5.0.10 (Bruker Daltonics), are composed of between 256,000 and 1 million data points, and are an average of between 32 and 128 scans. All spectra were internally calibrated by using known compounds.
Mouse Infections.
Bacteria were grown to log phase, cup-sonicated by using a Branson (Danbury, CT) Sonifier 250 at 90% for 15 s, spun for 5 min at 40 × g to remove clumps, and diluted to the desired inoculum in PBS. Bacteria were administered to BALB/c mice via nebulization for 15 min by using a custom-built aerosolization chamber (Mechanical Engineering Shops, University of Wisconsin, Madison, WI). For infections, bacterial suspensions with an OD600 = 0.1 were used, resulting in an initial seeding of ≈250 bacteria per mouse. Organs from infected mice were homogenized and plated for analysis of cfu. Four or five mice were used per time point. All mice were housed and treated humanely as described in an animal care protocol approved by the University of California, San Francisco, Institutional Animal Care and Use Committee.
Note.
While this article was under review, Bhatt et al. (47) reported that M. tb mutants in papA2 and papA1 are deficient in SL-1 biosynthesis. The biochemical activities and biosynthetic order of the corresponding proteins were not reported.
Supplementary Material
Acknowledgments
We thank members of the C.R.B. group and Analisa Schelle for critical review of the manuscript and for helpful suggestions. This work was supported by National Institutes of Health Grants AI51622 (to C.R.B.) and AI51667 (to J.S.C.) and by The W. M. Keck Foundation (J.S.C.).
Abbreviations
- M. tb
Mycobacterium tuberculosis
- SL-1
Sulfolipid-1
- T2S
trehalose-2-sulfate
- PDIM
phthiocerol dimycocerosate
- FT-ICR
Fourier transform ion cyclotron resonance
- PCoA
14C-palmitoyl-CoA.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0611649104/DC1.
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