• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbcAbout JBCASBMBSubmissionsSubscriptionsContactJBCThis Article
J Biol Chem. Oct 2, 2009; 284(40): 27146–27156.
Published online Aug 6, 2009. doi:  10.1074/jbc.M109.022715
PMCID: PMC2785642

Identification of Apolipoprotein N-Acyltransferase (Lnt) in Mycobacteria*An external file that holds a picture, illustration, etc.
Object name is sbox.jpg

Abstract

Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether-bound diacylglycerol but differ by a fatty acid amide bound to the α-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared with the parental strain. Matrix-assisted laser desorption ionization time-of-flight/time-of-flight analyses of a trypsin digest of LppX proved the presence of the diacylglycerol modification in both strains, the parental strain and lnt::aph mutant. N-Acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid, whereas the two fatty acids of the diacylglycerol residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.

Proteins of various organisms are modified in numerous ways, one of them is lipidation. Lipid modification of proteins is common in eucaryal and bacterial organisms and can involve myristoyl, palmitoyl, and isoprenyl polymers of various lengths or aminoglycan-linked phospholipids (1, 2). Lipoprotein modifications investigated here are restricted to bacteria.

The lipoprotein biosynthesis pathway is a major virulence factor in Mycobacterium tuberculosis, the causative agent of human tuberculosis. Every year 1.6 million people fall prey to tuberculosis and one-third of the population of the world are infected. Thus, tuberculosis is responsible for 2.5% of deaths in the world, which is the highest rate claimed by a single infectious agent. An M. tuberculosis knock-out mutant deficient in lipoprotein signal peptidase lspA showed reduced multiplication in bone marrow-derived macrophages, complete absence of lung pathology and a 1000-fold reduced number of colony forming units in a mouse model of infection (3, 4). Likewise, lipoprotein synthesis contributes to virulence of other Gram-positive pathogens, Listeria, Staphylococci, and Streptococci (5).

Bacterial lipoproteins are a functionally diverse class of lipidated proteins involved in cell wall synthesis, nutrient uptake, adhesion, and transmembrane signaling (6) and about 2% of open reading frames encode this kind of proteins (7). Lipidation allows anchoring of these proteins to the cell surface. Lipoproteins are characterized by the presence of a consensus sequence, the “lipobox,” located in the C-terminal part of the leader sequence and consisting of four amino acids (LVI/ASTVI/GAS/C) (7). Precursor lipoproteins are mainly translocated in a Sec-dependent manner across the plasma membrane and modified subsequently on the universally conserved, essential cysteine residue located in the lipobox motif. The modifications taking place after translocation are consecutively mediated by three enzymes: 1) formation of a thioether linkage between the conserved cysteine residue and a diacylglycerol catalyzed by phosphatidylglycerol:pre-prolipoprotein diacylglycerol transferase (Lgt), 2) cleavage of the N-terminal signal peptide by the prolipoprotein signal peptidase/signal peptidase II (LspA), and 3) in the case of Gram-negative bacteria, aminoacylation of the N-terminal cysteine residue by phospholipid:apolipoprotein N-acyltransferase (Lnt) (68). In Escherichia coli, most of the mature triacylated lipoproteins are finally transported across the periplasm by the LolABCDE transport system (9). Homologues of the Lol-transport system are absent in Mycobacteria. Although lipoprotein modifying enzymes act sequentially, Lgt-independent LspA-mediated signal sequence cleavage has recently been demonstrated in Listeria monocytogenes (10). Although Lgt and LspA are universally present in both Gram-positive and Gram-negative bacteria, Lnt has been reported to be restricted to Gram-negative bacteria (11), although some indications for N-acylation in Bacillus subtilis and Staphylococcus aureus were reported (1215).

Mycobacterial lipoproteins are immunodominant antigens (16) and several manipulate innate immune mechanisms and antigen presenting cells (17). It is known that mycobacterial lipoproteins, e.g. the 19-kDa lipoprotein, activate Toll-like receptor 2 (TLR2) and co-receptors TLR1, which recognize triacylated peptides, but also TLR6, which recognize diacylated peptides (18, 19). However, the lipid linkage of mycobacterial lipoproteins has not been determined.

In this study, we show that Lnt activity is more widely distributed than previously assumed. We demonstrate apolipoprotein N-acyltransferase activity in a Gram-positive Mycobacterium and give complete structural information about the lipid modification of mycobacterial lipoproteins. Hereby, the functionality of Lnt homologues in actinomycetes is revealed (5). We show that mycobacterial lipoproteins are triacylated and carry mycobacteria-specific fatty acids.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Conditions

Mycobacterium smegmatis was grown on Middlebrook 7H10 agar supplemented with oleic acid albumin dextrose (OADC, Difco) or LB agar. Tween 80 (0.05%) was added to liquid broth to avoid clumping; when appropriate, antibiotics were added at the following concentrations: 50 μg ml−1 kanamycin, 100 μg ml−1 streptomycin, and 25 μg ml−1 hygromycin. Strain designations were as follows: M. smegmatis SmR5 (20), a derivative of M. smegmatis mc2 (21) carrying a non-restrictive rpsL mutation conferring streptomycin resistance (=parental strain); lnt::aph = lnt knock-out mutant; lnt::aph-lntMs = M. smegmatis lnt::aph transformed with complementing vector pMV361-hyg-lntMs; lnt::aph-ppm1Tb = M. smegmatis lnt::aph transformed with complementing vector pMV361-hyg-ppm1Tb.

Complementation of Conditional E. coli lnt Mutant PAP8508

LntMs was amplified by PCR and cloned into the EcoRI/BamHI sites of pUC18 resulting in pUC18-lntMs. Plasmids pUC18-lntMs323W, pUC18-lntMs477Y, and pUC18-lntMs323W/477Y were generated by standard mutagenesis PCR techniques.

The E. coli conditional lnt mutant PAP8508 and its parental strain PAP105 (a generous gift of N. Buddelmeijer) were used for complementation analysis (22). Strains were plated on LB agar supplemented with 1 mm isopropyl 1-thio-β-d-galactopyranoside, 100 μg/ml ampicillin, and either 0.4% (w/v) glucose or 0.2% (w/v) arabinose.

Disruption of lnt in M. smegmatis

A 3.8-kbp genomic fragment of M. smegmatis from position 3,929,396 to 3,933,223 spanning the entire lntMs gene was PCR amplified and cloned into pGem-T Easy (Promega) to result in pGem-T Easy-lntMs. For functional inactivation of lntMs, a 1.04-kbp SfiI/EcoRV fragment was replaced with a 1.4-kbp SnaBI/HpaI kanamycin resistance cassette from pUC4K (GE Healthcare) subcloned in pMCS5-Kan.2 Subsequently a 4.6-kbp PvuII fragment containing the inactivated lntMs allele (lntMs::aph) was inserted into the EcoRV site of ptrpA1-rpsL (20) to result in ptrpA1- rpsL-lntMs::aph. The lntMs::aph allele was substituted for lntMs in the M. smegmatis chromosome as described previously (23) and confirmed by Southern blot analyses with a 0.2-kbp SmaI/NcoI lntMs upstream probe.

For complementation with M. smegmatis lnt, a 4.3-kbp PvuII fragment from pGem-T Easy-lntMs comprising the entire lntMs gene was cloned into the HpaI site of plasmid pMV361-hyg (24) to result in pMV361-hyg-lntMs. For complementation with M. tuberculosis ppm1 a 6.3-kbp fragment from M. tuberculosis genomic position 2,306,187 to 2,312,526 spanning the entire ppm1 gene was cloned into pGem-T Easy to result in pGem-T Easy-ppm1Tb and subsequently subcloned as a 6.3-kbp EcoRI fragment into the HpaI site of plasmid pMV361-hyg (24) to result in pMV361-hyg-ppm1Tb. Complementation was confirmed by Southern blot analyses with a 0.2-kbp SmaI/NcoI lntMs upstream probe and a 0.2-kbp KpnI/HindIII ppm1Tb upstream probe.

Construction of Expression Vector pMV261-Gm-FusLppX

Plasmid pMV261-Gm, a derivative of pMV261, is a shuttle vector replicating in E. coli as well as in mycobacteria (25). M. tuberculosis LppX was amplified by PCR from genomic DNA and fused to the M. tuberculosis 19-kDa promoter. Two sequences encoding a hemagglutinin and a His6 epitope were fused to the 3′ part of the gene to facilitate subsequent purification and detection on Western blot and the insert was cloned into the EcoRI site to result in pMV261-Gm-FusLppX.

Preparation of Cell Extracts and Western Blot Analysis

Bacteria from 2-liter cultures were harvested, resuspended in phosphate-buffered saline containing Complete EDTA-free tablets (Roche) to inhibit protein degradation, and subjected to two French press cycles (American Instrument Co.) at 2 × 108 pascal. Extracts were treated with 2% sodium N-lauroylsarcosine for 1 h at room temperature and subsequently incubated at 4 °C overnight. Soluble and insoluble fractions were separated by centrifugation at 30,000 × g for 1 h at 4 °C. Extracts corresponding to 1–5 μg of total protein were separated by SDS-PAGE (12%) and analyzed by Western blot. Antiserum against the HA3 epitope (Roche) was diluted 1:300.

Fast Protein Liquid Chromatography Protein Purification

The soluble fraction of cell extracts was diluted with buffer containing 20 mm NaH2PO4, 0.5 m NaCl to 1% sodium N-lauroylsarcosine and loaded on a HisTrapTM HP column (GE Healthcare) equilibrated with buffer containing 20 mm NaH2PO4, 0.5 m NaCl, 0.2% sodium N-lauroylsarcosine, and 20 mm imidazole. Proteins were eluted with 0.125–0.5 m imidazole.

Thrombin Cleavage of RecLppX

Purified RecLppX was dialyzed against phosphate-buffered saline, pH 7.4, at 4 °C because imidazole can lower thrombin activity (26). About 0.1–1 μg of RecLppX was digested with 4–30 NIH units of thrombin from bovine plasma (Sigma) for 16 h at 37 °C with continuous shaking (25 rpm). The reaction was stopped through incubation at 95 °C for 5 min.

MALDI-TOF/TOF

Purified lipoprotein (100–200 pmol) were prepared and analyzed according to Ujihara et al. (27). After tryptic digestion samples were resuspended in 5 μl of 0.1% trifluoroacetic acid, 66% acetonitrile. 1.2 μl were loaded onto the target and covered with 1 μl of matrix (α-cyano-4-hydroxycinnamic acid (Bruker Daltonics), 5 mg/ml in 0.1% trifluoroacetic acid, 50% acetonitrile). Mass spectra were recorded on an Ultraflex II MALDI-TOF/TOF instrument with smartbeam laser upgrade (Bruker, Germany). The frequency-tripled Nd:YAG laser using a structured-focus profile (smartbeam, Bruker Daltonics) was set to a repetition rate of 100 Hz and the ion acceleration voltage was 29.5 kV. The mass measurements were performed in the positive ion reflector mode.

RESULTS

Mycobacterial Lipoproteins Are Modified at the N Terminus

We chose the well characterized M. tuberculosis lipoprotein LppX (28) as a model substrate for mycobacterial lipoprotein synthesis and generated the expression vector pMV261-Gm-FusLppX. Plasmid pMV261-Gm-FusLppX was transformed into M. smegmatis SmR5 (20), M. smegmatis ΔlspA, and complemented M. smegmatis ΔlspA-lspA.2 Whole cell extracts of these strains were subjected to Western blot analysis with anti-HA antibody. We observed bands with an apparent size of 23 kDa in parental and complemented strains and 26 kDa in the ΔlspA mutant and to a small amount also in the parental strain. After purification of LppX, the 26-kDa band was also detected to a higher amount in the parental strain. The 23-kDa band corresponds to the predicted mass of mature recombinant LppX-HA-His and 26-kDa band to the predicted mass of the prolipoprotein form of LppX-HA-His. These results indicate LspA-dependent signal peptide cleavage of recombinant LppX-HA-His in M. smegmatis and verify its post-translational modification within the lipoprotein synthesis pathway (Fig. 1a).

FIGURE 1.
Western blot analysis of LppX. a, total lysates of the M. smegmatis parental strain, ΔlspA, ΔlspA-lspA expressing LppX with HA and the His6 epitope analyzed with mouse anti-hemagglutinin monoclonal antibody; b, amino acid sequence of LppX-HA-His. ...

Purified LppX-HA-His from the M. smegmatis parental strain was subjected to protein sequence analysis. Edman degradation of the prolipoprotein revealed a sequence starting at the initial methionine of the signal peptide of LppX (Fig. 1b). In contrast, no sequence was obtained from the mature LppX indicating a modification of the N-terminal amino group.

Identification of Putative N-Acyltransferases in Bacterial Genomes

In E. coli, N-acylation of lipoproteins is conferred by Lnt (29). We performed a BLAST search analysis with E. coli Lnt as a query to investigate the distribution of Lnt homologues in the bacterial kingdom and to identify putative homologues in mycobacteria. Lnt homologues are widely distributed in Gram-negative bacteria (α, β, γ, δ, ϵ Proteobacteria, Spirochetes, Aquifex, Cytophaga, and Thermotoga), but absent from all classes (Clostridia, Mollicutes, and Bacilli) of low GC Gram-positive bacteria (Firmicutes), although some indications for N-acylation in low GC Gram-positive bacteria have been reported (1215). In contrast, Lnt homologues were identified in all classes of high GC Gram-positive bacteria (Actinobacteria, e.g. Streptomyces, Nocardia, Corynebacteria, and Mycobacteria) (Fig. 2a), but Lnt activity of those homologues could not be demonstrated (22). The cell envelope of the phylum Actinobacteria is more complex than the cell envelope of Firmicutes. In M. tuberculosis and M. smegmatis, Rv2051c (Ppm1) and MSMEG_3860 (Ppm2) have the highest similarity to E. coli Lnt. M. tuberculosis Rv2051c encodes a two-domain protein, of which the N-terminal part shows similarity to E. coli Lnt. The C-terminal part of the protein encodes a polyprenol-monophosphomannose (Ppm) synthase, an enzyme involved in lipomannan and lipoarabinomannan synthesis (30). MSMEG_3860 has been shown to stabilize M. smegmatis Ppm1 in the bacterial membrane and therefore has been annotated as Ppm2 (31). MSMEG_3860 will be referred to here as LntMs. Lnt homologues are also present in Mycobacterium avium and Mycobacterium leprae and are encoded by a separate open reading frame as in M. smegmatis. The genomic region surrounding Lnt homologues is conserved in mycobacteria (Fig. 2b).

FIGURE 2.
Lnt-BLASTp. a, E. coli Lnt was used as a query to identify homologues on the National Center for Biotechnology Information BLASTp server. The sequence filtering option was switched off and the expect value was set at 10, the cut-off value set at 10−4 ...

M. smegmatis Lnt Does Not Restore Growth of a Conditional E. coli lnt Mutant

All enzymes of the lipoprotein synthesis pathway are essential in Gram-negative bacteria. We intended to demonstrate mycobacterial Lnt activity by complementation of an E. coli conditional lnt mutant. Because LntMs is encoded by a separate open reading frame and not fused to a second domain as in M. tuberculosis, we chose M. smegmatis Lnt (MSMEG_3860) instead of M. tuberculosis Ppm1 (Rv2051c) for complementation. LntMs encodes a protein of 654 amino acids with a 25% identity and 40% similarity to E. coli and a 63% identity and 73% similarity to the N-terminal part of M. tuberculosis Rv2051c. LntMs was cloned into vector pUC18 (Fermentas) to result in pUC18-lntMs and transformed into the conditional E. coli lnt mutant PAP8508 (22). However, we could not restore growth of the PAP8508 mutant under restrictive conditions (data not shown). Seven amino acids (Trp-237, Glu-267, Lys-335, Glu-343, Cys-387, Tyr-388, and Glu-389) are reported to be essential for E. coli Lnt function (22). Five of these seven residues are conserved in LntMs, whereas two are altered (LntEc Trp-237 corresponds to LntMs Glu-323, LntEc Tyr-388 corresponds to LntMs Trp-477). We exploited site-directed mutagenesis to introduce these E. coli codons into the M. smegmatis sequence of pUC18-lntMs to result in pUC18-lntMs323W, pUC18-lntMs477Y, and pUC18-lntMs323W/477Y. However, transformation of none of these vectors complemented the conditional E. coli lnt mutant (data not shown).

Generation and Characterization of M. smegmatis lnt::aph Mutant

Because we were unable to complement an E. coli lnt mutant, we decided to investigate Lnt activity directly in mycobacteria by generating a M. smegmatis lnt deletion mutant. The deletion mutant was constructed by transformation of M. smegmatis SmR5 with the suicide plasmid ptrpA1-rpsL- lntMs::aph using rpsL counter-selection strategy (20). The mutant strain resulting from allelic replacement is here referred to as M. smegmatis lnt::aph. Deletion of lntMs was verified by Southern blot analysis using a 5′ lntMs DNA probe (supplemental Fig. S1). The probe hybridized to a 1.4-kbp fragment of the parental strain and a 6.4-kbp fragment of the lnt::aph mutant. The difference in size results from the deletion of a BstEII restriction site and insertion of a kanamycin resistance cassette. We cloned two complementation vectors (pMV361-hyg-lntMs and pMV361-hyg-ppm1Tb) expressing M. smegmatis Lnt and M. tuberculosis Ppm1 under control of their native promoters. Transformation of these plasmids into the M. smegmatis lnt::aph mutant resulted in strains M. smegmatis lnt::aph-lntMs and M. smegmatis lnt::aph-ppm1Tb.

Western blot analysis of extracts from M. smegmatis lnt::aph expressing LppX-HA-His revealed a molecular mass of the detected protein, which cannot be distinguished from that of LppX-HA-His expressed in the M. smegmatis parental strain. However, N-terminal sequencing revealed that LppX-HA-His purified from M. smegmatis lnt::aph is accessible to Edman degradation (sequence CSSP) indicating that the N-terminal amino group is not still blocked.

LntMs and Ppm1Tb Are Apolipoprotein N-Acyltransferases

Because fatty acids of membrane phospholipids are the substrates for N-acylation of lipoproteins in E. coli (3234), its lipoproteins are modified with myristic, palmitic, palmitoleic, oleic, or vaccinic acid (35). Phospholipids in mycobacteria mainly consist of palmitic, palmitoleic, oleic, and tuberculostearic acid (10-methyloctadecanoic acid) (36). Therefore we hypothesized that N-acylation of lipoproteins in mycobacteria increase the molecular mass by ~0.3 kDa. To differentiate between lipoproteins with a free or acylated N terminus, we cloned an additional expression vector, RecLppX. It differs from LppX-HA-His by a hemagglutinin epitope followed by a thrombin cleavage site inserted after amino acid Ala (+19) of the mature LppX (Fig. 1b). The thrombin cleavage site LVPRGS was inserted to produce a small N-terminal fragment of 33 residues (about 3.5 kDa) after thrombin cleavage. To ensure that the insertion of a HA epitope and a thrombin site does not abolish recognition of RecLppX as a lipoprotein, we analyzed total lysates of the M. smegmatis parental strain, M. smegmatis ΔlspA, and M. smegmatis ΔlspA-lspA by Western blot (Fig. 3a). Temperature-sensitive lspA mutants of E. coli and lspA knock-out mutants of Gram-positive bacteria accumulate prolipoproteins (37, 38). Immunoblotting of total lysates of the M. smegmatis parental strain, ΔlspA and ΔlspA-lspA with antiserum against the HA epitope (Roche) revealed the presence of a 25-kDa band in parental and complemented strains. In contrast, a band with a slightly larger size (approximately 27 kDa, the increase corresponds to the mass of the signal sequence) was observed in the ΔlspA strain. This result shows that insertion of a HA epitope and a thrombin cleavage site did not impair the recognition of RecLppX as a lipoprotein. We then investigated thrombin-digested RecLppX for Lnt-dependent modification by Western blot analyses (Fig. 3b). In the lnt::aph mutant we observed a slightly smaller size of the N-terminal part of RecLppX suggesting, that there are fewer modifications on the protein compared with the parental strain and both complemented strains lnt::aph-lntMs and lnt::aph-ppm1Tb. In all strains, we also found a double band of the N-terminal part of RecLppX, indicating partial modification of RecLppX by enzymes other than Lnt or LspA. We also observed a deviation from the calculated size of the N-terminal part of mature RecLppX. The molecular mass was calculated to be 3.5 kDa but in the parental strain we found a band corresponding to a size of about 6 kDa. This difference in size is probably due to an altered migration behavior because of lipid modifications. It can be excluded that these bands at 6 kDa are prolipoprotein forms, still containing the signal peptide, because the N-terminal part of pro-RecLppX from the ΔlspA mutant is running at about 8.5 kDa. These results show that RecLppX is modified by LspA as well as LntMs. Ppm1Tb is sufficient to replace LntMs implicating that similar lipoprotein modifications take place in M. tuberculosis.

FIGURE 3.
Western blot analysis of RecLppX. a, total lysates of M. smegmatis wild type (wt), ΔlspA, and ΔlspA-lspA expressing RecLppX with hemagglutinin and the His6 epitope analyzed with mouse anti-hemagglutinin monoclonal antibody. b, RecLppX ...

Recombinant M. tuberculosis LppX (FusLppX) was heterologously expressed and purified. Tryptic fragments of FusLppX were analyzed by MALDI-TOF/TOF mass spectrometry to characterize modifications taking place on lipoproteins in M. smegmatis at the molecular level. Purified mature LppX from parental strain, lnt::aph mutant, and lnt::aph-ppm1Tb was prepared for analysis according to Ujihara et al. (27). For identification of the modifications of the universally conserved cysteine, the structure of the N-terminal tryptic peptide was determined. Experimentally found m/z values are summarized and compared with calculated m/z values in Table 1. Trypsin cleavage sites of LppX are given in Fig. 1b. The expected monoisotopic molecular mass of the unmodified N-terminal tryptic peptide of LppX is 2963.46 Da. Instead, we found a [M + H]+ signal at m/z = 3795.42 for the N-terminal tryptic peptide of LppX from the parental strain and a signal at m/z = 3557.01 from the lnt::aph mutant. The N-terminal tryptic peptide from lnt::aph-ppm1Tb is a mixture and showed signals of m/z = 3795.32 and 3557.07 (Fig. 4). This indicates that the N-terminal peptide of LppX is modified in an LntMs-dependent manner. To identify the found modification, we calculated diacylglycerol modifications with all theoretical combinations of the four fatty acids found in mycobacterial phospholipids: palmitic acid (C16:0), palmitoleic acid (C16:1), oleic acid (C18:1), and tuberculostearic acid (C19:0). The difference in molecular mass between the peptide of the lnt::aph mutant and the unmodified peptide is 592.55 Da indicating a diacylglycerol modification with ester-linked C19:0 and C16:0 fatty acid. These fatty acids are most likely tuberculostearic acid and palmitic acid.

TABLE 1
Comparison of m/z values of LppX N-terminal tryptic peptides found in the different mutants
FIGURE 4.
MALDI-TOF analysis of a trypsin digest of purified LppX. MS analysis of LppX tryptic peptides purified from the M. smegmatis parental strain (1); M. smegmatis lnt::aph (2), and M. smegmatis lnt::aph-ppm1Tb (3). Filled triangle, diacylglycerol plus N-acyl ...

The difference in molecular mass of 238.41 Da between the lnt::aph mutant (m/z = 3557.07) and parental strain (m/z = 3795.42) indicates an additional modification with a C16:0 fatty acid in the parental strain. By thoroughly analyzing the signal at m/z = 3795.42 in high resolution, we also observed a minor signal (approximately 10%) at m/z = 3793.35. This indicates the presence of a C16:1 fatty acid in place of the C16:0 fatty acid, but only a small amount (data not shown). In the complemented mutant lnt::aph-ppm1Tb both forms of the N-terminal tryptic peptide of LppX were found. This finding indicates partial complementation of LntMs by M. tuberculosis Ppm1.

In the lnt::aph and the lnt::aph-ppm1Tb mutant we additionally found an N-terminal tryptic peptide at m/z = 3739.18 and 3739.32, respectively. But no evidence for an aminoacyl modification was found in the MS/MS fragmentation pattern. In contrast, the release of 183 Da most likely corresponds to a covalent modification of the free N-terminal amino group of LppX in these two strains with 4-(2-aminoethyl)-benzenesulfonyl fluoride, a component of the protease inhibitor mixture that has been used. 4-(2-Aminoethyl)-benzenesulfonyl fluoride is known to modify hydroxylated amino acids and to a lesser extent also free amino groups.

To obtain information about linkage of the modifications, the structure of the triacylated N-terminal tryptic peptide of LppX (m/z = 3795.42) was investigated by MS/MS (Figs. 5 and supplemental S2). A summary of all found eliminations in the three strains are given in Table 2. The ions at m/z = 3539.59 and 3496.52 correspond to the neutral loss of a C16 (Δ = 255.82 Da) and a C19:0 fatty acid (Δ = 298.89 Da), respectively. The most intense fragment ion at m/z = 3169.21 corresponds to the elimination of a diacylthioglycerol carrying both O-linked C19:0 and C16 fatty acids (Δ = 626.22 Da). In addition, the release of 370.38 Da from the ion at m/z = 3539.59 corresponds to the elimination of a C19:0 fatty acid α-thioglycerol ester and the release of 327.31 Da from the ion at m/z = 3496.52 corresponds to the elimination of a C16 fatty acid α-thioglycerol ester. This fragmentation pattern shows that the +1 cysteine is modified at the sulfhydryl group by a diacylglycerol residue carrying ester-bound C16 fatty acid and C19:0 fatty acid. Whether the C19:0 fatty acid is in the Sn1 or Sn2 position cannot be determined. The release of 255.08 Da from the ion at m/z = 3169.21 indicates the release of a palmitamide derived from an amide-bound C16 fatty acid. The MS/MS fragmentation pattern of the LppX N-terminal tryptic peptide of the lnt::aph mutant (m/z = 3557.07) showed only the elimination of the diacylthioglycerol, but no release of palmitamide, as expected (Fig. 5).

FIGURE 5.
MALDI-TOF-TOF analysis of the N-terminal peptides of LppX. MS/MS analysis of N-terminal peptides of LppX purified from M. smegmatis parental strain (1), M. smegmatis lnt::aph (2), and M. smegmatis lnt::aph-ppm1Tb (3). A schematic drawing of the modified ...
TABLE 2
Comparison of experimentally determined eliminations from N-terminal tryptic peptides of LppX in the MALDI-TOF/TOF spectra of the different mutants with theoretically calculated eliminations

To verify the diacylglycerol modification, we also analyzed tryptic peptides of LppX from the ΔlspA mutant by MALDI-TOF/TOF mass spectrometry (Fig. 6). Experimentally found m/z values are summarized and compared with calculated m/z values in Table 3. Trypsin cleavage sites of pro-LppX are given in Fig. 1b. We found three m/z signals corresponding to the tryptic peptide containing the +1 cysteine. The signal at m/z = 4887.30 corresponds to the peptide with a disulfide bridge between the two present cysteines (position −8 and +1). The signal at m/z = 5041.23 corresponds to the peptide with both cysteines being modified by β-mercaptoethanol, a buffer component used for SDS-PAGE. The signal at m/z = 5558.12 corresponds to the peptide with one cysteine being modified with a β-mercaptoethanol but the other being modified with the diacylglycerol carrying O-linked C16 and C19:0 fatty acids also found in the previously analyzed strains (Fig. 6). This result shows that LppX purified from the ΔlspA mutant is a mixture of pre-pro-LppX and pro-LppX.

FIGURE 6.
MALDI-TOF analysis of peptides resulting from trypsin digestion of LppX from ΔlspA mutant. MS analysis of LppX tryptic peptides purified from the M. smegmatis ΔlspA mutant. Asterisk, N-terminal peptide without fatty acid modifications; ...
TABLE 3
Comparison of m/z values of tryptic peptides of pro-LppX containing the +1 cysteine found in the ΔlspA mutant

Taken together the results show that the universally conserved cysteine of M. tuberculosis LppX is modified with a thioether-linked diacylglycerol residue carrying an ester-bound C19:0 and an ester-bound C16 fatty acid. In addition, it is modified with an amide-linked third C16 fatty acid. The C19:0 fatty acid corresponds most likely to the mycobacterial specific tuberculostearic acid. It is also proved that LntMs is an N-acyltransferase and M. tuberculosis Ppm1 is able to complement the M. smegmatis lnt::aph mutant and therefore Ppm1 seems to be a bifunctional protein. Within this protein the N-terminal domain presumably exhibits N- acyltransferase activity (our data) and the C-terminal domain exhibits mannosyltransferase activity (30).

DISCUSSION

The lipoprotein biosynthesis pathway consisting of the three enzymes Lgt, LspA, and Lnt has been intensively studied in E. coli and has been shown to be essential and necessary for transport of lipoproteins to the outer membrane of Gram-negative bacteria (11, 39, 40). In mycobacteria, little is known about synthesis and localization of lipoproteins, only a few lipoproteins are functionally characterized and annotation is mainly based on theoretical considerations instead of experimental evidence. However, consistent with the biosynthetic pathway in E. coli, putative lgt (Rv1614) and lsp (Rv1539) genes have been identified in the M. tuberculosis genome (41). In previous studies (3, 4) we showed that in mycobacteria the lipoprotein pathway is a major virulence factor. For fundamental knowledge and further investigations, we were interested in how lipoproteins are modified in mycobacteria. In the present study, we investigated the lipid moieties of a representative mycobacterial lipoprotein.

We identified Lnt homologues in mycobacteria, corynebacteria, and streptomyces species. In low GC Gram-positive bacteria Lnt homologues are completely absent (Fig. 2), but in 1985 the first indirect detection of N-acylation in the Gram-positive B. subtilis was published and in S. aureus triacylation of lipoprotein SitC was recently reported (14, 15), whereas another lipoprotein (SAOUHSC_02699) was only found to be diacylated (13). The protein responsible for attaching the third fatty acid to lipoproteins in S. aureus has not been identified. It may be differentially expressed depending on culture conditions or may have a narrow substrate specificity. In M. tuberculosis the Lnt homologue found is annotated as Rv2051c. This open reading frame was originally annotated as a two-domain enzyme with a putative N-terminal Lnt domain and a C-terminal polyprenol monophosphomannose synthase (Ppm1) domain and was characterized as the latter one (30). Although the putative Lnt domain is not needed for Ppm1 activity, on overexpression in M. smegmatis it appeared to enhance the mannosyltransferase activity. Interestingly, the two domains of M. tuberculosis Ppm1 are encoded by separate, adjacent open reading frames in the genomes of other mycobacteria (Fig. 2b).

Previous attempts to complement a conditional E. coli lnt mutant with Lnt homologues from other bacterial species corresponding to the order Actinomycetales (Streptomyces and Corynebacterium) failed (22). Likewise we were unable to complement this E. coli strain with a mycobacterial Lnt homologue. Even after exchange of the two essential amino acids differing between M. smegmatis and E. coli, complementation of the E. coli lnt mutant failed. LntMs as E. coli Lnt attaches a C16 fatty acid to the free amino group of the universally conserved cysteine. Therefore the failure of complementation is not due to the absence of fatty acid substrates. Rather mycobacterial lipoproteins are modified with a diacylglycerol carrying mycobacterial specific fatty acids. Failure of complementation of PAP8508 therefore is probably due to the fact that LntMs recognizes only lipoproteins modified with a diacylglycerol residue carrying at least one ester-bound mycobacterial specific fatty acid. This implies that LntMs does not recognize lipoproteins modified with diacylglycerol residues carrying only small fatty acids like palmitic or palmitoleic acid. Specificity could be tested in an in vitro assay system. Alternatively, the expression level or enzymatic activity of mycobacterial Lnt homologues may not sustain growth of fast growing E. coli.

We then investigated LntMs and M. tuberculosis Ppm1 activity in a mycobacterial background. As lntMs is not an essential gene in mycobacteria, we generated an isogenic M. smegmatis lnt::aph mutant. After thrombin cleavage, the recombinant lipoprotein (RecLppX) extracted from the M. smegmatis lnt::aph mutant showed a faster running behavior on SDS-PAGE than RecLppX extracted from the parental strain. The size was about 0.3 kDa smaller corresponding to fewer modifications of RecLppX in the lnt::aph mutant. We also recognized a double band of digested RecLppX in all strains used as well as a discrepancy between the calculated and the apparent molecular mass. The altered running behavior is probably due to the modifications on the small N-terminal fragment and the observed double band indicates partial processing of RecLppX by enzymes other than Lgt, LspA, or Lnt. Glycosylation of RecLppX is one possibility, but information about the structure, function, and biosynthetic pathways of prokaryotic glycoproteins is scarce. Glycosylation of M. tuberculosis lipoproteins has been confirmed for the 45/47 kDa protein, SodC, and for the Mycobacterium bovis MPB83 protein (Rv2873) (4244). Glycosylation of SodC influences its ultimate subcellular localization and also its proteolytic processing.

By performing MALDI-TOF/TOF analyses of a trypsin digest of purified LppX we unambiguously identified modifications at the universally conserved cysteine. After trypsin cleavage of LppX, the N-terminal peptide from the parental strain has a mass of 3794.41 Da instead of 2963.46 Da predicted for the unmodified peptide, whereas the N-terminal peptide from the isogenic lnt::aph mutant showed a mass of 3556.01 Da. This strongly indicates that LntMs covalently links a C16 fatty acid (C16:0 or C16:1) to the N terminus of the peptide. The additional increase by 592 Da corresponds to a diacylglycerol residue with C16 fatty acid and a C19:0 fatty acid, which corresponds most likely to tuberculostearic acid (10-methyloctadecanoic acid), forming a thioether linkage to the sulfhydryl group of the cysteine. Whether the C19:0 fatty acid is in the Sn1 or Sn2 position cannot be determined. The same diacylglycerol modification was also found in the ΔlspA mutant. The loss of the N-acyl modification in the lnt::aph mutant is complemented by the M. tuberculosis homologue Ppm1 suggesting that mature M. tuberculosis lipoproteins are N-acylated. N-Acylation affects the interaction of lipoproteins with innate immune receptors (45). Ppm1 was shown to exhibit polyprenol-monophosphomannose synthetase activity (30) although the major part of the protein has homology to E. coli Lnt. Both masses of the N-terminal tryptic peptide (3556.07 and 3794.32 Da) were found in the complemented mutant indicating that not all apolipoprotein was converted to mature lipoprotein by Ppm1. As M. tuberculosis has a generation time of about 24 h and M. smegmatis only of about 3 h, it is possible that M. tuberculosis Ppm1 has a lower enzymatic activity than LntMs. Alternatively, the expression level of M. tuberculosis Ppm1 is lower than that of M. smegmatis Lnt. The identification of O-linked tuberculostearic acid shows that mycobacterial lipoproteins are modified with mycobacterial specific fatty acids and differ from lipoproteins modified in E. coli.

In this study we directly show that Gram-positive mycobacteria synthesize triacylated lipoproteins. This is the first time to our knowledge that responsible genes for Lnt activity are assigned in Gram-positive bacteria. LntMs and M. tuberculosis Ppm1 are functional homologues of E. coli Lnt as they catalyze the transfer of the third acyl moiety to the free α-amino group of the N-terminal amino acid of lipoproteins. Most likely mycobacterial Lnt homologues differ in substrate specificity from E. coli Lnt. N-Acylation is a prerequisite for transport of E. coli lipoproteins to the outer membrane (46). Likewise, N-acylation of mycobacterial lipoproteins may be required for transport to the outer most lipid layer of mycobacteria, which according to recent investigations resembles the outer membrane of Gram-negative bacteria (47, 48).

Supplementary Material

Supplemental Data:

*This work was supported in part by the Swiss National Science Foundation Grant 3100A0-120326 and the European Union TB-Drug Grant LSHP-CT-2006-037217.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpg The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

2A. Tschumi, C. Nai, P. Keller, T. Grau, and P. Sander, unpublished data.

3The abbreviations used are:

HA
hemagglutinin
Ppm
polyprenol-monophosphomannose
MALDI-TOF
matrix-assisted laser desorption ionization time-of-flight.

REFERENCES

1. Eichler J., Adams M. W. (2005) Microbiol. Mol. Biol. Rev. 69, 393–425 [PMC free article] [PubMed]
2. Nadolski M. J., Linder M. E. (2007) FEBS J. 274, 5202–5210 [PubMed]
3. Rampini S. K., Selchow P., Keller C., Ehlers S., Böttger E. C., Sander P. (2008) Microbiology 154, 2991–3001 [PubMed]
4. Sander P., Rezwan M., Walker B., Rampini S. K., Kroppenstedt R. M., Ehlers S., Keller C., Keeble J. R., Hagemeier M., Colston M. J., Springer B., Böttger E. C. (2004) Mol. Microbiol. 52, 1543–1552 [PubMed]
5. Hutchings M. I., Palmer T., Harrington D. J., Sutcliffe I. C. (2009) Trends Microbiol. 17, 13–21 [PubMed]
6. Sutcliffe I. C., Harrington D. J. (2004) FEMS Microbiol. Rev. 28, 645–659 [PubMed]
7. Babu M. M., Priya M. L., Selvan A. T., Madera M., Gough J., Aravind L., Sankaran K. (2006) J. Bacteriol. 188, 2761–2773 [PMC free article] [PubMed]
8. Rezwan M., Grau T., Tschumi A., Sander P. (2007) Microbiology 153, 652–658 [PubMed]
9. Yakushi T., Masuda K., Narita S., Matsuyama S., Tokuda H. (2000) Nat. Cell Biol. 2, 212–218 [PubMed]
10. Baumgärtner M., Kärst U., Gerstel B., Loessner M., Wehland J., Jänsch L. (2007) J. Bacteriol. 189, 313–324 [PMC free article] [PubMed]
11. Wu H. C. (1996) in Escherichia coli and Salmonella typhimurium ( Neidhardt F. C., Curtis R. 3rd, Ingraham J. L., Lin E. C. C., Low K. B., Magasanik B., Reznikoff W. S., Riley M., Schaechter M., Umbarger H. E., editors. eds) pp. 1005–1014, American Society for Microbiology, Washington, D. C.
12. Navarre W. W., Daefler S., Schneewind O. (1996) J. Bacteriol. 178, 441–446 [PMC free article] [PubMed]
13. Tawaratsumida K., Furuyashiki M., Katsumoto M., Fujimoto Y., Fukase K., Suda Y., Hashimoto M. (2009) J. Biol. Chem. 284, 9147–9152 [PMC free article] [PubMed]
14. Hayashi S., Chang S. Y., Chang S., Giam C. Z., Wu H. C. (1985) J. Biol. Chem. 260, 5753–5759 [PubMed]
15. Kurokawa K., Lee H., Roh K. B., Asanuma M., Kim Y. S., Nakayama H., Shiratsuchi A., Choi Y., Takeuchi O., Kang H. J., Dohmae N., Nakanishi Y., Akira S., Sekimizu K., Lee B. L. (2009) J. Biol. Chem. 284, 8406–8411 [PMC free article] [PubMed]
16. Young D. B., Garbe T. R. (1991) Res. Microbiol. 142, 55–65 [PubMed]
17. Pecora N. D., Gehring A. J., Canaday D. H., Boom W. H., Harding C. V. (2006) J. Immunol. 177, 422–429 [PubMed]
18. Drage M. G., Pecora N. D., Hise A. G., Febbraio M., Silverstein R. L., Golenbock D. T., Boom W. H., Harding C. V. (2009) Cell. Immunol. 258, 29–37 [PMC free article] [PubMed]
19. Buwitt-Beckmann U., Heine H., Wiesmüller K. H., Jung G., Brock R., Akira S., Ulmer A. J. (2006) J. Biol. Chem. 281, 9049–9057 [PubMed]
20. Sander P., Meier A., Böttger E. C. (1995) Mol. Microbiol. 16, 991–1000 [PubMed]
21. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R., Jr. (1990) Mol. Microbiol. 4, 1911–1919 [PubMed]
22. Vidal-Ingigliardi D., Lewenza S., Buddelmeijer N. (2007) J. Bacteriol. 189, 4456–4464 [PMC free article] [PubMed]
23. Sander P., Springer B., Böttger E. C. (2001) in Mycobacterium tuberculosis Protocols (Parish T., Stoker N. G., editors. eds) pp. 93–104, Humana Press, Totowa, NJ
24. Sander P., Prammananan T., Meier A., Frischkorn K., Böttger E. C. (1997) Mol. Microbiol. 26, 469–480 [PubMed]
25. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H., Hatfull G. F., et al. (1991) Nature 351, 456–460 [PubMed]
26. Isaacs R. C., Solinsky M. G., Cutrona K. J., Newton C. L., Naylor-Olsen A. M., Krueger J. A., Lewis S. D., Lucas B. J. (2006) Bioorg. Med. Chem. Lett. 16, 338–342 [PubMed]
27. Ujihara T., Sakurai I., Mizusawa N., Wada H. (2008) Anal. Biochem. 374, 429–431 [PubMed]
28. Sulzenbacher G., Canaan S., Bordat Y., Neyrolles O., Stadthagen G., Roig-Zamboni V., Rauzier J., Maurin D., Laval F., Daffé M., Cambillau C., Gicquel B., Bourne Y., Jackson M. (2006) EMBO J. 25, 1436–1444 [PMC free article] [PubMed]
29. Gupta S. D., Gan K., Schmid M. B., Wu H. C. (1993) J. Biol. Chem. 268, 16551–16556 [PubMed]
30. Gurcha S. S., Baulard A. R., Kremer L., Locht C., Moody D. B., Muhlecker W., Costello C. E., Crick D. C., Brennan P. J., Besra G. S. (2002) Biochem. J. 365, 441–450 [PMC free article] [PubMed]
31. Baulard A. R., Gurcha S. S., Engohang-Ndong J., Gouffi K., Locht C., Besra G. S. (2003) J. Biol. Chem. 278, 2242–2248 [PubMed]
32. Lai J. S., Wu H. C. (1980) J. Bacteriol. 144, 451–453 [PMC free article] [PubMed]
33. Gupta S. D., Dowhan W., Wu H. C. (1991) J. Biol. Chem. 266, 9983–9986 [PubMed]
34. Jackowski S., Rock C. O. (1986) J. Biol. Chem. 261, 11328–11333 [PubMed]
35. Lin J. J., Kanazawa H., Wu H. C. (1980) J. Biol. Chem. 255, 1160–1163 [PubMed]
36. Goren M. B., Brennan P. J. (1979) in Tuberculosis (Youmans G. P., editor. ed) pp. 63–193, The W. B. Saunders Co., Philadelphia, PA
37. Venema R., Tjalsma H., van Dijl J. M., de Jong A., Leenhouts K., Buist G., Venema G. (2003) J. Biol. Chem. 278, 14739–14746 [PubMed]
38. Sankaran K., Wu H. C. (1995) Methods Enzymol. 248, 169–180 [PubMed]
39. Robichon C., Bonhivers M., Pugsley A. P. (2003) Mol. Microbiol. 49, 1145–1154 [PubMed]
40. Robichon C., Vidal-Ingigliardi D., Pugsley A. P. (2005) J. Biol. Chem. 280, 974–983 [PubMed]
41. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S., Barry C. E., 3rd, Tekaia F., Badcock K., Basham D., Brown D., Chillingworth T., Connor R., Davies R., Devlin K., Feltwell T., Gentles S., Hamlin N., Holroyd S., Hornsby T., Jagels K., Krogh A., McLean J., Moule S., Murphy L., Oliver K., Osborne J., Quail M. A., Rajandream M. A., Rogers J., Rutter S., Seeger K., Skelton J., Squares R., Squares S., Sulston J. E., Taylor K., Whitehead S., Barrell B. G. (1998) Nature 393, 537–544 [PubMed]
42. Dobos K. M., Khoo K. H., Swiderek K. M., Brennan P. J., Belisle J. T. (1996) J. Bacteriol. 178, 2498–2506 [PMC free article] [PubMed]
43. Michell S. L., Whelan A. O., Wheeler P. R., Panico M., Easton R. L., Etienne A. T., Haslam S. M., Dell A., Morris H. R., Reason A. J., Herrmann J. L., Young D. B., Hewinson R. G. (2003) J. Biol. Chem. 278, 16423–16432 [PubMed]
44. Sartain M. J., Belisle J. T. (2009) Glycobiology 19, 38–51 [PMC free article] [PubMed]
45. Akira S. (2003) Curr. Opin. Immunol. 15, 5–11 [PubMed]
46. Fukuda A., Matsuyama S., Hara T., Nakayama J., Nagasawa H., Tokuda H. (2002) J. Biol. Chem. 277, 43512–43518 [PubMed]
47. Hoffmann C., Leis A., Niederweis M., Plitzko J. M., Engelhardt H. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 3963–3967 [PMC free article] [PubMed]
48. Zuber B., Chami M., Houssin C., Dubochet J., Griffiths G., Daffé M. (2008) J. Bacteriol. 190, 5672–5680 [PMC free article] [PubMed]

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...