Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB′ mutant

The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by complex lipids and amongst these, lipoglycans are of particular interest. The disruption of NCgl2106 in C. glutamicum resulted in a mutant devoid of monoacylated phosphatidyl-myo-inositol dimannoside (Ac1PIM2) resulting in the accumulation of Ac1PIM1 and cessation of phosphatidyl-myo-inositol (PI) based lipomannan (Cg-LM, now also termed ‘Cg-LM-A’) and lipoarabinomannan (Cg-LAM) biosynthesis. Interestingly, SDS-analysis of the lipoglycan fraction from the mutant revealed the synthesis of a single novel lipoglycan, now termed ‘Cg-LM-B’. Further chemical analyses established the lipoglycan possessed an α-d-glucopyranosyluronic acid-(1 → 3)-glycerol (GlcAGroAc2) based anchor which was then further glycosylated by 8–22 mannose residues, with Man12–20GlcAGroAC2 molecular species being the most abundant, to form a novel lipomannan structure (Cg-LM-B). The deletion of NCgl2106 in C. glutamicum has now provided a useful strain, in addition with a deletion mutant of NCgl0452 in C. glutamicum for the purification of Cg-LM-A and Cg-LM-B. Interestingly, both Cg-LM species induced a similar production of TNF-α by a human macrophage cell line suggesting that the phospho-myo-inositol residue of the PI-anchor does not play a key role in lipoglycan pro-inflammatory activity.

The core mannan backbone is glycosylated by Rv2181 and results in the synthesis of a(1 ? 2)-Manp linked branches, characteristic of the mannan backbone in LM and LAM . Recently, Mishra et al. (2007) and Kaur et al. (2007) reported a novel a-mannosyltransferase, MptA (Rv2174), involved in the latter stages of LM/LAM biosynthesis in Corynebacterineae. The mature LM is then elaborated with arabinose by EmbC to form LAM (Berg et al. 2005). Recently a novel mannosyltransferase, MT1671 (and Rv1635c), has been shown to add terminal Manp residues to the mature LAM in M. tuberculosis to form ManLAM Appelmelk et al. 2007).
In this study, we have examined an NCgl2106 null mutant of C. glutamicum and established that NCgl2106 is a phosphatidyl-myo-inositol monomannoside mannopyranosyltransferase exclusively involved in the synthesis of Ac 1 PIM 2 from Ac 1 PIM 1 , now termed Cg-pimB 0 (Lea-Smith et al. 2008) (Fig. 1). In addition, the utilisation of this mutant strain allowed the isolation and chemical analysis of a novel lipoglycan, now termed Cg-LM-B, based on a GlcAGroAc 2 anchor rather than a PI-based anchor, which typifies Corynebacterineae LM and LAM. Moreover, purification of Cg-LM-A and Cg-LM-B, has allowed an evaluation of their pro-inflammatory properties.

Strains and culture conditions
The wild type of Corynebacterium glutamicum, ATCC 13032, was grown on either the complex medium Brain Heart Infusion (BHI) (Difco) or a salt medium CGXII (Eggeling and Bott 2005) at 30°C. The Escherichia coli strain DH5 was grown on LB at 37°C. Kanamycin and ampicillin were used at a concentration of 25 or 50 lg/ml, wherever appropriate. Samples for lipid analysis were prepared by harvesting the cells at an optical density 600 nm of 10-15 followed by a saline wash and freeze drying.

Construction of plasmids and strains
To construct the deletion vector pK19mobsacBD2106 crossover PCR was applied with primer pairs Nout2106/Nin2106 (Nout2106, AATCGGAGATCC GAGACCGGG; Nin2106, CCCATCCACTAAACTT AAACATTTTCGGGATGCAGACACAAAGA) (all primers in 5 0 -3 0 direction) and Cout2106/Cin2106 (Cout2106, ACCCAGTTGTCAGCGCCTTGAG; Cin 2106, TGTTTAAGTTTAGTGGATGGGCGGTTG ACCAATATTTTGCAGAG) with C. glutamicum genomic DNA as template. Both amplified products were used in a second PCR with primer pairs Nout2106/Cout2106 to generate a 1025 bp fragment consisting of sequences adjacent to NCgl2106, which was made blunt, phosphorylated and ligated with SmaI-cleaved pK19mobsacB. The chromosomal deletion of NCgl2106 was performed as described previously using two rounds of positive selection (Schafer et al. 1994), and its successful deletion verified by use of two different primer pairs. Plasmids were introduced into C. glutamicum by electroporation with selection to kanamycin resistance (25 lg/ml) on BHI. To enable expression of NCgl2106 in the deletion mutant, NCgl2106 was amplified using the primer pair CGCGGATCCAAGGAG ATATAGATA TGTCTGCATCCCGAAAAACTCTC and CGCGAA TTCTCATCGTGGTTCACTCTGC. The purified PCR fragment was digested with BamHI-EcoRI and ligated with pEKEx2 (Eikmanns et al. 1991). All cloned fragments were verified by sequencing.

Permethylation of Cg-LM-B prior to MALDI-TOF analysis
Permethylation was performed using the sodium hydroxide procedure as described previously ). MALDI-TOF and TOF/TOF MS data on permethylated samples were acquired in the positive ion mode (M + Na) + using a 4800 (Applied Biosystems) mass spectrometer in the reflector mode with delayed extraction. The collision energy was set to 1 kV, and argon was used as collision gas for MS/MS data collection. Samples were dissolved in methanol, and 1 ll was mixed at a 1:1 ratio (v/v) with 2,5dihydrobenzoic acid (20 mg/ml in 70% methanol in water) as matrix.
Gas chromatography-mass spectrometry linkage analysis Partially methylated alditol acetates were prepared from permethylated samples for gas chromatography-mass spectrometry (GC-MS) linkage analysis as described ). The partially methylated alditol acetates were analyzed using a PerkinElmer Clarus 500 instrument fitted with a RTX-5 column (30 m 9 0.25-mm internal diameter, Restek Corp.).
The sample was dissolved in hexane and injected onto the column at 65°C. The column was maintained at this temperature for 1 min and then heated to 290°C at a rate of 8°C per min. TNF-a production by human macrophage cell line THP-1 human monocyte/macrophage cell line was maintained in continuous culture with RPMI 1640 medium (Lonza), 10% fetal calf serum (Lonza) in an atmosphere of 5% CO 2 at 37°C, as non-adherent cells. Lipoglycans at concentrations of 5 or 15 lg/ml were added in triplicate to cells (10 5 cells/well) in 96-well culture plates and then incubated for 16 h at 37°C. Supernatants were assayed for TNF-a by sandwich ELISA using commercially available kits and according to manufacturer's instructions (R&D Systems).

Construction and growth of C. glutamicumDpimB 0
We found that the gene product of NCgl0452 of C. glutamicum, respectively its ortholog Rv0557 in M. tuberculosis, originally termed PimB and now termed MgtA ), primarily acts as an a-mannosyltransferase to add mannose to GlcAGro-Ac 2 ). This study also highlighted the presence of a lipomannan, now termed Cg-LM-B, based on a GlcAGroAc 2 anchor rather than a PI-anchor. Further characterization of Cg-LM-B was hampered due to co-migration and co-elution following size exclusion chromatography with the PI-based Cg-LM-A ). To investigate the structure and function of Cg-LM-B further we adopted a strategy based on the existence of two pathways to lipoglycan synthesis (Fig. 1) and to identify Cg-pimB 0 involved in Ac 1 PIM 2 synthesis whereby disruption would block C. glutamicum LM/LAM synthesis while Cg-LM-B would be unaffected. We searched for unknown glycosyltransferases using nucleotide-activated sugars. Amongst others we identified NCgl2106 in the genome of C. glutamicum with orthologs present in all Corynebacterianeae including Mycobacterium species.
In order to delete NCgl2106 in the wild type of C. glutamicum, we constructed the non-replicative plasmid pK19mobsacBD2106. This was used to transform C. glutamicum to kanamycin resistance, indicating integration in its chromosome (Fig. 2a). Loss of vector was obtained by selection for sucroseresistance yielding clones with NCgl2106 deleted. A PCR analysis with primers hybridizing outside of the sequences used for strain construction resulted in the expected fragment of 2159 bp for the wild type and of 1088 bp for the deletion mutant, which was termed C. glutamicumDpimB 0 .

Polar lipid analysis
Lyophilised cells were extracted using petroleum-ether and methanolic saline to recover apolar lipids. Further processing of the methanolic extract afforded the polar lipid fraction, which was examined by two-dimensional thin-layer chromatography (2D-TLC). The extract from wild type C. glutamicum showed the presence of ManGlcAGroAc 2 , GlcAGroAc 2 (Gl-A) and Ac 1 PIM 2 , by a-naphthol/sulfuric acid staining (Fig. 2b). Surprisingly, while the synthesis of ManGlcAGroAc 2 and GlcAGroAc 2 remained unaffected the component corresponding to Ac 1 PIM 2 identified by negative ion mode MALDI-MS at m/z 1398 [M-H]with the fatty acyl groups C 16 and C 18:1 ) was absent in C. glutamicumDpimB 0 with the appearance of a new product, which was sugar (Fig. 2b) and phosphate positive (data not shown) by specific staining. This predominant lipid spot was purified and corresponded to Ac 1 PIM 1 , which was confirmed by negative ion mode MALDI-MS analyses due to the characteristic ion at m/z 1236 (M-H) -(data not shown). Complementation of C. glutamicumDpimB 0 by pEKEx2-Cg-pimB 0 restored the wild type phenotype (Fig. 2b). Altogether, the data indicated that Ac 1 PIM 1 in C. glutamicumDpimB 0 occurs possibly as a result of inactivation of a phosphatidylmyo-inositol mannosyltransferase, presumably which transfers a mannopyranosyl (Manp) residue from GDP-Mannose to the 6-position of Ac 1 PIM 1 . In addition this data also shed further light on the acylation step in PIM biosynthesis in Corynebacterianeae. The accumulation of Ac 1 PIM 1 in C. glutamicumDpimB 0 showed that the acylation step (Kordulakova et al. 2003) precedes the second mannosylation step in PIM biosynthesis, and results in the formation of Ac 1 PIM 2 (Schaeffer et al. 1999).
Lipoglycans were extracted by refluxing delipidated cells in ethanol, followed by hot-phenol extraction, protease digestion and dialysis to remove impurities. The extracted lipoglycans were examined initially on 15% SDS-PAGE (Fig. 2c). Extracts from wild type C. glutamicum showed the presence of Cg-LAM, Cg-LM-A and Cg-LM-B ). As expected the lipoglycan extract from C. glutamicumDpimB 0 showed the absence of Cg-LAM and Cg-LM-A, and the presence of a single species (Cg-LM-B) by chemical characterization as described below. Complementation of C. glutamicumDpimB 0 by transformation with plasmid pEKEx2-Cg-pimB 0 restored the wild type phenotype of Cg-LAM, Cg-LM-A and Cg-LM-B (Fig. 2c). As predicated, the inactivation of Cg-PimB 0 abolished the synthesis of Cg-LAM and Cg-LM-A, and provided a strain that allowed the purification of Cg-LM-B for further chemical and functional characterisation. Cg-LM-B from C. glutamicumDpimB 0 and Cg-LM-A from C. glutamicumDpimB 0 ) were purified by hydrophobic interaction chromatography (HIC) and size exclusion chromatography.

Analysis of Cg-LM-B by mass spectrometry
The Cg-LM-B was permethylated prior to detailed mass spectrometric analysis. During the permethylation step the acyl groups from the lipoglycan are liberated and the glycerol hydroxyl groups are methylated. The MALDI-TOF MS profile of the permethylated sample in the positive mode showed no signals for ManGlcAGro (m/z 579) but, interestingly, a series of peaks (M + Na + ) corresponding to the addition of 4-22 hexose residues to the GlcAGro core structure. In the lower mass region of the spectrum, signals consistent with the elongation of the GlcAGro core were observed at m/z 1191.7, 1395.8 and 1600.0 corresponding to the compositions Hex 4 GlcAGro, Hex 5 GlcAGro and Hex 6 GlcAGro, respectively (data not shown). Peaks corresponding to Hex 7 GlcAGro and Hex 9 GlcAGro were absent from the spectrum. At higher mass, signals were observed corresponding to a series of Hex n GlcAGro units containing even numbers of hexoses at m/z 2008. 2, 2416.4, 2824.6, 3232.8, 3641.0, 4049.2, 4457.2 and 4865.6 with Hex 12 GlcAGro (m/z 2824.6) being most abundant (Fig. 3a). The low abundance of signals attributable to components carrying an odd number (Hex11,13,15,17,19 and 21) of hexoses (m/z 2620.5, 3028.7, 3436.9, 3849.0, 4252.2 and 4660.4), as compared to even numbered, suggests that the hexose polymer is branched rather than linear. In addition, there are also signals present in the MS spectrum attributable to hexose oligomers. This may result from partial degradation of the sample during preparation but could also be due to contamination. Each of the major signals in the spectrum was subjected to collision induced decomposition MS/MS (CID-MS/MS) analysis to establish their structures. For example, MS/MS spectra of the signal at m/z 2416, which has the composition Hex 10 GlcAGro, showed peaks at m/z 2198, 1585.6, 1381.5, 1177 which are due to the loss of 1, 2, 4, 5, 6, 8 and 10 hexose units from the molecular ion. The inset to Fig. 3b shows the likely sequence that is consistent with this set of fragment ions.
In order to confirm the nature of the hexose units and linkages, GC/MS analysis of partially methylated alditol acetates was carried out. This revealed the presence of terminal mannose, 2 linked mannose, 2,6 linked mannose (data not shown). The presence of t-Man and 2,6-Man convincingly establishes that the backbone of the oligomannans is heavily branched. The relative abundances of t-Man (1.0), 6-Man (0.5) and 2,6-Man (0.98) are consistent with single mannose residues being appended at O-2 to the 6-linked mannosyl backbone. TNF-a production by human macrophage cell line in response to Cg-LMs PI-anchored lipoglycans, and most particularly LMs, isolated from other actinomycete genera, including diamond-glucuronic acid mycobacteria, have previously been shown to induce cytokine production by phagocytic cells (Garton et al. 2002;Gibson et al. 2004;Quesniaux et al. 2004). We thus investigated the potency of both types of Cg-LMs to stimulate the release of TNF-a using a human macrophage cell line. As expected, the PI-anchored LM, Cg-LM-A, elicited a dose-dependent production of the cytokine (Fig. 4). However interestingly, the GlcAGroAc 2 -anchored LM, Cg-LM-B, was also found to be as stimulatory as the PI-anchored LM, suggesting that the phosphomyo-inositol unit of the anchor in LM does not play a key role in lipoglycan pro-inflammatory activity.

Discussion
SDS-PAGE analysis of purified lipoglycans from C. glutamicumDpimB 0 showed the presence of a single species, which migrated akin to Cg-LM. Chemical characterization of this novel species using MS-MS established it as an oligomer of hypermannosylated oligosaccharides linked to GlcAGroAc 2 . It was previously established that these glycosylated diacylglycerols function as precursors/anchors for hyperglycosylated variants, such as the lipomannans, as found in the case of dimannosyl diacylglycerols in Micrococcus and lipoteichoic acids (Pakkiri and Waechter 2005). Moreover, related di-and monoacylglycerols containing glucuronosyl residues have been well reported in Pseudomonas sp. (Wilkinson 1968) Bacillus cereus T (Minnikin et al. 1971), and halotolerant bacteria (Stern and Tietz 1971). The data in relation to Mycobacterium are sparse in regards to the presence of glycosyl diacylglycerols, except for a few limited cases, such as a diglucosyl diacylglycerol (Hunter et al. 1986). In Mycobacterium smegmatis, a minor glucuronosyl diacylglycerol glycolipid has been reported (Wolucka et al. 1993), and uronosylcontaining glycopeptidolipids in Mycobacterium avium (Chatterjee et al. 1987) and Mycobacterium habana (Khoo et al.1996). Our results also support our initial hypothesis that ManGlcAGroAc 2 also participates in the biosynthesis of a novel Cg-LMlike molecule and that the Cg-LM most likely consists of two components, a minor Cg-LM based on ManGlcAGroAc 2 (Cg-LM-B) and a more abundant component akin to the characteristic dominant mycobacterial PI-based LM (Cg-LM-A) . The presence of a Cg-LM-B type species may exist in Mycobacterium, based on the identification as mentioned earlier of a minor glucuronosyl diacylglycerol glycolipid (Wolucka et al. 1993), and further experiments are needed to address this question. It is also interesting to also note that Cg-LM-A is arabinosylated, yet Cg-LM-B, which possesses a similar mannan is not arabinosylated. A possibility for this structural difference maybe that Cg-LM-B is more heavily branched than Cg-LM-A thereby removing potential sites for arabinosylation leading to Cg-LAM.
LM-like molecules are powerful pro-inflammatory lipoglycans found in the cell-walls of mycobacteria and some related actinomycetes genera (Garton et al. 2002;Gibson et al. 2004;Quesniaux et al. 2004), including Corynebacterium ). They are composed of two parts, a PI anchor to which is attached an a-D-mannan domain, that both play a key role in their ability to induce cytokine production by phagocytic cells. The PI-acylation pattern is also critical for activity (Gilleron et al. 2006) and the latter is abrogated after deacylation of LM by alkaline treatment (Gibson et al. 2005). But the structure of the glycosidic moiety has also been shown to modulate the pro-inflammatory activity of Fig. 4 TNF-a production by human macrophage cell line in response to Cg-LMs. Cg-LM-A was isolated and purified as described previously ) and Cg-LM-B (this study) were tested at 5 (grey bars) and 15 (black bars) lg/ml the molecule. Indeed, the presence of an arabinan domain such as in the case of LAM-like molecules undermines lipoglycan activity by masking the mannan domain (Vignal et al. 2003;Gibson et al. 2004). In addition, LM activity seems also to be modulated by the presence or not of side chains on the mannan domain as well as the size of these chains (Gibson et al. 2005). We have shown that C. glutamicum contains two types of LM that share a similar mannan domain but differ by their lipidic anchor, one bearing a classical PI anchor and the other one based on a GlcAGroAc 2 anchor ). In the present study, aided by two different mutants, which are selectively affected in the biosynthesis pathway of each type of LM, we have managed to separate them. We found that both LMs induced a similar amount of cytokine secretion by a human macrophage cell line, indicating that the PI part of Cg-LM-A does not play a key role in lipoglycan proinflammatory activity. Since the completion of these studies and preparation of manuscript, Lea-Smith et al. (2008) have reported similar findings on the role of Cg-PimB 0 . However, their study did not analyze the structural basis of Cg-LM-B in detail and report on the pro-inflammatory properties of this lipoglycan in comparison to Cg-LM-A, which is now reported in this present study. Finally, a proposed hypothetical pathway that takes into account the potential role of MgtA and PimB 0 , the accumulation of Ac 1 PIM 1 and Cg-LM-B, and abrogation of ManGlcAGroAc 2 ) and Ac 1 PIM 2 is presented in Fig. 1.