1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India
2 TB Center, The Public Health Research Institute at the International Center for Public Health, 225 Warren Street, Newark, NJ 07103-3535, USA
3 Department of Organic Chemistry, Indian Institute of Science, Bangalore-560012, India
Correspondence
Dipankar Chatterji
dipankar{at}mbu.iisc.ernet.in
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ABSTRACT |
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INTRODUCTION |
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GPLs are among the major antigens that are non-covalently attached to the cell surface. They contain a tetrapeptide-amino alcohol core (D-phe-D-allothr-D-ala-L-alaninol), linked to a 3-hydroxy or 3-methoxy, C26C34 fatty acyl chain at the N-terminal of D-Phe through an amide bond (Brennan & Goren, 1979). The best known serovar-nonspecific GPLs (nsGPLs), which are found in all species of the M. avium complex, consist of a 2,3,4-tri-O-methylrhamnose or a 3,4-di-O-methylrhamnose, linked to the terminal L-alaninol, along with a glycosylated 6-deoxytalose unit at the D-allothreonine (Brennan & Goren, 1979
; Belisle & Brennan, 1989
). However, these nsGPLs are further glycosylated at the 6-deoxytalose with an oligosaccharide appendage, to produce the serovar-specific polar GPLs (ssGPLs). The ssGPLs are sometimes associated with smooth colony morphologies in some M. avium strains (Barrow et al., 1980
). M. smegmatis produce only a simple range of nsGPLs, which are of four major types. The species vary in the degree of methylation of the fatty acyl chain and of the rhamnose (Billman-Jacobe et al., 1999
). As M. smegmatis does not possess ssGPLs, it has been exploited as a natural mutant and used for the identification of genes encoding specific glycosyltransferases from M. avium required for the synthesis of haptenic disaccharides linked to 6-deoxytalose (Belisle et al., 1991
). It was also shown that the simpler GPLs found in M. smegmatis could serve as intermediates in the biosynthesis of recombinant ssGPLs in M. smegmatis (Eckstein et al., 1998
). This led to the characterization of the ser2 gene cluster, encoding enzymes like rhamnosyltransferase (encoded by ser2A), required for biosynthesis of GPLs in M. avium (Belisle et al., 1991
; Eckstein et al., 1998
; Maslow et al., 2003
). Subsequently, the GPL-biosynthesis gene cluster of M. smegmatis was found to encode a peptidylsynthetase, four methyltransferases and three putative glycosyltransferases among other proteins (Billman-Jacobe et al., 1999
; Patterson et al., 2000
; Jeevarajah et al., 2002
, 2004
).
We have previously reported on the conditional synthesis of a novel polar GPL in carbon-starved cultures of M. smegmatis, and have speculated that the normal apolar GPL species are hyperglycosylated (Ojha et al., 2002). In this paper, we describe the complete structure of a novel class of GPLs. We also propose that one of the glycosyltransferases in the GPL locus is regulated in response to environmental signals.
This work describes an approach using MALDI-TOF-MS and an HPLC ESI-MS technique, along with 13C-NMR, to obtain the primary sequence and glycosidic composition of this polar GPL.
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METHODS |
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Chemical reagents.
All chemicals, including L-rhamnose monohydrate and D-talose, were obtained at the highest grade from Aldrich unless otherwise specified. Milli-Q (Millipore) water was used for all chemical reactions.
Purification and analysis of GPLs.
The GPLs from M. smegmatis mc2155, its recombinant clones and mutants were purified as mentioned previously (Khoo et al., 1999). Briefly, cells were harvested at exponential and stationary phases of growth, and separated from the culture medium by centrifuging at 4000 r.p.m. for 15 min. Lipids were extracted with CHCl3/CH3OH (2 : 1, v/v) at room temperature for 24 h. The organic supernatant was dried and dissolved in CHCl3/CH3OH (2 : 1, v/v), and deacylated by treating with an equal volume of 0·2 M NaOH in CH3OH at 37 °C for 30 min, then neutralized with a few drops of glacial acetic acid. After drying to remove the solvents, lipids were dissolved in CHCl3/CH3OH/H2O (4 : 2: 1, by vol.) and centrifuged. The aqueous layer was discarded, and the organic layer containing the lipids was washed with supersaturated brine and concentrated. The deacylated lipids were spotted onto silica-coated TLC plates (Merck) and developed with CHCl3/CH3OH (9 : 1, v/v). The sugar-containing lipids were visualized by spraying the plates with 10 % H2SO4 in ethanol, followed by charring the separated spots at 120 °C for 10 min.
Spectroscopy.
13C-NMR spectra were obtained in CD3OD at 300 K on a Bruker AMX-400 instrument using the SEFT (spin echo Fourier transform) pulse programme (Brown et al., 1981). Tetramethylsilane (
ppm=0) was used as an internal calibrant.
Mass spectrometry.
Each GPL species was eluted from preparative TLC silica plates (20x20 cm) by dissolving the GPLs in CHCl3/CH3OH (2 : 1, v/v). Samples were mixed with an equal volume of matrix solution (dihydroxybenzoic acid in ethanol), and then allowed to crystallize at room temperature. MALDI-TOF-MS and MS-MS spectra were acquired on a Bruker Daltonics ULTRAFLEX TOF-TOF instrument equipped with a pulsed N2 laser, and analysed in the reflectron mode using a 90 ns time delay, and a 25 kV accelerating voltage in the positive ion mode. To improve the signal-to-noise ratio a mean of 500 shots were taken for each spectrum. External calibration was done to a spectrum acquired for a mixture of peptides with masses ranging from 10462465 Da. MS-MS spectra were acquired by selecting the precursor mass with a 10 Da window, and fragments were generated by collision-induced dissociation using He. In this case 1000 laser shots were acquired and averaged to generate the MS-MS spectra (Mechref et al., 2003).
Analytical procedures.
The N-acyl-phenylalanyl methyl ester was produced from the pure native GPL by strong-acid methanolysis with anhydrous 1·5 M CH3OH/HCl for 16 h at 80 °C, as described previously (Villeneuve et al., 2003). Then this N-acyl-phenylalanyl methyl ester was permethylated (Ojha et al., 2002
). Briefly, 0·5 ml anhydrous DMSO was mixed with one pellet of NaOH and the slurry was added to a vial containing the N-acyl-phenylalanyl methyl ester. To this mixture 0·5 ml CH3I was added and stirred at room temperature for 10 min. This reaction was quenched by the slow addition of 1 ml water. The permethylated product was extracted by adding 2 ml CHCl3 and washing the CHCl3 layer three times with water. The organic phase was dried and concentrated. After every step, the mass of the compounds was checked by MALDI-TOF-MS.
Carbohydrate composition analysis.
For monosaccharide composition analysis, pure GPL species were hydrolysed in 2 M trifluoroacetic acid for 2 h at 100 °C as described previously (Billman-Jacobe et al., 1999), and the acid was removed at 40 °C under a high vacuum. 2-Propanol (0·5 ml) was added to the dried tube and evaporated. For HPLC-MS analysis of the released monosaccharides, the trifluoroacetic acid hydrolysate as well as the standard monosaccharides were passed through a Phenomenex LUNA/NH2 column, with 2 % water in acetonitrile as a mobile phase at a flow rate of 0·3 ml min1 at 20 °C, in a Waters HPLC instrument equipped with a refractive index detector. The eluted fractions were analysed by ESI-MS and ESI MS-MS, at a 270 °C nebulizer temperature with N2 as the nebulizer and dry gas in a Bruker Daltonics ESQUIRE 3000 mass spectrometer equipped with an ion trap mass detector.
Preparation of partially O-methylated monosaccharides.
For the synthesis of partially methylated rhamnose, L-rhamnose monohydrate was taken as a starting material; mono-O-benzylated rhamnose was produced by the reaction scheme as depicted in Fig. 1(a). This mono-O-benzylated rhamnose was then methylated with one, two and three equivalents of CH3I to produce mono-O-methylated, di-O-methylated and tri-O-methylated rhamnose. In addition, methylation was carried out at the C-4 position after protecting the C-2 and the C-3 positions as benzylidine acetal. After the desired synthetic manipulations, the protected, methylated monosaccharides were deprotected using H2/Pd under pressure, and purified to homogeneity in a silica column by adsorption chromatography. A mixture of mono- and di-O-methylrhamnose obtained upon methylation with CH3I (Fig. 1a
) was also separated by silica column. 6-Deoxytalose was prepared using D-talose as the starting material, following the reaction scheme shown in Fig. 1(b)
. O-Benzylated talose was tosylated at the C-6 position, and then reduced with NaBH4 to generate 6-deoxytalose. This was deprotected under H2/Pd to obtain 6-deoxytalose. In each case the purity of the product was checked by TLC and by elemental analysis (Thermo Finnigan; FlashEA 1112 CHNS).
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Creation of an M. smegmatis sigB mutant.
A 2·2 kb fragment of the M. smegmatis chromosome containing sigB was cloned into plasmid pAlter, resulting in plasmid pSM140. Then a kanamycin-resistance cassette was cloned into the BamHI site of sigB, in pSM140, inactivating this gene, to create plasmid pSM187. This plasmid was linearized and was used to transform M. smegmatis mc2155, with selection for kanamycin resistance. Disruption of sigB in one of the transformants was verified by Southern hybridization (data not shown), and this strain, SM140, was used in the studies described here.
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RESULTS AND DISCUSSION |
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Cell surface composition and colony morphology in the sigB mutant and SigB-overexpressing M. smegmatis
The presence of an extra rhamnose unit, as discussed in the previous section, strongly suggests that the gene responsible for rhamnose transfer is upregulated during exponential growth in limiting carbon, or during stationary phase in high-carbon-containing cultures of M. smegmatis as these conditions show the accumulation of this polar GPL (Fig. 2b). We were interested to find out whether such regulation is dependent on SigB, thought to be a sigma factor with an important role in the stationary phase (Gomez & Smith, unpublished data). For this purpose, an M. smegmatis sigB mutant was constructed as described in Methods, and a strain that overexpressed SigB was also constructed. For these experiments we used the M. tuberculosis SigB, which has 93 % identity to M. smegmatis SigB. Interestingly, overexpression of SigB in mc2155 cells, i.e. strain PMVSigB, caused a prolonged generation time concomitant with a marked change in the colony morphology (Fig. 7
). We observed small, round and smooth colonies on 7H9 plates, in contrast to the normal wild-type colonies, which are large and have non-uniform margins with rugose morphology. However, the sigB-deleted mc2155 cells showed the usual wild-type phenotype. The GPL composition of strain PMVSigB was found to be altered. The PMVSigB clones produced the hyperglycosylated polar species of GPLs of masses 1334 and 1320 for spot 1 and spot 0 (which is 14 Da smaller than spot 1) respectively (Fig. 8
), at the exponential phase of growth. This is the same polar GPL whose structure has been established here. The sigB-deleted M. smegmatis showed the absence of any hyperglycosylated or undermethylated species at its exponential phase of growth. It was difficult to grow this strain in 0·02 % glucose medium, and at very late stationary phase in 2 % glucose only a faint band at the spot 1 position appeared. We believe this observation will lead to further understanding of the network of regulation of gene expression in mycobacteria during nutrient starvation.
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ACKNOWLEDGEMENTS |
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Received 21 January 2005;
revised 29 March 2005;
accepted 29 March 2005.
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