Department of Medicine, Pulmonary and Critical Care Division, Atlanta VA Medical Center, Emory University School of Medicine, Room 12C 106, 1670 Clairmont Rd, Decatur, GA 30033, USA1
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA2
Author for correspondence: Carlos A. Rivera-Marrero. Tel: +1 404 321 6111 ext. 7178. Fax: +1 404 728 7750. e-mail: crivera{at}emory.edu
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ABSTRACT |
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Keywords: Mycobacterium, gene expression, glycolipid sulfotransferase, sulfolipid
Abbreviations: PAPS, 3'-phosphoadenosine-5'-phosphosulfonate; SL, sulfolipid; ST, sulfotransferase; sulfatide, 3'-sulfate galactosylceramide
The GenBank/EMBL/DDBJ accession number for the sequence (gene Rv1373) reported in this paper is Z81011.
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INTRODUCTION |
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Sulfated glycolipids of the cell wall of M. tuberculosis were implicated several years ago in virulence. Early studies showed that virulent strains could be distinguished cytochemically by their staining with the cationic dye neutral red (Dubois & Middlebrook, 1948 ), leading to the discovery of the neutral-red-reactive sulfolipids (SLs) (Middlebrook et al., 1959
). Since then, several studies have established a correlation between the presence of sulfated glycolipids and the degree of virulence of M. tuberculosis strains (Gangadharam et al., 1963
; Mitchison, 1964
; Goren et al., 1974
, 1982
). It has also been proposed that sulfated glycolipids may be involved in intracellular survival of virulent M. tuberculosis by their interaction with phagosomes and prevention of lysosomal fusion (Goren et al., 1976
; Goren, 1977
; DArcy Hart & Young, 1988
; Fujiwara, 1997
). More recently, it was shown that M. tuberculosis sulfolipid-I (SL-I) blocked the LPS and gamma interferon (INF-
) activation of human macrophages for enhanced release of superoxide (Pabst et al., 1988
; Brosna et al., 1991
). In neutrophils however, SL-1 stimulated superoxide production and primed neutrophil responses to several metabolic agonists such as N-formyl methionyl-leucyl-phenylalanine (FMLP) and phorbolmyristate acetate (PMA) (Zhang et al., 1988
, 1991
). Although these studies strongly implicate SLs as potential virulence factors, the role of SLs in virulence, immune modulation and pathogenicity of tuberculosis remains unclear.
The major sulfated glycolipids of M. tuberculosis consist of a family of trehalose-2-sulfate esters with an array of acyl fatty acids (phthioceranate, hydroxyphthioceranate, palmitate, stearate) at various positions of the trehalose molecule (Goren, 1984 ). SL-1, the principal sulfatide of M. tuberculosis, has been identified as 2,3,6,6'-tetraacyl-
,
'-D-trehalose-2'-sulfate (Goren, 1970a
, b
). Although the structure of M. tuberculosis trehalose SLs has been well characterized, their biosynthetic pathways, including enzymes such as sulfotransferases (STs) possibly involved in their biosynthesis and regulation, are not known. The identification of genes encoding these enzymes and the generation of allelic knockout mutants is crucial for determining their role in the biology and pathogenesis of tuberculosis.
In this study we demonstrate that gene Rv1373, a putative aryl-ST gene in M. tuberculosis, encodes a novel glycolipid ST with activity towards the eukaryotic glycolipids galactosylceramide and glucosylceramide, and endogenous mycobacterial glycolipids. This is the first report of a mycobacterial glycolipid ST possibly involved in the biosynthesis of the biologically relevant trehalose SLs of the cell wall of M. tuberculosis.
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METHODS |
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Metabolic labelling and glycolipid isolation.
Mycobacterium species M. avium smooth opaque and transparent, M. bovis BCG, M. smegmatis mc2155, and M. tuberculosis strains H37Ra, H37Rv and Erdman were grown in 50 ml cultures of 7H9 broth containing 100 µCi 35S (as sulfate; 3·7 MBq; 37 TBq mmol-1; Amersham Pharmacia) for 57 days. Cultures were centrifuged at 3000 g for 10 min, the bacterial pellets were washed in PBS, resuspended in 1·0 ml PBS and sonicated extensively (10 times, 20 s pulses). Glycolipids were extracted by a modification of the Folch method (Morrison, 1994 ). Two volumes of chloroform/methanol (2:1) were added, the samples were sonicated (20 s), centrifuged (9500 g for 1 min), and the upper (aqueous) and lower (organic) phases saved. The upper phase contains gangliosides and neutral lipids with long carbohydrate chains and contaminating protein; the lower phase contains the neutral glycolipids with short carbohydrate chains, neutral lipids and phospholipids. The lower organic phase, containing insoluble material, was extracted sequentially in chloroform/methanol/water (4:8:3), chloroform/methanol (1:1), chloroform/water (2:1) and 100% ethanol. After extraction, the pellet of insoluble material was discarded and the extracted aqueous and organic phases were combined, dried in vacuo and resuspended in 50100 µl chloroform/methanol (2:1). Glycolipid samples (approx. 10000 c.p.m.) were separated by TLC on Silica gel 60 plates (EM Science) with chloroform/methanol/water (65:25:4), allowed to dry and exposed to X-ray film or a scanning phosphorimager. TLC plates were also stained with orcinol ferric chloride spray (Sigma) and heated at 100 °C until the brownish colour bands of glycolipids were revealed.
Gene cloning.
The 0·98 kb putative aryl-ST gene homologue Rv1373, in M. tuberculosis cosmid SCY02B12 (accession no. z81011) (Philipp et al., 1996 ; Cole et al., 1998
), was cloned by PCR amplification of M. tuberculosis genomic DNA. A set of forward (5'-CCCCGAATTCGATGAATTCAGAACACCCGAT-3') and reverse (5'-CCCCAAGCTTTCAGTTGGCCGGGTCGTATC-3') oligonucleotide primers, with EcoRI and HindIII restriction sites (underlined) incorporated at the 5' ends, were utilized for PCR amplification. PCR amplification was done in 50 µl reaction mixtures under the action of Taq and Pwo DNA polymerases and the Expand Long Template PCR System (Roche). Amplification conditions were as follows: 1 cycle of 94 °C for 2 min; 5 cycles of 94 °C for 45 s, 65 °C for 45 s, 68 °C for 5 min; 35 cycles of 94 °C for 45 s, 55 °C for 45 s, 68 °C for 5 min; and a final extension of 68 °C for 7 min. The reaction product was separated by low-melting-point agarose electrophoresis and visualized by ethidium bromide staining. The discrete 1012 bp band was excised from the gel and isolated by spin filtration. After digestion with EcoRI and HindIII, the gene was subcloned into the EcoRI/HindIII site of expression vectors pET-23b (Novagen) and pTRcHisC (Invitrogen). The resulting construct STpET contains the full length M. tuberculosis aryl-ST gene with a 6-histidine fusion at the carboxyl terminus, while the STpTRcHis contains a 6-histidine peptide and an enterokinase cleavage sequence at the amino terminal end of the protein. The M. tuberculosis aryl-ST gene was also subcloned out-of-frame (construct OF-STpTRcHis) to generate an inactive his-tagged protein for control experiments. Sequence analysis of the M. tuberculosis aryl-ST gene plasmid constructs was done in a 373 ABI Sequencer by the Taq dye-deoxy terminator method using the T7 promoter and T7 terminator primers and gene-specific primers based on published genomic DNA sequence. Sequence data were analysed using GenBank and the NCBI BLAST server (Altschul et al., 1990
).
Expression and purification of recombinant gene Rv1373.
Expression of M. tuberculosis gene Rv1373 as a recombinant fusion protein in Escherichia coli was conducted following Novagen protocols. Plasmid constructs STpET, STpTRcHis and OF-STpTRcHis were introduced into the BL21(DE3) host E. coli strain and T7 RNA polymerase gene-specific transcription induced by addition of 0·8 mM IPTG for 3 h at 37 °C. After induction, cell extracts were prepared by lysis under non-denaturing conditions and separated by metal chelation affinity chromatography using His-Trap resin (Amersham Pharmacia). Histidine-bound proteins were eluted stepwise with 60 mM, 300 mM and 500 mM imidazole buffer (all in 20 mM phosphate, 0·5 M NaCl), pH 7·5. The eluted proteins were dialysed against PBS pH 7·2, concentrated in a Centriprep-10 filter (Amicon), and analysed by SDS-PAGE and Coomassie blue staining. Purified recombinant fusion proteins were stored at -70 °C until tested for ST activity.
ST assays.
These were conducted with sonicated cell extracts of M. tuberculosis bacilli and with the purified recombinant M. tuberculosis aryl-ST. Assays with M. tuberculosis extracts consisted of 100 µg protein (containing enzyme and acceptor substrate), 1·0 µCi (37 kBq) donor substrate 3'-phosphoadenosine-5'-[35S]phosphosulfonate, PAP35S, (NEN Life Science Products; 96·2 GBq mmol-1), in 100 µl reaction buffer (40 mM MES/NaOH pH 7·0, 0·05 M NaCl, 0·2% Triton X-100, 5 mM EDTA). Assays with purified recombinant protein from clones STpET (his-tag at C terminus), STpTRcHis (his-tag at N terminus) and OF-STpTRcHis (out-of-frame) contained 100 µg protein and 100 µg acceptor substrates galactosylceramide, glucosylceramide or sulfatide (Sigma). Assays with M. tuberculosis glycolipids as substrate contained 50100 µg total glycolipid. Reaction mixtures were incubated for 6 h at 37 °C, then 900 µl water was added to stop the reactions and the samples were separated by reverse phase chromatography in a Sep-Pak C:18 column pre-equilibrated in water. The column was washed with 15x1 ml aliquots of water to remove hydrophilic molecules and eluted with 10x1 ml aliquots of methanol to elute the hydrophobic lipids. The radiolabelled SL products eluted were determined by scintillation counting, and by TLC and autoradiography on Silica gel 60 plates (EM Science) with chloroform/methanol/water (65:25:4). ST assays were done in duplicate and controls without enzyme or acceptor substrate were included.
Ceramide glycanase digestion.
The 35S-labelled glycolipid fractions (approx. 1000020000 c.p.m.) obtained by elution of C:18 columns in methanol were dried in Eppendorf tubes, and resuspended in 35 µl water, 10 µl 250 mM phosphate buffer pH 5·0 and 1 µl (0·24 mU) ceramide glycanase (Sigma). Enzyme digestions were done for 5 h or overnight at 37 °C. After digestion, reaction mixtures were diluted in 1 ml water and separated by reverse phase chromatography. The 35S-labelled digestion products were determined by liquid scintillation counting, and by TLC and autoradiography as described.
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RESULTS |
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To confirm that the sulfate residue was added to the saccharide moiety of the glycolipid acceptors, the 35S-labelled glycolipids (1000020000 c.p.m.) were treated with ceramide glycanase and the digestion products separated by reverse-phase chromatography. Treatment of 35S-galactosylceramide products (GalCer Type I and II) with ceramide glycanase resulted in the release of 35S-labelled material ( 2030% of total radioactivity) that eluted with the aqueous phase (unbound) instead of the lipid phase (bound) (Fig. 6a
). Analysis of the aqueous (unbound) phase and the lipid (bound) phases by TLC and autoradiography revealed that the ceramidase-digested aqueous material migrated close to the origin as free 35S-galactose, while the lipid phase consisted of undigested 35S-GalCer Type I and II products (Fig. 6b
). A second digestion of the ceramidase-resistant material resulted in the release of more 35S-galactose (2030%) in the aqueous phase, suggesting that the ceramide glycanase utilized has relatively low activity toward monosaccharide glycosphingolipids such as galactosyl- and glycosylceramide. Nevertheless, these results demonstrate that the activity of the M. tuberculosis ST is towards the sugar moiety (galactose or glucose) of typical eukaryotic glycolipid substrates.
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DISCUSSION |
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Since the structure of M. tuberculosis trehalose SLs is well characterized, but most of their biosynthetic enzymes are not known, we tested for the presence of SL STs in cell free lysates of M. tuberculosis. ST assays utilizing PAP35S as the donor substrate and cell lysates of H37Ra, containing enzyme and acceptor lipid substrates, showed ST activity towards four endogenous lipids. One of the sulfated products is the abundant and relatively non-polar SL-I present in virulent strains but absent in H37Ra after metabolic labelling. This suggests that sulfation of this particular glycolipid may be regulated in vivo at either the enzyme or the substrate level. We also found that assays with cell lysates of H37Rv and Erdman typically showed lower activity than those with H37Ra. We attributed these differences to the availability of unsulfated glycolipid substrates in the cell lysates of H37Ra in comparison to H37Rv and Erdman. Nevertheless, the fact that soluble ST activity was identified in crude cell lysates prompted us to search for ST gene sequences in M. tuberculosis.
The M. tuberculosis gene Rv1373 showed slight similarity (24%) to eukaryotic aryl-STs but it contains the highly conserved signature sequences regions I and IV, which are involved in PAPS binding and transfer of sulfate (Weinshilboum et al., 1997 ). Gene Rv1373 showed no homology to a Klebsiella aryl-ST (Baek et al., 1996
) and is the only sequence in the M. tuberculosis genome with similarity to eukaryotic aryl-STs (Cole et al., 1998
). Other putative ST genes in M. tuberculosis, such as genes Rv2392 (cysH), Rv31173118 (cysA3sseC2), and Rv0815c0814c (cysA2sseC), show similarity to prokaryotic cysteine thio-STs (Cole et al., 1998
) and are possibly involved in the assimilatory pathway of cysteine biosynthesis (unpublished data). Interestingly, a BLAST analysis of Rv1373 against the M. bovis virulent strain AF2122/97 sequence database from the Sanger Centre (www.sanger.ac.uk) showed a gene homologue in Contig 281 with 99% identity. However, this sequence shows nucleotide changes such as an extra C at position 455 and a C to T change (Pro to Leu) at position 692. The extra nucleotide change would result in a frameshift at amino acid 155, a premature termination after residue 264 and truncation of 31 aa at the carboxyl terminus of the protein. This truncation would possibly result in inactivation of the enzyme since this region contains the highly conserved signature sequence region IV. Since we showed that BCG is almost devoid of sulfated glycolipids, future studies are designed to determine if the lack of SLs is due to a mutated Rv1373 gene homologue.
Gene Rv1373 was cloned by PCR, expressed in E. coli as a recombinant histidine-fusion protein and isolated by affinity chromatography. The purified fusion protein was first tested for PAP35S ST activity towards known substrates of eukaryotic glycolipid STs. The eukaryotic ceramide glycolipids were used since they are simple glycolipids that are commercially available in sulfated or desulfated form. We demonstrated that the recombinant M. tuberculosis aryl-ST protein was able to transfer sulfate to glucosylceramide and galactosylceramide (Type I and II) but not sulfatide (3'-sulfate GalCer). The inability to sulfate sulfatide suggests that sulfation of galactosylceramide may occur at carbon 3 of the galactose. In addition, treatment of sulfated products with ceramide glycanase showed that sulfation was associated with the sugar moiety (galactose or glucose) of the glycolipid. To further determine the function of the recombinant M. tuberculosis aryl-ST, assays were performed with total glycolipids extracted from H37Ra bacilli. The M. tuberculosis aryl-ST was capable of efficiently transferring sulfate to several glycolipids in a crude mixture. Comparison of the mobility of sulfated products in the TLC to that of typical SLs in H37Rv, obtained after metabolic labelling with 35S, showed that the main product corresponds to SL-IV, while the minor bands correspond to SLs III, V and VI. Interestingly, the most abundant SL (SL-I) was not produced. We showed that SL-I is not found in H37Ra after metabolic labelling (Fig. 1), but can be produced in PAPS assays with cell free lysates of H37Ra (Fig. 2
). This suggests that synthesis of SL-I requires other STs or factors only present in the cell lysates. Also, it is possible that the acceptor substrate required for SL-I synthesis is present in limited quantities in H37Ra in comparison to the virulent strains H37Rv and Erdman. Taken together, these studies show that gene Rv1373 encodes a novel glycolipid ST in M. tuberculosis with broad specificity and that could be involved in the biosynthesis of trehalose SLs in M. tuberculosis.
In mammals, cytosolic aryl-STs, such as the hydroxysteroid, phenol, monoamine and oestrogen-sulfating types, function in the detoxification of xenobiotic molecules and the metabolism of steroids and bile acids (Falany, 1997 ). The other class of STs are membrane-bound Golgi STs that are responsible for sulfation of glycosaminoglycans (GAGs), glycoproteins and glycolipids (Falany, 1997
). The M. tuberculosis ST is a novel enzyme since it is cytosolic, shows similarity to eukaryotic cytosolic aryl-STs but not to glycolipid STs and is active towards cerebroside glycolipids. There are few examples of prokaryotic glycolipid STs. The soil proteobacterium Rhizobium sp. secretes specific lipo-chitooligosaccharide signals called Nod factors that are required for infection and nodulation of legumes (Varin et al., 1997
). Sulfation of these glycolipids is catalysed by STs encoded by the nodH (Bourdineaud et al., 1995
; Ehrhardt et al., 1995
; Schultze et al., 1995
) and noeE (Hanin et al., 1997
) genes, which are also required for host specificity and biological activity. The NodH ST has specificity toward terminal N-acetylglucosamine residues of lipochitooligosaccharides (Roche et al., 1991
), while the NodE ST is fucose specific (Hanin et al., 1997
; Quesada-Vincens et al., 1998
). In M. tuberculosis, sulfation of cell wall glycolipids has been implicated in evasion of bacilli from the phagolysosomal compartment of the macrophage (Goren, 1977
; Goren et al., 1982
, 1987
). However, because of the lack of knowledge about the genes and biosynthetic pathways involved, their function in virulence and pathogenicity has not been defined. Recently, a gene locus encoding polyketide synthase genes (pks2) was shown to encode the synthase for hepta- and octamethyl-branched fatty acids in SLs. A gene knockout of pks2 resulted in mutants of H37Rv lacking SLs (Sirakova et al., 2001
). This finding, and the present work showing the first glycolipid ST in M. tuberculosis are important steps towards defining the biological role of SLs. Other aspects of their biosynthesis such as the synthesis of trehalose, the synthesis of sulfate donor substrates (PAPS), the transfer of fatty acids to trehalose, and their translocation and assembly into the cell wall, are equally important. Further studies are under way to fully characterize the M. tuberculosis glycolipid ST, determine the molecular structure of its endogenous substrates, generate gene knockout mutants, and define its role in the biology of M. tuberculosis.
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ACKNOWLEDGEMENTS |
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Received 18 September 2001;
accepted 1 November 2001.