Analysis of a New Mannosyltransferase Required for the Synthesis of Phosphatidylinositol Mannosides and Lipoarbinomannan Reveals Two Lipomannan Pools in Corynebacterineae
2008; Elsevier BV; Volume: 283; Issue: 11 Linguagem: Inglês
10.1074/jbc.m707139200
ISSN1083-351X
AutoresDavid J. Lea‐Smith, Kirstee L. Martin, James Pyke, Dedreia Tull, Malcolm J. McConville, Ross L. Coppel, Paul K. Crellin,
Tópico(s)Pneumocystis jirovecii pneumonia detection and treatment
ResumoThe cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum ΔNCgl2106 was restored by complementation with M. tuberculosis Rv2188c. In vivo labeling of the mutant with [3H]Man and in vitro labeling of membranes with GDP-[3H]Man confirmed that NCgl2106/Rv2188c catalyzed the second mannose addition in PIM biosynthesis, a function previously ascribed to PimB/Rv0557. The C. glutamicum ΔNCgl2106 mutant lacked mature LAM but unexpectedly still synthesized the major pool of LM. Biochemical analyses of the LM core indicated that this lipopolysaccharide was assembled on Gl-X. These data suggest that NCgl2106/Rv2188c and the previously studied PimB/Rv0557 transfer mannose residues to distinct mannoglycolipids that act as precursors for LAM and LM, respectively. The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum ΔNCgl2106 was restored by complementation with M. tuberculosis Rv2188c. In vivo labeling of the mutant with [3H]Man and in vitro labeling of membranes with GDP-[3H]Man confirmed that NCgl2106/Rv2188c catalyzed the second mannose addition in PIM biosynthesis, a function previously ascribed to PimB/Rv0557. The C. glutamicum ΔNCgl2106 mutant lacked mature LAM but unexpectedly still synthesized the major pool of LM. Biochemical analyses of the LM core indicated that this lipopolysaccharide was assembled on Gl-X. These data suggest that NCgl2106/Rv2188c and the previously studied PimB/Rv0557 transfer mannose residues to distinct mannoglycolipids that act as precursors for LAM and LM, respectively. The Corynebacterineae, a suborder of the Actinomycetales, includes bacterial pathogens of medical and veterinary importance, most notably Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium subspecies paratuberculosis, and Corynebacterium diphtheriae. The growing rise of drug-resistant strains of the devastating pathogen M. tuberculosis, the causative agent of human tuberculosis (TB), 7The abbreviations used are:TBtuberculosisGC-MSgas chromatographymass spectrometryMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightLMlipomannanLAMlipoarabinomannanPIMphosphatidylinositol mannosideHPTLChigh performance thin layer chromatographyPIphosphatidylinositolBHIbrain heart infusion media.7The abbreviations used are:TBtuberculosisGC-MSgas chromatographymass spectrometryMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightLMlipomannanLAMlipoarabinomannanPIMphosphatidylinositol mannosideHPTLChigh performance thin layer chromatographyPIphosphatidylinositolBHIbrain heart infusion media. represents a global health emergency (1Raviglione M.C. Tuberculosis (Edinb.). 2003; 83: 4-14Crossref PubMed Scopus (247) Google Scholar). Because existing anti-TB drugs target enzymes involved in the formation of the Corynebacterineae cell wall, the characterization of novel cell wall enzymes as potential drug targets is an area of intense interest. tuberculosis gas chromatographymass spectrometry matrix-assisted laser desorption/ionization time-of-flight lipomannan lipoarabinomannan phosphatidylinositol mannoside high performance thin layer chromatography phosphatidylinositol brain heart infusion media. tuberculosis gas chromatographymass spectrometry matrix-assisted laser desorption/ionization time-of-flight lipomannan lipoarabinomannan phosphatidylinositol mannoside high performance thin layer chromatography phosphatidylinositol brain heart infusion media. The cell walls of all Corynebacterineae comprise a giant macromolecule of covalently linked type 4 peptidoglycan, arabinogalactan, and long chain mycolic acids (2Brennan P.J. Tuberculosis (Edinb.). 2003; 83: 91-97Crossref PubMed Scopus (623) Google Scholar). A diverse range of glycolipids coat the surface of this structure and/or are linked to the underlying plasma membrane. The most abundant and highly conserved of these are the phosphatidylinositol mannosides (PIM) and their hyperglycosylated derivatives lipomannan (LM) and lipoarabinomannan (LAM) (2Brennan P.J. Tuberculosis (Edinb.). 2003; 83: 91-97Crossref PubMed Scopus (623) Google Scholar). In pathogenic mycobacteria, these glycolipids are important virulence factors by acting as ligands for host macrophage receptors, preventing maturation of macrophage phagosomes, inhibiting early apoptosis in infected macrophages, and modulating the adaptive immune response (reviewed in Ref. 3Koul A. Herget T. Klebl B. Ullrich A. Nat. Rev. Microbiol. 2004; 2: 189-202Crossref PubMed Scopus (305) Google Scholar). Considerable progress has been made in delineating steps and genes involved in PIM, LM, and LAM biosynthesis (reviewed in Ref. 4Berg S. Kaur D. Jackson M. Brennan P.J. Glycobiology. 2007; 17: 35-56Crossref PubMed Scopus (167) Google Scholar). Most Corynebacterineae (including Corynebacterium glutamicum and pathogenic mycobacteria) accumulate the PIM species AcPIM2 that is synthesized via the sequential transfer of two mannose residues and one fatty acyl chain to phosphatidylinositol. The first and second mannose additions are thought to be catalyzed by two separate mannosyltransferases, PimA and PimB (5Kordulakova J. Gilleron M. Mikusova K. Puzo G. Brennan P.J. Gicquel B. Jackson M. J. Biol. Chem. 2002; 277: 31335-31344Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6Schaeffer M.L. Khoo K.H. Besra G.S. Chatterjee D. Brennan P.J. Belisle J.T. Inamine J.M. J. Biol. Chem. 1999; 274: 31625-31631Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Both mannosyltransferases lack detectable signal sequences and utilize GDP-Man as the mannose donor, suggestive of localization on the cytoplasmic side of the plasma membrane. AcPIM2 is subsequently elongated with two α1–6-linked mannose residues to form AcPIM4, a likely branch point intermediate in polar PIM and LM/LAM biosynthesis (7Morita Y.S. Patterson J.H. Billman-Jacobe H. McConville M.J. Biochem. J. 2004; 378: 589-597Crossref PubMed Google Scholar, 8Kovacevic S. Anderson D. Morita Y.S. Patterson J. Haites R. McMillan B.N. Coppel R. McConville M.J. Billman-Jacobe H. J. Biol. Chem. 2006; 281: 9011-9017Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Polar PIMs and the mannan backbone of LM/LAM are formed by the addition of one to two α1–2-linked mannose residues or long (>20) chains of α1–6-mannose residues to AcPIM4, respectively (9Khoo K.H. Dell A. Morris H.R. Brennan P.J. Chatterjee D. 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Puhler A. Rey D.A. Ruckert C. Rupp O. Sahm H. Wendisch V.F. Wiegrabe I. Tauch A. J. Biotechnol. 2003; 104: 5-25Crossref PubMed Scopus (761) Google Scholar). Importantly, C. glutamicum mutants lacking cell wall components essential for mycobacterial survival are often viable. Using this approach we identified a new gene in C. glutamicum that is required for the conversion of AcPIM1 to AcPIM2. This enzyme, encoded by the ortholog of the M. tuberculosis gene Rv2188c, has been named PimB′. Remarkably, loss of function of PimB′ results in loss of synthesis of LAM but not the major pool of LM. Our data provide strong evidence that Corynebacteria contain two pathways for lipopolysaccharide biosynthesis. Bioinformatics Analyses—M. tuberculosis strain H37Rv sequences were obtained from the Tuberculist World-Wide Web Server at the Institut Pasteur. C. glutamicum ATCC 13032 Kitasato sequences, in addition to protein alignments, were obtained at The Institute for Genomic Research (TIGR) Comprehensive Microbial Resource web site. Amino acid similarity percentage values were assigned according to the results found in the protein versus all alignment menu located for each gene in the above C. glutamicum ATCC 13032 Kitasato web site. Sequences were compared utilizing ClustalW. Strains and Culture Conditions—E. coli DH5α was cultured in Luria-Bertani media at 37 °C. All C. glutamicum strains, including the wild-type strain ATCC 13032, were grown in brain heart infusion media (BHI) (Oxoid) at 30 °C. 15 g/liter of agar was used for preparation of solid media, and 10% sucrose (w/v) was added when necessary. C. glutamicum electrocompetent cells were prepared according to a protocol (27der Rest van M.E. Lange C. Molenaar D. Appl. Microbiol. Biotechnol. 1999; 52: 541-545Crossref PubMed Scopus (367) Google Scholar) except electroporation was performed at the following settings: 2.5 kV, 200 ohms, and 25 microfarads. Kanamycin (30 μg/ml) and ampicillin (100 μg/ml) were added to media when necessary. Construction of the C. glutamicum ΔNCgl2106 Mutant and Complementation Plasmids—Gene deletion was performed by amplifying a 2.41-kb fragment containing the entire NCgl2106 gene and flanking sequences. PCR was performed by standard procedures using Proofstart DNA polymerase (Qiagen) and the primers NCgl2106for, incorporating an XbaI site (5′-CCGTCTAGAACAACACGCAAATGACCAAA-3′) and NCgl2106rev, incorporating a HindIII site (5′-GCAAAGCTTCCACAAAACGTGATTGATCG-3′). The PCR product was inserted into the XbaI/HindIII sites of pUC18 (28Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11458) Google Scholar), sequenced, and a 680-bp fragment excised from NCgl2106 by NruI/SphI digestion. The remaining 1.73-kb fragment was then excised and inserted into the XbaI/HindIII sites of the suicide plasmid pK18mobsacB (29Schafer A. Tauch A. Jager W. Kalinowski J. Thierbach G. Puhler A. Gene (Amst.). 1994; 145: 69-73Crossref PubMed Scopus (2170) Google Scholar). Following electroporation, kanamycin-resistant transformants were tested for single homologous recombination events by Southern blot hybridization using a probe prepared from the digoxigenin hybridization kit (Roche Applied Science). A verified single crossover clone was cultured on BHI plates containing 10% sucrose to select for double homologous recombination events. Gene deletion was screened by PCR using the primers NCgl2106for and NCgl2106rev and then confirmed by Southern blot hybridization analysis. One confirmed deletion strain, ΔNCgl2106, was selected for further analysis. Complementation was performed by inserting the NCgl2106 gene into plasmid pSM22 containing the Corynebacterium origin of replication repA and the kanamycin resistance gene aphA3 (30McKean S. Davies J. Moore R. Microbes Infect. 2005; 7: 1352-1363Crossref PubMed Scopus (43) Google Scholar). The entire Rv2188c gene was cloned into pUC18, sequenced, and subcloned into pSM22. The complementation plasmid and empty plasmid control were then electroporated into C. glutamicum ΔNCgl2106 and transformants selected on kanamycin plates. Extraction of Cell Wall Glycolipids/Lipopolysaccharides—C. glutamicum strains were grown to exponential phase (A600 nm = 7) and cell wall lipids extracted in chloroform:methanol (2:1 v/v) and chloroform:methanol:water (1:2:0.8 v/v) (31Billman-Jacobe H. McConville M.J. Haites R.E. Kovacevic S. Coppel R.L. Mol. Microbiol. 1999; 33: 1244-1253Crossref PubMed Scopus (101) Google Scholar). After removal of insoluble material by centrifugation (15,000 × g, 10 min), extracts were dried under nitrogen and subjected to biphasic partitioning in 1-butanol and water (2:1 v/v). The organic phase was dried, and lipids were resuspended in 1-butanol prior to analysis by HPTLC. Glycolipids were analyzed by either one- or two-dimensional HPTLC as indicated in the text using aluminum-backed silica gel sheets (Merck) and stained with orcinol:H2SO4. One-dimensional HPTLCs were developed in chloroform, methanol, 13 m NH3, 1 m ammonium acetate, water (180:140:9:9:23 v/v), whereas two-dimensional TLCs were developed in the first dimension using chloroform: methanol:water (65:25:4 v/v) and chloroform:acetic acid:methanol:water (40:25:3:6 v/v) in the second dimension. Individual glycolipid species were extracted from silica scrapings from one-dimensional HPTLCs using chloroform:methanol:water (10:10:3 v/v), and the supernatant was dried under nitrogen and desalted by 1-butanol:water biphasic partitioning (2:1 v/v). The 1-butanol phase was dried, and purified lipids were resuspended in a minimal volume of 1-butanol. LM and LAM were extracted from delipidated cell pellets by reflux in 50% ethanol (8Kovacevic S. Anderson D. Morita Y.S. Patterson J. Haites R. McMillan B.N. Coppel R. McConville M.J. Billman-Jacobe H. J. Biol. Chem. 2006; 281: 9011-9017Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). LMs and LAM were further purified by octyl-Sepharose chromatography. Dried extracts were suspended in 0.1 m NH4OAc containing 5% 1-propanol and loaded onto an octyl-Sepharose column using the same buffer. Following elution of unbound material, LM and LAM were eluted with 30 and 40% 1-propanol. LMs and LAMs were analyzed by SDS-PAGE and periodic acid-Schiff silver staining using reagents from the GelCode SilverSNAP stain kit 2 (Pierce). LAM/LM fractions were exhaustively digested with jack bean α-mannosidase (Sigma) in 0.1 m sodium acetate buffer, pH 5.0, in the presence of 0.1% taurodeoxycholate. The released mannose residues were separated from the residual glycolipid anchor by 1-butanol:water partitioning (2:1 v/v), and the organic phase was dried and resuspended in a minimal volume of 1-butanol. The glycolipids were analyzed by one-dimensional HPLTC using chloroform:methanol:water (60:35:8 v/v) and visualized using orcinol:H2SO4. Analytical Procedures—The monosaccharide composition of the purified glycolipids was determined after solvolysis (0.5 m methanolic HCl, 100 °C 16 h) and conversion of the released sugar methyl esters to their trimethylsilyl derivatives with N-methyl-N-(trimethylsilyl) trifluoroacetamide containing 1% trichloromethylsilane (Pierce). The trimethylsilyl derivatives were analyzed by GC-MS (32McConville M.J. Thomas-Oates J.E. Ferguson M.A. Homans S.W. J. Biol. Chem. 1990; 265: 19611-19623Abstract Full Text PDF PubMed Google Scholar). LM and LAM fractions were permethylated as described previously (32McConville M.J. Thomas-Oates J.E. Ferguson M.A. Homans S.W. J. Biol. Chem. 1990; 265: 19611-19623Abstract Full Text PDF PubMed Google Scholar). For linkage analysis, permethylated LM/LAM were hydrolyzed in 2 m trifluoroacetic acid (100 °C, 2 h), and released permethylated sugars were reduced with NaB2H4 and acetylated prior to GC-MS analysis (32McConville M.J. Thomas-Oates J.E. Ferguson M.A. Homans S.W. J. Biol. Chem. 1990; 265: 19611-19623Abstract Full Text PDF PubMed Google Scholar). For analysis by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF), samples were loaded onto a metal plate pre-loaded with the matrix α-cyano-4-hydroxycinnamic acid (Sigma) in 60% 1-propanol. Samples were analyzed using a Voyager-DE STR mass spectrophotometer, with an accelerating voltage of 21,000 V, grid voltage of 69%, and guide wire voltage of 0.05%. Isoglobotrihexosylceramide (IGb3, Sapphire Bioscience) was used as the external standard. In Vivo Enzyme Assays—Mid-log C. glutamicum cells were collected by centrifugation (4,000 × g, 10 min), washed with glucose-free Middlebrook 7H9 media, and resuspended in the same media at 1 g wet weight cells per ml of media. The cells were pulse-labeled with 50 μCi/ml [2-3H]mannose (21 Ci/mmol; ICN) at 37 °C for 5 min with shaking (180 rpm). The cell suspension was then diluted 10-fold with BHI media to initiate chase labeling. Aliquots were removed at various time points and rapidly chilled by dilution with ice-cold phosphate-buffered saline to quench the labeling reaction, and the cells were collected by centrifugation (4000 × g, 10 min, 4 °C). Lipids were extracted and separated by one-dimensional HPTLC using the same methods described for characterization of cell wall glycolipids. Labeled lipids were detected by autoradiography after spraying the plate with En3Hance (PerkinElmer Life Sciences). In Vitro Enzyme Assays—Mid-log cells were collected by centrifugation (4000 × g, 10 min) and washed with 50 mm HEPES, pH 7.4. The cells were resuspended in lysis buffer (50 mm HEPES, pH 7.4, 5 mm MgCl2) and lysed by probe sonication (MSE Soniprep 150 instrument; 4 × 45 s at 15 μm, 4 °C). Cellular debris and unbroken cells were removed by centrifugation (4000 × g, 10 min, 4 °C), and the membranes were then collected by centrifugation of the supernatant (100,000 × g, 1 h, 4 °C). The pelleted membranes were resuspended in 50 mm HEPES, 5 mm MgCl2. To initiate the assay, 0.25 μCi of GDP-[2-3H]mannose was added and the reaction mixture incubated at 37 °C for 1 h. The reaction was terminated, and lipids were extracted through the addition of chloroform:methanol (1:1 v/v) to achieve a final concentration of chloroform:methanol: water of 10:10:3 (v/v). The supernatant was dried under nitrogen, and the lipids were desalted by 1-butanol:water partitioning (2:1 v/v) and separated by HPTLC, with detection by autoradiography as described above. Rv2188c Is a Putative Glycosyltransferase Highly Conserved within Corynebacterineae—As corynebacteria and mycobacteria contain structurally related PIMs, LM and LAM, we undertook a bioinformatics search for genes that were highly conserved in both groups of bacteria. All proteins predicted to be encoded by the C. glutamicum ATCC 13032 Kitasato genome were compared with those encoded by the M. tuberculosis H37Rv genome using the BLAST algorithm (33Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70308) Google Scholar). Conserved genes of unknown function that are essential in M. tuberculosis, as determined by the transposon mutagenesis studies of Sassetti et al. (34Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1998) Google Scholar), were considered candidates for cell wall biosynthesis genes of major structural components, because the majority of characterized cell wall genes are refractory to deletion in mycobacteria. Inspection of our list of 83 conserved genes led to our selection of the putative glycosyltransferase Rv2188c for genetic disruption in C. glutamicum. Rv2188c is a putative CAZy Family 4 glycosyltransferase containing an EXXGXXXXE motif, representing a potential GDP-mannose-binding site, as well as a conserved lysine residue characteristic of many members of this family (Fig. 1). The M. tuberculosis ortholog is predicted to consist of 385 amino acid residues and be 41 kDa in size. In addition, Rv2188c shares some sequence homology, and several potential motifs, with the mannosyltransferases PimA and MgtA. The orthologs of Rv2188c in M. leprae, M. avium subspecies paratuberculosis, C. glutamicum, and C. diphtheriae display 81, 81, 65, and 64% amino acid similarity, respectively, to the M. tuberculosis protein (Fig. 1). Inactivation of the C. glutamicum NCgl2106 Gene—Because Rv2188c is an essential gene in M. tuberculosis (34Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (1998) Google Scholar), we targeted its ortholog in C. glutamicum, NCgl2106, for disruption using a two-step recombination strategy. The suicide plasmid pK18mobsacBΔNCgl2106, containing the NCgl2106 sequence and flanking regions but devoid of 680 bp of the 1145-bp gene, was constructed. Following electroporation into C. glutamicum, kanamycin-resistant transformants were selected and examined for the genomic integration of the suicide plasmid via a homologous recombination event using Southern blot hybridizations (data not shown). One of these single crossover recombinants was chosen to generate a knock-out strain by selecting for a second recombination event on sucrose, which is toxic to bacteria carrying the sacB gene. After 3 days of growth on BHI:sucrose plates, hundreds of colonies were obtained. A large number were examined by PCR using the NCgl2106for and NCgl2106rev primers, and all were found to be wild-type revertants. After growth for a further 5 days, a second population of colonies was evident, indicating a growth defect. These small colonies were examined by PCR and displayed the expected knock-out profile (data not shown). Deletion of NCgl2106 in these strains was confirmed by Southern blot hybridization following digestion of genomic DNA with ClaI. Bands of 1.9 and 6.0 kb in the wild-type sample hybridized to the probe, whereas a single band of 7.2 kb was observed for the potential knock-outs, because a ClaI site was present in the excised region of NCgl2106 (Fig. 2, A and B). These results confirmed inactivation of NCgl2106. Complementation of the mutant by electroporation with the plasmids pSM22:NCgl2106 and pSM22: Rv2188c, but not with the empty pSM22 vector, restored the colony size to wild-type dimensions (data not shown). This slow growth phenotype was maintained in liquid BHI media and was also restored to wild-type levels by both complementation plasmids (Fig. 2C). Characterization of PIMs in C. glutamicum ΔNCgl2106 and Complementation Strains—Two-dimensional HPTLC analysis of the lipid fractions of wild-type C. glutamicum and the ΔNCgl2106 mutant strain revealed a marked change in the steady state levels of two glycolipid species (Fig. 3), provisionally identified as AcPIM1 and AcPIM2, based on their co-migration with authentic standards. Cellular levels of the putative AcPIM1 were markedly elevated in the ΔNCgl2106 mutant, whereas the predominant AcPIM2 was missing. A third glycolipid species, with a slightly faster HPTLC mobility than AcPIM2, was expressed at similar levels in both wild-type and mutant strain. The HPTLC mobility of this glycolipid was consistent with it being the recently characterized Gl-X glycolipid. The synthesis of the putative AcPIM2 species in ΔNCgl2106 was restored by complementation of the mutant with either NCgl2106 or Rv2188c, but not with empty pSM22 vector (Fig. 3). The identities of each of these glycolipid species were confirmed by monosaccharide analysis and MALDI-TOF of one-dimensional HPTLC-purified species (see Fig. 4A for an example of one-dimensional HPTLC). Monosaccharide analysis of the putative AcPIM1 bands from both wild type and the ΔNCgl2106 mutant revealed only mannose and myo-inositol in a 1:1 molar ratio (data not shown). Negative ion MALDI-TOF analysis of these fractions gave pseudomolecular ions at m/z 1239 consistent with the presence of an AcPIM1 species with C16:0 and C18:1 fatty acids (Fig. 4B). Comparable analysis of the glycolipid doublet (comprising putative AcPIM2 and Gl-X) from wild-type cells revealed the presence of mannose, glucuronic acid, and myo-inositol in the molar ratio 1:0.5:1. MALDI-TOF analysis in positive mode clearly showed that this fraction comprised two
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