A Lipid Profile Typifies the Beijing Strains of Mycobacterium tuberculosis
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.041939
ISSN1083-351X
AutoresGaëlle Huet, Patricia Constant, Wladimir Malaga, Marie‐Antoinette Lanéelle, Kristin Kremer, Dick van Soolingen, Mamadou Daffé, Christophe Guilhot,
Tópico(s)Pneumocystis jirovecii pneumonia detection and treatment
ResumoThe Mycobacterium tuberculosis Beijing strains are a family highly prevalent in Asia and have recently spread worldwide, causing a number of epidemics, suggesting that they express virulence factors not found in other M. tuberculosis strains. Accordingly, we looked for putative characteristic compounds by comparing the lipid profiles of several Beijing and non-Beijing strains. All the Beijing strains analyzed were found to synthesize structural variants of two well known characteristic lipids of the tubercle bacillus, namely phthiocerol dimycocerosates (DIM) and eventually phenolglycolipids (PGL). These variants were not found in non-Beijing M. tuberculosis isolates. Structural elucidation of these variants showed that they consist of phthiotriol and glycosylated phenolphthiotriol dimycocerosates, eventually acylated with 1 mol of palmitic acid, in addition to the conventional acylation of the β-diol by mycocerosic acids. We demonstrated that this unusual lipid profile resulted from a single point mutation in the Rv2952 gene, which encodes the S-adenosylmethionine-dependent methyltransferase participating to the O-methylation of the third hydroxyl of the phthiotriol and phenolphthiotriol in the biosynthetic pathway of DIM and PGL. Consistently, the mutated enzyme exhibited in vitro a much lower O-methyltransferase activity than did the wild-type Rv2952. We finally demonstrated that the structural variants of DIM and PGL fulfill the same function in the cell envelope and virulence than their conventional counterparts. The Mycobacterium tuberculosis Beijing strains are a family highly prevalent in Asia and have recently spread worldwide, causing a number of epidemics, suggesting that they express virulence factors not found in other M. tuberculosis strains. Accordingly, we looked for putative characteristic compounds by comparing the lipid profiles of several Beijing and non-Beijing strains. All the Beijing strains analyzed were found to synthesize structural variants of two well known characteristic lipids of the tubercle bacillus, namely phthiocerol dimycocerosates (DIM) and eventually phenolglycolipids (PGL). These variants were not found in non-Beijing M. tuberculosis isolates. Structural elucidation of these variants showed that they consist of phthiotriol and glycosylated phenolphthiotriol dimycocerosates, eventually acylated with 1 mol of palmitic acid, in addition to the conventional acylation of the β-diol by mycocerosic acids. We demonstrated that this unusual lipid profile resulted from a single point mutation in the Rv2952 gene, which encodes the S-adenosylmethionine-dependent methyltransferase participating to the O-methylation of the third hydroxyl of the phthiotriol and phenolphthiotriol in the biosynthetic pathway of DIM and PGL. Consistently, the mutated enzyme exhibited in vitro a much lower O-methyltransferase activity than did the wild-type Rv2952. We finally demonstrated that the structural variants of DIM and PGL fulfill the same function in the cell envelope and virulence than their conventional counterparts. The factors contributing to the development of tuberculosis after infection with the etiologic agent of this disease, Mycobacterium tuberculosis, are not completely understood. It is currently thought that the genetic traits of both host and pathogen influence the outcome of the disease (1Caws M. Thwaites G. Dunstan S. Hawn T.R. Lan N.T. Thuong N.T. Stepniewska K. Huyen M.N. Bang N.D. Loc T.H. Gagneux S. van Soolingen D. Kremer K. van der Sande M. Small P. Anh P.T. Chinh N.T. Quy H.T. Duyen N.T. Tho D.Q. Hieu N.T. Torok E. Hien T.T. Dung N.H. Nhu N.T. Duy P.M. van Vinh Chau N. Farrar J. PloS Pathog. 2008; 4: e1000034Crossref PubMed Scopus (372) Google Scholar, 2Malik A.N. Godfrey-Faussett P. Lancet Infect. Dis. 2005; 5: 174-183Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). On the pathogen side, molecular epidemiological studies have differentiated clinical isolates of M. tuberculosis into defined genetically related lineages (3Gagneux S. Small P.M. Lancet Infect. Dis. 2007; 7: 328-337Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). One of the most important worldwide lineages, the "Beijing" lineage, was initially described by van Soolingen et al. (4van Soolingen D. Qian L. de Haas P.E. Douglas J.T. Traore H. Portaels F. Qing H.Z. Enkhsaikan D. Nymadawa P. van Embden J.D. J. Clin. Microbiol. 1995; 33: 3234-3238Crossref PubMed Google Scholar) who showed that more than 85% of M. tuberculosis isolates from the Beijing region belong to this genetically conserved genotype. The Beijing strains were grouped together on the basis of their highly similar multicopy IS6110 restriction fragment polymorphism patterns, the deletion of spacers 1–34 in the direct repeat region found in all M. tuberculosis strains, and the insertion of IS6110 into the dnaA-dnaN locus (5Kremer K. Glynn J.R. Lillebaek T. Niemann S. Kurepina N.E. Kreiswirth B.N. Bifani P.J. van Soolingen D. J. Clin. Microbiol. 2004; 42: 4040-4049Crossref PubMed Scopus (192) Google Scholar). This genotype family of M. tuberculosis is highly prevalent in Southeast Asia, where more than 50% of the strains isolated correspond to the Beijing genotype (6European Concerted Action on New Generation Genetic Markers and Techniques for the Epidemiology and Control of TuberculosisEmerg. Inf. Dis. 2006; 12: 736-743Crossref PubMed Scopus (228) Google Scholar). However, these strains have spread worldwide and have been responsible for major outbreaks of tuberculosis. The "W" strain responsible for a multidrug-resistant tuberculosis in the New York region, for example, belongs to this genotype (7Bifani P.J. Plikaytis B.B. Kapur V. Stockbauer K. Pan X. Lutfey M.L. Moghazeh S.L. Eisner W. Daniel T.M. Kaplan M.H. Crawford J.T. Musser J.M. Kreiswirth B.N. JAMA. 1996; 275: 452-457Crossref PubMed Google Scholar, 8Bifani P.J. Mathema B. Liu Z. Moghazeh S.L. Shopsin B. Tempalski B. Driscol J. Frothingham R. Musser J.M. Alcabes P. Kreiswirth B.N. JAMA. 1999; 282: 2321-2327Crossref PubMed Scopus (141) Google Scholar). A Beijing strain was introduced into Gran-Canaria Island in 1993 and rapidly spread; this strain accounted for 27.1% of tuberculosis cases on the island in 1996 (9Caminero J.A. Pena M.J. Campos-Herrero M.I. Rodríguez J.C. García I. Cabrera P. Lafoz C. Samper S. Takiff H. Afonso O. Pavón J.M. Torres M.J. van Soolingen D. Enarson D.A. Martin C. Am. J. Resp. Crit. Care Med. 2001; 164: 1165-1170Crossref PubMed Scopus (168) Google Scholar). This spread of Beijing strains from the region of the world in which they are endemic to other areas in which they have become epidemic suggests that these strains may be more virulent than others. Consistent with this hypothesis, several studies have demonstrated that mice infected with Beijing strains die more rapidly than mice infected with other strains (10López B. Aguilar D. Orozco H. Burger M. Espitia C. Ritacco V. Barrera L. Kremer K. Hernandez-Pando R. Huygen K. van Soolingen D. Clin. Exp. Immunol. 2003; 133: 30-37Crossref PubMed Scopus (361) Google Scholar, 11Dormans J. Burger M. Aguilar D. Hernandez-Pando R. Kremer K. Roholl P. Arend S.M. van Soolingen D. Clin. Exp. Immunol. 2004; 137: 460-468Crossref PubMed Scopus (144) Google Scholar, 12Manca C. Tsenova L. Bergtold A. Freeman S. Tovey M. Musser J.M. Barry 3rd, C.E. Freedman V.H. Kaplan G. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 5752-5757Crossref PubMed Scopus (469) Google Scholar, 13Reed M.B. Domenech P. Manca C. Su H. Barczak A.K. Kreiswirth B.N. Kaplan G. Barry 3rd, C.E. Nature. 2004; 431: 84-87Crossref PubMed Scopus (592) Google Scholar). This hypervirulence phenotype has been associated with a failure of Beijing strains to induce or to maintain an adequate Th1-type immune response (10López B. Aguilar D. Orozco H. Burger M. Espitia C. Ritacco V. Barrera L. Kremer K. Hernandez-Pando R. Huygen K. van Soolingen D. Clin. Exp. Immunol. 2003; 133: 30-37Crossref PubMed Scopus (361) Google Scholar, 12Manca C. Tsenova L. Bergtold A. Freeman S. Tovey M. Musser J.M. Barry 3rd, C.E. Freedman V.H. Kaplan G. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 5752-5757Crossref PubMed Scopus (469) Google Scholar) and with more severe pneumonia resulting in the death of the infected animals. A recent study suggests that patients infected with Beijing strains are more likely to develop extrathoracic tuberculosis than people infected with strains of other lineages (14Kong Y. Cave M.D. Zhang L. Foxman B. Marrs C.F. Bates J.H. Yang Z.H. J. Clin. Microbiol. 2007; 45: 409-414Crossref PubMed Scopus (111) Google Scholar). This observation is consistent with the results obtained in a rabbit model with the Beijing strain HN878, which was found more likely to infect the brain and to disseminate (15Tsenova L. Ellison E. Harbacheuski R. Moreira A.L. Kurepina N. Reed M.B. Mathema B. Barry 3rd, C.E. Kaplan G. J. Infect. Dis. 2005; 192: 98-106Crossref PubMed Scopus (192) Google Scholar). A recent study in Vietnam pointed out that the typical lineage of Beijing genotype strains is more frequently found in patients who received a Bacillus Calmette-Guérin vaccination. This suggests that bacteria of this lineage are more capable of circumventing Bacillus Calmette-Guérin-induced immunity (16Kremer K. van der Werf M.J. Au B.K. Kam K.M. van Doorn H.R. Borgdroff M.W. van Soolingen D. Emerg. Inf. Dis. 2009; 15: 335-339Crossref PubMed Scopus (81) Google Scholar). In the case of the HN878 strain, the production of a phenolic glycolipid (PGL) 3The abbreviations used are: PGLphenolglycolipidDIMphthiocerol dimycocerosatesHyghygromycinKmkanamycinMALDI-TOFmatrix-assisted laser desorption-ionization time-of-flightcfucolony-forming unitELISAenzyme-linked immunosorbent assayTNF-αtumor necrosis factor-αILinterleukinBMDMbone marrow-derived macrophageMICminimal inhibitory concentration. 3The abbreviations used are: PGLphenolglycolipidDIMphthiocerol dimycocerosatesHyghygromycinKmkanamycinMALDI-TOFmatrix-assisted laser desorption-ionization time-of-flightcfucolony-forming unitELISAenzyme-linked immunosorbent assayTNF-αtumor necrosis factor-αILinterleukinBMDMbone marrow-derived macrophageMICminimal inhibitory concentration. was shown to be important for the hypervirulent phenotype. The disruption of pks15/1, a PGL biosynthetic gene (17Constant P. Perez E. Malaga W. Lanéelle M.A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), in strain HN878 increased the release of pro-inflammatory cytokines by infected murine bone marrow macrophages and decreased the virulence of the bacterium in the mouse and rabbit disease models (13Reed M.B. Domenech P. Manca C. Su H. Barczak A.K. Kreiswirth B.N. Kaplan G. Barry 3rd, C.E. Nature. 2004; 431: 84-87Crossref PubMed Scopus (592) Google Scholar, 15Tsenova L. Ellison E. Harbacheuski R. Moreira A.L. Kurepina N. Reed M.B. Mathema B. Barry 3rd, C.E. Kaplan G. J. Infect. Dis. 2005; 192: 98-106Crossref PubMed Scopus (192) Google Scholar). PGL are very unusual molecules with a lipid core and a saccharide domain. The lipid core is formed by a long chain β-diol, naturally occurring as a diester of polymethyl-branched fatty acids and common to phthiocerol dimycocerosates (DIM), another group of lipids found in the cell envelope of M. tuberculosis and a few other slow-growing Mycobacterium species (18Daffé M. Laneelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar) (Fig. 1A). In the major form of PGL from M. tuberculosis (PGL-tb), this lipid core is ω-terminated by an aromatic nucleus glycosylated with a 2,3,4-tri-O-methyl-l-fucopyranosyl-(α1→3)-l-rhamnopyranosyl-(α1→3)-2-O-methyl-l-rhamnopyranosyl-(α1→) (19Daffé M. Lacave C. Lanéelle M.A. Lanéelle G. Eur. J. Biochem. 1987; 167: 155-160Crossref PubMed Scopus (116) Google Scholar) (Fig. 1A). This structure is specific to M. tuberculosis and Mycobacterium canettii (18Daffé M. Laneelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar, 20Malaga W. Constant P. Euphrasie D. Cataldi A. Daffé M. Reyrat J.M. Guilhot C. J. Biol. Chem. 2008; 283: 15177-15184Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Only a few M. tuberculosis strains synthesize PGL-tb because of a natural frameshift mutation in the pks15/1 gene (17Constant P. Perez E. Malaga W. Lanéelle M.A. Saurel O. Daffé M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Thus, based on the results of Reed et al. (13Reed M.B. Domenech P. Manca C. Su H. Barczak A.K. Kreiswirth B.N. Kaplan G. Barry 3rd, C.E. Nature. 2004; 431: 84-87Crossref PubMed Scopus (592) Google Scholar), it was thought that the production of this glycolipid was the principal cause of hypervirulence in Beijing strains. However, even within the Beijing lineage, only a minority of isolates produce PGL-tb (21Reed M.B. Gagneux S. Deriemer K. Small P.M. Barry 3rd, C.E. J. Bacteriol. 2007; 189: 2583-2589Crossref PubMed Scopus (188) Google Scholar, 22Sinsimer D. Huet G. Manca C. Tsenova L. Koo M.S. Kurepina N. Kana B. Mathema B. Marras S.A. Kreiswirth B.N. Guilhot C. Kaplan G. Infect. Immun. 2008; 76: 3027-3036Crossref PubMed Scopus (106) Google Scholar). In addition, complementation of the laboratory strain H37Rv with a functional pks15/1 allele, allowing the production of PGL-tb, does not significantly increase the virulence of this strain in the mouse and rabbit models (22Sinsimer D. Huet G. Manca C. Tsenova L. Koo M.S. Kurepina N. Kana B. Mathema B. Marras S.A. Kreiswirth B.N. Guilhot C. Kaplan G. Infect. Immun. 2008; 76: 3027-3036Crossref PubMed Scopus (106) Google Scholar). The molecular reasons for the higher virulence of strains of the Beijing lineage therefore remain unclear. phenolglycolipid phthiocerol dimycocerosates hygromycin kanamycin matrix-assisted laser desorption-ionization time-of-flight colony-forming unit enzyme-linked immunosorbent assay tumor necrosis factor-α interleukin bone marrow-derived macrophage minimal inhibitory concentration. phenolglycolipid phthiocerol dimycocerosates hygromycin kanamycin matrix-assisted laser desorption-ionization time-of-flight colony-forming unit enzyme-linked immunosorbent assay tumor necrosis factor-α interleukin bone marrow-derived macrophage minimal inhibitory concentration. In this study, we identified a mutation specific to the Beijing lineage that affected the activity of an enzyme involved in PGL-tb and DIM biosynthetic pathways. We analyzed the impact of this mutation on the structure of these compounds and explored the potential changes in cell envelope function and virulence induced by this mutation. M. tuberculosis Beijing strains were obtained from the collection of the National Institute for Public Health and the Environment (Bilthoven, The Netherlands), with the exception of Beijing strains HN878 and 1237 that were kindly provided by Prof. Clifton Barry III (Tuberculosis Research Section, National Institutes of Health, Rockville, MD) and Prof. Carlos Martin (Grupo de Genetica de Micobacterias, Universidad de Zaragoza, Spain) respectively. M. tuberculosis strains were grown at 37 °C in Middlebrook 7H9 broth (Invitrogen) containing ADC (0.2% dextrose, 0.5% bovine serum albumin fraction V, 0.0003% beef catalase) and 0.05% Tween 80 when necessary, and on solid Middlebrook 7H11 broth containing OADC (ADC plus 0.005% oleic acid). When required, kanamycin (Km) and hygromycin (Hyg) were used at concentrations of 40 and 50 μg·ml−1, respectively. Plasmids were propagated at 37 °C in Escherichia coli DH5α in LB broth or LB agar (Invitrogen) supplemented with either Km (40 μg·ml−1) or Hyg (200 μg·ml−1). Genomic DNA was used as the template for PCR amplification of the Rv2952 gene with specific primers binding 91 bp upstream and 64 bp downstream from the Rv2952 gene as follows: 2952 forward (5′-GGATGTCATGGCATCGACCG-3′) and 2952 reverse (5′-ATATAGAACGATGTCGAACTCG-3′). The PCR was performed on a GeneAmp PCR System 2700 thermocycler (Applied Biosystems) in a final volume of 50 μl containing 2.5 units of TaqDNA polymerase (New England Biolabs), 10% DMSO, and 1 μm of each primer. Reactions were initiated by denaturation for 5 min at 94 °C, and primer extension was then carried out over 30 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 57 °C, and extension for 1 min at 72 °C. A final extension of 10 min at 72 °C was then applied. PCR products were analyzed by electrophoresis in a 0.8% agarose gel. The 968-bp fragment was purified with the QIAquick purification kit (Qiagen, Courtaboeuf, France) and inserted into the pGEM®-T vector (Promega). Sequencing was carried out by Millegen (Labège, France) with the SP6 and T7 primers. A region covering the Rv2952 gene, from 500 bp upstream from the start codon to 8 bp downstream from the stop codon, was amplified by PCR from the M. tuberculosis H37Rv and HN878 strains with the primers Rv2952M (5′-TATATGACGTCGGATTGCGCCGACCTGCA-3′) and Rv2952N (5′-TATATACTAGTCAGTCCTTGGTGAAGCAGTA-3′). The PCR products were purified, digested with AatII and SpeI, and inserted between the AatII and SpeI sites of pMIP12d vector (20Malaga W. Constant P. Euphrasie D. Cataldi A. Daffé M. Reyrat J.M. Guilhot C. J. Biol. Chem. 2008; 283: 15177-15184Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The inserts were recovered from these plasmids as AatII-NheI fragments containing the transcription terminator ESAT 6. These fragments were inserted into AatII + NheI-digested pMV361 vector to give rise to p2952wt and p2952mu. These plasmids were transferred alone or with pPET1 into H37Rv, HN878, and 94-1707 cells by electroporation. Transformants were selected onto 7H11 plates supplemented with OADC, Km, and Hyg when required. The Rv2952 or Rv2952 G176R proteins were produced in E. coli using a T7 promoter expression system. The corresponding genes were cloned upstream in a His tag coding sequence in plasmid pET26b (Novagen). E. coli BL21(DE3) strains were transformed with each construct and the empty vector and grown in 10 ml of LB medium supplemented with antibiotics. At an A600 nm of 0.4, expression of Rv2952 (or the mutated protein) was induced by adding 0.1 mm isopropyl 1-thio-β-d-galactopyranoside and a further incubation for 6 h at 20 °C. Cells were collected by centrifugation, and stored at −20 °C. After thawing at room temperature, cells were resuspended in 1 ml of buffer (50 mm Tris-HCl, pH 8) and sonicated using a Vibra cell apparatus (Bioblock Scientific), three times for 20 s (microtip 4, 50% duty cycle), in ice. The cleared lysates were obtained following centrifugation at 12,000 × g for 15 min. Protein concentration was assayed using the method of Bradford (Bio-Rad protein assay), and the amounts of Rv2952 and Rv2952 G176R proteins in these extracts were evaluated by Western blot using anti-His antibodies (Rockland), revelation with ECL Western blotting detection system (GE Healthcare), and quantification of the signal using GS800 calibrated densitometer and Quantity One 4.6.3 software (Bio-Rad). Two substrates were used for O-methyltransferase activity as follows: the phthiotriol dimycocerosates prepared from M. tuberculosis, the natural substrate of the enzyme, and the 2-hexadecanol (Aldrich) chosen for its structural analogy with phthiocerol and relatives, especially phthiocerol B (18Daffé M. Laneelle M.A. J. Gen. Microbiol. 1988; 134: 2049-2055PubMed Google Scholar) and for the putative solubility problem with very long chain compounds as the C80–90 DIM-like compounds in in vitro tests. The lipids were dissolved at 60 mm for 2-hexadecanol and 1.5 mm for phthiotriol dimycocerosates in dimethyl sulfoxide, and S-[methyl-14C]adenosyl-l-methionine, specific activity 50.43 mCi·mmol −1 (PerkinElmer Life Sciences), was used as a tracer. The assay buffer was 50 mm Tris-HCl, pH 8. Briefly, in a final volume of 275 μl, 50–200 μl of protein crude extracts (0.5 mg·ml−1) were incubated at 37 °C for 1 h under agitation (200 rpm) in the presence of lipid substrates (10 μl) and S-[methyl-14C]adenosyl-l-methionine (28 μl). The reaction was stopped by adding a few drops of 20% H2SO4, and the reaction products were extracted three times with diethyl ether. The upper organic phases were pooled, washed with water until neutrality, and dried under air stream. The whole resulting material, dissolved in diethyl ether (30 μl), was spotted on TLC and run with petroleum ether/ether 9:1 (v/v) before exposure to Typhoon Imager (Amersham Biosciences) for the detection of labeled lipids. In initial experiments, kinetics of product formation was determined by stopping the reaction at 5, 10, 15, 20, 30, and 60 min and 6 h (data not shown). The incubation time (1 h) of subsequent experiments was chosen because the reaction was in initial velocity phase. Each strain was cultured (16 ml) to exponential growth phase and labeled by incubation with 0.625 μCi·ml−1 [1-14C]propionate (specific activity of 54 Ci·mol−1) for 24 h with continuous shaking. Lipids were extracted by adding 2 volumes of CH3OH and 1 volume of CHCl3 to 0.8 volume of culture, to yield a homogeneous one-phase mixture. After incubation for 2 days, the mixture was partitioned into two phases by adding 1 volume of H2O/CHCl3 (1:1, v/v). The organic phase was recovered. The extraction was repeated once by adding 1 volume of CHCl3 to the aqueous phase. The organic phases were pooled, washed at least twice with water, and dried before analysis. The dried lipid extracts from each culture were dissolved in 100 μl of CHCl3, and 30 μl of this preparation were spotted onto a Silica Gel 60 TLC plate (20 × 20 cm; Merck). The TLC plate was run in CHCl3/CH3OH (95:5, v/v) or in petroleum ether/diethyl ether (90:10, v/v) for PGL and DIM analysis, respectively. Labeled lipids were visualized with a Typhoon PhosphorImager (Amersham Biosciences). The production of various PGL and DIM forms was quantified by comparing the number of counts/min obtained for the lipid of interest with the total number of counts/min spotted. As the various PGL and DIM forms produced in the same batch of culture are expected to be labeled at the same position and with the same efficiency, comparison of this ratio provided a good evaluation of the relative amount of each DIM and PGL variant. For nonradioactive analysis, bacterial cells were harvested from 125-ml cultures in Sauton's medium, and lipids were extracted by a first incubation in CHCl3/CH3OH (1:2, v/v) for 2 days at room temperature and then in CHCl3/CH3OH (2:1, v/v). The two organic phases were pooled, washed twice with water, and dried before analysis. Extracted mycobacterial lipids were analyzed by TLC after resuspension in CHCl3 at a final concentration of 20 mg·ml−1. Equivalent amounts of each extract were spotted on TLC plates. DIM and PGL were visualized by spraying the plates with 10% phosphomolybdic acid in ethanol or with a 0.2% anthrone solution in concentrated H2SO4, respectively, followed by heating. For structural analysis, lipids were fractionated by chromatography on a Florisil column. Increasing concentrations of diethyl ether in petroleum ether or CH3OH in CHCl3 were used as eluents to obtain various members of the DIM and PGL family, respectively. The fractions containing the product of interest were then pooled and separated on preparative TLC. An enriched fraction was then finally recovered by scraping the silica gel from the plates. Purified molecules were analyzed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, as described previously (23Laval F. Lanéelle M.A. Déon C. Monsarrat B. Daffé M. Anal. Chem. 2001; 73: 4537-4544Crossref PubMed Scopus (101) Google Scholar). Spectra were acquired in reflectron mode, with an Applied Biosystems 4700 analyzer mass spectrometer (Applied Biosystems, Framingham) equipped with an Nd:YAG laser (wavelength 355 nm; pulse <500 ps; repetition rate 200 Hz). A total of 2500 shots were accumulated in positive ion mode, and mass spectrometry data were acquired with the default calibration for the instrument. NMR spectroscopy experiments were carried out at 295 K on a Bruker AVANCE spectrometer operating at 600,13 MHz with a 5-mm triple resonance TCI 1H 13C 15N pulsed field z-gradient cryoprobe. Samples were dissolved in 99.9% CDCl3. Chemical shifts are expressed in parts/million using the chloroform signal as an internal reference (7.23 ppm). Cultures of strains used for SDS resistance assays were grown to mid-exponential growth phase in 7H9 supplemented with ADC, 0.05% Tween 80, and Km when necessary. Cultures were centrifuged at 10,000 × g for 10 min at room temperature in a Jouan CR412 centrifuge with a T4 swing out rotor. The cells were then resuspended in 7H9-ADC without Tween 80 and incubated at 37 °C for 48 h. Bacterial concentration was evaluated by measuring the absorbance at 600 nm, and 10-ml cultures were inoculated to a final A600 of 0.02. SDS was added to each culture at a final concentration of 0.1%. Aliquots were collected after 0, 1, 4, and 8 days of growth, and the number of viable bacteria remaining was evaluated by plating serial dilutions on solid medium. Bacteria were prepared 48 h before plating from frozen stocks of known concentrations. Stocks were centrifuged and bacteria were resuspended in 7H9 and incubated at 37 °C for 2 days. Serial dilutions were then plated on 7H11 containing various concentration of rifampicin, isoniazid, or ethambutol and incubated for 3–4 weeks at 37 °C. For all the drugs tested, the minimal inhibitory concentration (MIC) corresponded to the minimal drug concentrations resulting in decreases of more than 90% in cfu counts. For the mixed infection experiments in mice, the M. tuberculosis HN878 and 94-1707 wild-type strains were transformed with the modified mycobacterial plasmid pMV361Hy derived from the pMV361 plasmid in which the Km resistance cassette was replaced by a Hyg resistance marker. The inoculum was prepared by mixing HN878:pMV361Hy cells with HN878:p2952wt cells, or 94-1707:pMV361Hy/94-1707:p2952wt, at a final concentration of 2.5 × 106 cfu·ml−1 for each strain. The concentration of the inoculum was determined by plating serial dilutions on 7H11 plates containing either Km or Hyg. Fifteen BALB/c mice were infected intranasally with 20 μl (1 × 105 cfu) of the inoculum, and for each infection group, five mice from each group were killed 1, 21, and 42 days after infection, and the numbers of colony-forming units present in lungs and spleen (except for day 1 after infection) were determined by plating serial dilutions on 7H11 plates containing either Km or Hyg serial dilutions of homogenates obtained from the organs by homogenizing tissues in 5 ml of NaCl/Pi supplemented with 0.05% Tween 80. All investigations in mice were carried out in accordance with CNRS guidelines for animal experimentation and were approved by the "Comité Regional d'Ethique pour l'Expérimentation Animale." We generated bone marrow-derived macrophages (BMDM) by flushing bone marrow cells from the femurs of ∼8-week-old B6D2 F1 mice into high glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% heat-inactivated fetal calf serum (Dutscher), 10% L929 conditioned medium, and 2 mm glutamine. For the infection assays, mouse bone marrow cells were seeded at a density of 2 × 105 cells per well in 24-well plates and allowed to differentiate for 7 days. Bacteria used for the infection were freshly prepared in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum and 2 mm glutamine. Bacterial concentration was adjusted to a final concentration of 3.3 × 106 bacteria per ml on the basis of absorbance 600 nm. An aliquot of the M. tuberculosis suspensions used to infect macrophages was spread on 7H11 plates to determine the number of bacteria in the inoculum more precisely. Infection assays were carried out as follows. The culture medium of each well was removed, and the cells were washed once with 500 μl of phosphate-buffered saline. We then added 300 μl of the mycobacterial suspension to obtain a multiplicity of infection of 5:1 (bacilli/BMDM). Control wells containing uninfected macrophages were filled with 300 μl of fresh medium. Infected and uninfected cultures were incubated for 4 h at 37 °C in an atmosphere containing 5% CO2. Infection was terminated by removing the overlaying medium and washing each well three times with 500 μl of Dulbecco's modified Eagle's medium before adding 400 μl of fresh culture medium per well. At 5 and 24 h the supernatants were recovered and analyzed for the presence of TNF-α, IL-10, IL-12p70, and MCP-1 by enzyme-linked immunosorbent assay (ELISA) (R & D Systems). Statistical analysis of the data were carried out with a one-way analysis of variance test to determine the significance of differences between the cytokine levels. M. tuberculosis strains are known to produce species-specific lipids that are critical for their virulence (24Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar, 25Guilhot C. Chalut C. Daffé M. The Mycobacterial Cell Envelope.in: Daffé M. Reyrat J.M. American Society for Microbiology, Washington, D. C.2008: 273-289Google Scholar); among these are the phthiocerol dimycocerosates (DIM A) and relatives (DIM B) (Fig. 1A). Some strains also elaborate the structurally related glycosylated phenolphthiocerol dimycocerosates (PGL-tb; Fig. 1A). These variants have different mobilities on TLC, and thus their production can be analyzed by this method. TLC analysis of the lipids of various clinical isolates of M. tuberculosis showed a special lipid profile for the Beijing isolates, particularly in the migration area of DIM (Fig. 1B) and PGL (data not shown). Phthiocerol dimycocerosate (D
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