The Role of MmpL8 in Sulfatide Biogenesis and Virulence of Mycobacterium tuberculosis
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m400324200
ISSN1083-351X
AutoresPilar Domenech, Michael B. Reed, Cynthia S. Dowd, Claudia Manca, Gilla Kaplan, Clifton E. Barry,
Tópico(s)Diagnosis and treatment of tuberculosis
ResumoTo study the role of MmpL8-mediated lipid transport in sulfatide biogenesis, we insertionally inactivated the mmpL8 gene in Mycobacterium tuberculosis. Characterization of this strain showed that the synthesis of mature sulfolipid SL-1 was interrupted and that a more polar sulfated molecule, termed SL-N, accumulated within the cell. Purification of SL-N and structural analysis identified this molecule as a family of 2,3-diacyl-α,α′-d-trehalose-2′-sulfates. This structure suggests that transport and biogenesis of SL-1 are coupled and that the final step in sulfatide biosynthesis may be the extra-cellular esterification of two trehalose 6-positions with hydroxyphthioceranic acids. To assess the effect of the loss of this anionic surface lipid on virulence, we infected mice via aerosol with the MmpL8 mutant and found that, although initial replication rates and containment levels were identical, compared with the wild type, a significant attenuation of the MmpL8 mutant strain in time-to-death was observed. Early in infection, differential expression of cytokines and cytokine receptors revealed that the mutant strain less efficiently suppresses key indicators of a Th1-type immune response, suggesting an immunomodulatory role for sulfatides in the pathogenesis of tuberculosis. To study the role of MmpL8-mediated lipid transport in sulfatide biogenesis, we insertionally inactivated the mmpL8 gene in Mycobacterium tuberculosis. Characterization of this strain showed that the synthesis of mature sulfolipid SL-1 was interrupted and that a more polar sulfated molecule, termed SL-N, accumulated within the cell. Purification of SL-N and structural analysis identified this molecule as a family of 2,3-diacyl-α,α′-d-trehalose-2′-sulfates. This structure suggests that transport and biogenesis of SL-1 are coupled and that the final step in sulfatide biosynthesis may be the extra-cellular esterification of two trehalose 6-positions with hydroxyphthioceranic acids. To assess the effect of the loss of this anionic surface lipid on virulence, we infected mice via aerosol with the MmpL8 mutant and found that, although initial replication rates and containment levels were identical, compared with the wild type, a significant attenuation of the MmpL8 mutant strain in time-to-death was observed. Early in infection, differential expression of cytokines and cytokine receptors revealed that the mutant strain less efficiently suppresses key indicators of a Th1-type immune response, suggesting an immunomodulatory role for sulfatides in the pathogenesis of tuberculosis. Despite more than 100 years of research, tuberculosis continues to be a serious global health problem, and the bacterial factors that facilitate the intracellular survival and pathogenesis of this disease remain largely unknown. The unique mycobacterial envelope, rich in diverse biologically active lipids, not only provides a physical barrier from environmental factors and host damage but also presents multiple lipid species that can contribute directly to the pathology of mycobacterial disease (1Daffe M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar, 2Manca C. Tsenova L. Barry III, C.E. Bergtold A. Freeman S. Haslett P.A. Musser J.M. Freedman V.H. Kaplan G. J. Immunol. 1999; 162: 6740-6746PubMed Google Scholar, 3Manca C. Tsenova L. Bergtold A. Freeman S. Tovey M. Musser J.M. Barry III, C.E. Freedman V.H. Kaplan G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5752-5757Crossref PubMed Scopus (471) Google Scholar).The genome of Mycobacterium tuberculosis (MTb) 1The abbreviations used are: MTb, Mycobacterium tuberculosis; RND, resistance, nodulation, and cell division; mmpL, mycobacterial membrane protein large; pks, polyketide synthase; PDIM, phthiocerol dimycocerosate; SL, sulfolipid; kbp, kilobase pair(s); TLC, thin layer chromatography; GC-MS, gas chromatography-mass spectrometry; EIMS, electrospray ionization-mass spectrometry; MS/MS, tandem mass spectrometry; CFU, colony-forming unit(s). 1The abbreviations used are: MTb, Mycobacterium tuberculosis; RND, resistance, nodulation, and cell division; mmpL, mycobacterial membrane protein large; pks, polyketide synthase; PDIM, phthiocerol dimycocerosate; SL, sulfolipid; kbp, kilobase pair(s); TLC, thin layer chromatography; GC-MS, gas chromatography-mass spectrometry; EIMS, electrospray ionization-mass spectrometry; MS/MS, tandem mass spectrometry; CFU, colony-forming unit(s). contains 12 genes that encode RND (resistance, nodulation, and cell division) proteins designated MmpL (Mycobacterial membrane protein Large) (4Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulson J.E. Taylor K. Whithead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6450) Google Scholar). These proteins are characterized by the presence of 12 transmembrane domains and two extracytoplasmic loops and have been reported in the genomes of organisms from all major kingdoms of life. In Gram-negative bacteria, these proteins facilitate the transport of a large variety of drugs, heavy metals, aliphatic and aromatic solvents, bile salts, fatty acids, detergents, and dyes (5Tseng T.T. Gratwick K.S. Kollman J. Park D. Nies D.H. Goffeau A. Saier Jr., M.H. J. Mol. Microbiol. Biotechnol. 1999; 1: 107-125PubMed Google Scholar). In Gram-positive bacteria, an ActII-ORF3 mutant (a member of the same family of proteins) in Streptomyces coelicolor has been shown to be impaired for γ-actinorhodin production (6Bystrykh L.V. Fernandez-Moreno M.A. Herrema J.K. Malpartida F. Hopwood D.A. Dijkhuizen L. J. Bacteriol. 1996; 178: 2238-2244Crossref PubMed Google Scholar). In this case, both synthesis and transport of this complex polyketide were affected. The co-localization of some of the mmpL genes with genes involved in polyketide biosynthesis (pks genes) and genes involved in lipid metabolism (papA and fadD) suggests a similar role of these proteins in complex lipid transport in MTb (7Tekaia F. Gordon S.V. Garnier T. Brosch R. Barrell B.G. Cole S.T. Tuber. Lung Dis. 1999; 79: 329-342Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 8Minnikin D.E. Kremer L. Dover L.G. Besra G.S. Chem. Biol. 2002; 9: 545-553Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Indeed, the MmpL7 protein has been shown to be involved in transport of phthiocerol dimycocerosate (PDIM) (9Camacho L.R. Constant P. Raynaud C. Laneelle M.A. Triccas J.A. Gicquel B. Daffe M. Guilhot C. J. Biol. Chem. 2001; 276: 19845-19854Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 10Cox J.S. Chen B. McNeil M. Jacobs Jr., W.R. Nature. 1999; 402: 79-83Crossref PubMed Scopus (609) Google Scholar).One of the mmpL genes, mmpL8, is positioned 8 kbp downstream from the pks2 gene. Pks2 is involved in the synthesis of heptamethyl- and octamethyl-branched fatty acids (known as phthioceranic acids) present in the major sulfolipid of M. tuberculosis, SL-1 (11Sirakova T.D. Thirumala A.K. Dubey V.S. Sprecher H. Kolattukudy P.E. J. Biol. Chem. 2001; 276: 16833-16839Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Structural analysis of SL-1 was performed by Goren and co-workers (12Goren M.B. Biochim. Biophys. Acta. 1970; 210: 116-126Crossref PubMed Scopus (94) Google Scholar, 13Goren M.B. Brokl O. Das B.C. Lederer E. Biochemistry. 1971; 10: 72-81Crossref PubMed Scopus (65) Google Scholar, 14Goren M.B. D'Arcy Hart P. Young M.R. Armstrong J.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2510-2514Crossref PubMed Scopus (218) Google Scholar), who identified it as 2-palmitoyl(stearoyl)-3-phthioceranol, 6,6′-bis(hydroxyphthioceranoyl) trehalose 2′-sulfate. The low abundance of this molecule in cultured MTb, its unique presence in the pathogenic human tubercle bacillus, and numerous experimental studies over the past 40 years strongly suggest a role for SL-1 in virulence. Some studies have reported a significant correlation between virulence of different strains of M. tuberculosis in guinea pigs and the amount of SL-1 produced by these strains cultured in vitro (15Goren M.B. Brokl O. Schaefer W.B. Infect. Immun. 1974; 9: 142-149Crossref PubMed Google Scholar, 16Gangadharam P.R. Cohn M.L. Middlebrook G. Tubercle. 1963; 44: 452-455Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Administration of M. tuberculosis sulfatides to cultured macrophages prevents phagosome-lysosome fusion (14Goren M.B. D'Arcy Hart P. Young M.R. Armstrong J.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2510-2514Crossref PubMed Scopus (218) Google Scholar), although this effect has been questioned since many anionic lipids could interact similarly with cationic sites on lysosomal hydrolases with resultant immobilization and/or inactivation of the enzymes (15Goren M.B. Brokl O. Schaefer W.B. Infect. Immun. 1974; 9: 142-149Crossref PubMed Google Scholar). A role for SL-1 in blocking human macrophage and neutrophil activation by modulation of superoxide release and secretion of IL-1β and TNF-α also has been observed (17Zhang L. English D. Andersen B.R. J. Immunol. 1991; 146: 2730-2736PubMed Google Scholar, 18Zhang L. Gay J.C. English D. Andersen B.R. J. Biomed. Sci. 1994; 1: 253-262PubMed Google Scholar, 19Zhang L. Goren M.B. Holzer T.J. Andersen B.R. Infect. Immun. 1988; 56: 2876-2883Crossref PubMed Google Scholar, 20Pabst M.J. Gross J.M. Brozna J.P. Goren M.B. J. Immunol. 1988; 140: 634-640PubMed Google Scholar, 21Brozna J.P. Horan M. Rademacher J.M. Pabst K.M. Pabst M.J. Infect. Immun. 1991; 59: 2542-2548Crossref PubMed Google Scholar). In addition, in vivo and in vitro studies have shown that SL-1 and cord factor may synergize in terms of mouse toxicity and attack on mitochondrial structure and function (22Kato M. Goren M.B. Infect. Immun. 1974; 10: 733-741Crossref PubMed Google Scholar). In contrast to these studies, two different groups (23Converse S.E. Mougous J.D. Leavell M.D. Leary J.A. Bertozzi C.R. Cox J.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6121-6126Crossref PubMed Scopus (197) Google Scholar, 24Rousseau C. Turner O.C. Rush E. Bordat Y. Sirakova T.D. Kolattukudy P.E. Ritter S. Orme I.M. Gicquel B. Jackson M. Infect. Immun. 2003; 71: 4684-4690Crossref PubMed Scopus (65) Google Scholar) have recently reported that pks2 disruption and SL-1 deficiency do not significantly affect the replication, persistence, or pathogenicity of M. tuberculosis in mice, guinea pigs, or cultured macrophages.In this study, we confirm and extend the recent observation by Converse et al. (23Converse S.E. Mougous J.D. Leavell M.D. Leary J.A. Bertozzi C.R. Cox J.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6121-6126Crossref PubMed Scopus (197) Google Scholar) that the synthesis of SL-1 is interrupted in an MTb mmpL8 mutant. This mutant accumulates a more polar molecule, termed SL-N, which is a likely precursor of SL-1. As a consequence, the cell surface charge appears to have been significantly altered. Purification and extensive analytical characterization of SL-N lead us to propose that, in contrast with the structure suggested by Converse et al. (23Converse S.E. Mougous J.D. Leavell M.D. Leary J.A. Bertozzi C.R. Cox J.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6121-6126Crossref PubMed Scopus (197) Google Scholar), this molecule is a family of 2,3-diacyl-α,α′-d-trehalose-2′-sulfates. Finally, we show that although loss of the MmpL8 protein does not affect in vivo replication rate or bacterial numbers during chronic infection, it does attenuate virulence of MTb in murine survival studies.EXPERIMENTAL PROCEDURESBacterial Strains, Culture Conditions, and Plasmids—The Electro-MAX DH5α Escherichia coli strain (Invitrogen) used for cloning was grown in Luria-Bertani medium with hygromycin (200 μg/ml) (Invitrogen) or gentamicin (Invitrogen) (5 μg/ml) when indicated. MTb strains were grown in Middlebroook 7H9 broth (Difco) supplemented with ADC (NaCl, 8.1 g/liter; bovine albumin fraction V (Calbiochem), 50 g/liter; d-glucose, 20 g/liter), 0.02% glycerol, and 0.05% Tween 80 (Sigma) or on Middlebroook 7H11 agar (Difco) supplemented with OADC enrichment (as ADC but including also 0.6 ml/liter oleic acid (ICN Biochemicals) and 3.6 mm sodium hydroxide). Where indicated hygromycin (50 μg/ml) or 2% sucrose was added (25Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (385) Google Scholar). H37Rv (Pasteur) was used as the parental strain of the H37Rv mmpL8::hyg mutant and H37Rv (ATCC, Manassas, VA) for the generation of the pks2::hyg.Nucleic Acid Techniques—E. coli transformations, cloning, and PCR were based on standard conditions (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Southern blotting and hybridization procedures were performed as described previously (27Domenech P. Menendez M.C. Garcia M.J. FEMS Microbiol. Lett. 1994; 116: 19-24Crossref PubMed Scopus (49) Google Scholar). Mycobacterial DNA was isolated using the protocol of Pelicic et al. (25Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (385) Google Scholar). Transformation of MTb was carried out as described previously (28Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Mol. Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (998) Google Scholar).Construction of the mmpL8 and pks2 Disrupted Mutants—Generation of the MTb disrupted mutants (mmpL8::hyg and pks2::hyg) was accomplished by homologous recombination using the system developed by Pelicic et al. (25Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (385) Google Scholar). A 2.1-kbp fragment containing the mmpL8 gene (nucleotide positions 538-2686) was generated by PCR and cloned into the SpeI site of the vector pcDNA2.1 (Invitrogen). A 1.6-kbp fragment carrying the hyg gene was cloned into the mmpL8 gene at the HpaI site (position 1656). Finally the 3.7-kbp fragment harboring the mmpL8::hyg gene was excised and cloned into the mycobacterial shuttle vector pPR27 (25Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (385) Google Scholar). The pks2 mutant was produced by PCR amplification of a 2-kbp fragment containing the pks2 gene (positions 3302-5310). This fragment was cloned into the vector pCRBlunt (Invitrogen), and a 1.6-kbp fragment carrying the hyg gene was substituted for a 1-kbp internal NheI (position 4457)-BglII (position 4775) segment. The 2.6-kbp fragment containing the disrupted pks2 sequence and hygromycin resistance determinant was excised and cloned into the mycobacterial shuttle vector pPR27 (25Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (385) Google Scholar). Transformations were plated on 7H11 with 50 μg/ml hygromycin at 32 °C for 5 weeks. The resulting colonies were grown at 32 °C in 10 ml of 7H9 containing 50 μg/ml hygromycin and subsequently were plated on 7H11 with 50 μg/ml hygromycin and 2% sucrose at 39 °C. DNA from HygR, SucR, and TR (hygromycin-, sucrose-, and temperature-resistant) colonies was digested with XhoI, transferred to Hybond-N nylon membrane (Amersham Biosciences) by Southern blot, and hybridized with a 2.1-kbp fragment probe of the mmpL8 gene of MTb H37Rv Pasteur. The pks2::hyg deletion-replacement was confirmed through Southern analysis of EcoRI- or EcoRV-restricted DNA that was hybridized with both the 2-kbp pks2 fragment generated by PCR (see above) and the 1-kbp fragment released prior to insertion of the hygromycin cassette.Lipid Analysis and Sulfolipid Purification—100-ml cultures of the different MTb strains (wild type, mmpL8::hyg, and pks2::hyg mutants) were grown to an OD650 of 0.3. Metabolic labeling of the methyl-branched fatty acids was achieved by incubating these cultures in the presence of 1 μCi/ml sodium [1-14C]propionate (American Radiolabeled Chemicals, specific activity ≈ 56 mCi/mmol) for 24 h prior to lipid extraction. Similarly, sulfated lipids were labeled by adding 3.3 μCi/ml [35S]Na2SO4 (Amersham Biosciences, specific activity ≈ 100 mCi/mmol) and incubated for 96 h. Mycobacteria were harvested from the media by centrifugation (750 g/15 min), and prior to lipid extraction the culture supernatants were sterilized by filtration through a 0.2-μm-pore size membrane. Bacteria-associated lipids were extracted by two different methods. Bacteria-associated total lipids were extracted using the Folch method (29Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), which includes two extractions with chloroform: methanol (2:1) and three washes with chloroform, methanol, and 0.58% NaCl in water at ratios 3:48:47. Alternatively, separation of bacteria-associated apolar and polar lipids was accomplished by two petroleum ether extractions (apolar) prior to chloroform:methanol extraction, following the procedures described by Slayden and Barry (30Slayden R.A. Barry III, C.E. Parish T. Stoker N.G. Mycobacterium tuberculosis Protocols. 54. Humane Press Inc., Totowa, NJ2001: 229-245Google Scholar). Apolar lipids present in the culture supernatant were extracted with 2 volumes of petroleum ether. Polar lipids in the supernatant were extracted as described elsewhere (31Constant P. Perez E. Malaga W. Laneelle M.A. Saurel O. Daffe M. Guilhot C. J. Biol. Chem. 2002; 277: 38148-38158Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Thin layer chromatography (TLC) was performed using 250-μm silica gel 60 plates (EM SCIENCE) with chloroform:methanol:H2O (65:25:4) as the developing solvent. TLC plates were visualized using a Storm 860 PhosphorImager (Amersham Biosciences).For purification of SL-1 and SL-N, 4-liter cultures of H37Rv and mmpL8::hyg were labeled with 1 mCi of [35S]Na2SO4 (Amersham Biosciences, 100 mCi/mmol) as is indicated above. Bacteria-associated lipids (apolar for SL-1, polar for SL-N, prepared as above) were passed over a silica gel 50 column and eluted with 9:1 chloroform:methanol (3 volumes), 5:1 chloroform:methanol (3 volumes), and 1:1 chloroform: methanol (6 volumes). Fractions containing sulfolipids were identified by TLC and autoradiography and were then pooled and dried. Next, the pooled fractions were applied to an anion exchange column (PS-DVB DEAE, 10 mm × 100 mm, 8-μm beads, Vydac) on a Waters 2690 HPLC. The column was activated prior to injection of the sample by equilibrating with chloroform:methanol:acetic acid (800:200:0.6) at a flow rate of 2 ml/min. Elution of the lipids was done using a 0-35% gradient over 40 min with chloroform:methanol:triethylamine (800:200:1.39) as the eluting solvent. 2-ml fractions were collected and analyzed by liquid scintillation counting and TLC. Purity of samples was assessed by staining TLC plates with 5% phosphomolybdic acid (Sigma) and charring.Neutral Red Assay—Chemical staining of H37Rv and the mmpL8::hyg strain was carried out following the protocol described by Soto et al. (32Soto C.Y. Andreu N. Gibert I. Luquin M. J. Clin. Microbiol. 2002; 40: 3021-3024Crossref PubMed Scopus (22) Google Scholar).GC-MS of Fatty Acid Methyl Esters—Fatty acid methyl esters from purified SL-1 and SL-N were prepared following the protocol described previously (30Slayden R.A. Barry III, C.E. Parish T. Stoker N.G. Mycobacterium tuberculosis Protocols. 54. Humane Press Inc., Totowa, NJ2001: 229-245Google Scholar). GC-MS was carried out on a Hewlett Packard 5890 instrument operated in splitless mode using an HP-5MS column (30 m × 0.25 mm × 0.25 μm). The injection port temperature was 310 °C, and the column temperature was ramped from 180 to 310 °C at 10 °C/min followed by an additional 15 min at 310 °C with 8 p.s.i. helium.Mass Spectrometry—Mass spectral analyses were performed at the Yale Cancer Center Mass Spectrometry Resource and the W. M. Keck Foundation Biotechnology Resource Laboratory. For both electrospray ionization-mass spectrometry (EI-MS) and tandem mass spectrometry (MS/MS), purified SL-N was dissolved in chloroform and then diluted with methanol, 1% ammonium hydroxide (for negative ion analysis) or methanol, 1% formic acid (for positive ion analysis) to a useable concentration. The samples were analyzed on a Q-Tof1 (Waters/Micromass) mass spectrometer using the nanospray technique for both positive and negative modes. For the MS/MS spectra, the collision cell was pressurized with argon, and the collision energy was adjusted to give the optimal spectrum, which was -4 electron volts for positive ion spectra and -70 electron volts for negative ion spectra. The spectra were calibrated using sodium iodide in either the positive or negative ion mode.Proton and COSY NMR Spectra—The 1H (one-dimensional) and COSY spectra of 35S-labeled SL-N were obtained on a Varian VXR-500S NMR spectrometer at 30 °C in CDCl3:CD3OD (2:1).Mouse Experiments—Prior to infection, well dispersed liquid cultures were adjusted to an OD650 nm of 0.5 and stored at -70 °C as 20% glycerol stocks. Inocula were prepared by diluting these stocks to 4 × 106 colony-forming units (CFU)/ml in PBS/Tween 80 (0.05%). Eight-week-old C57Bl/6 or B6D2/F1 mice (Taconic) were infected using a BioAerosol nebulizing generator (CH Technologies Inc., Westwood, NJ) for 10 min. Bacterial numbers were enumerated at 1, 14, 49, 63, 98, 182, and 205 days post-infection (4 mice/time point) by homogenizing the lungs and spleens of infected mice in 1 ml of 7H9 medium and plating 10-fold serial dilutions on 7H11 medium. An additional 12 mice/group were used in survival studies. Survival fractions were calculated using the Kaplan-Meier method (33Kaplan E.L. Meier P. J. Am. Stat. Assoc. 1958; 53: 457-481Crossref Scopus (47684) Google Scholar), and the log-rank test was used to determine statistical significance of observed survival differences (GraphPad Prism version 3.0; GraphPad Software, San Diego, CA).Cytokine Expression Studies—At 14 days post-infection, lungs of euthanized mice (4 mice/group) were removed and immediately snap-frozen on dry ice/ethanol. Tissues were homogenized in 3 ml of RNAzolB (Cinna/BiotecX, Houston, TX) using a tissue Polytron homogenizer. RNA was extracted according to the manufacturer's instructions, and 5 μg of RNA from each sample was reverse transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase (Ambion). Biotinylated cDNA probes were hybridized to cytokine-chemokine cDNAs spotted on GEArray membranes according to the manufacturer's directions (SuperArray, Bethesda, MD). Arrays were developed with CDP-Star chemiluminescence substrate and recorded with x-ray film. The ScanAlyze 2 program was used for image analysis, and the GEArray-Analyzer was used to process the raw data. Gene expression was normalized to the signal derived from β-actin. 2-fold difference in mRNA expression between strains was considered significant.RESULTSDisruption of the MTb mmpL8 Gene Interrupts SL-1 Biosynthesis—Disruption of the mmpL8 gene in MTb H37Rv was accomplished by insertion of a hygromycin resistance cassette within the coding sequence of a cloned copy of this gene followed by homologous recombination of the inactivated allele onto the chromosome of MTb using a plasmid based on the pPR27 vector (temperature-sensitive mycobacterial origin of replication and sacB negative selection marker) (25Pelicic V. Jackson M. Reyrat J.M. Jacobs Jr., W.R. Gicquel B. Guilhot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10955-10960Crossref PubMed Scopus (385) Google Scholar). Fig. 1A shows the results of Southern blot analysis of chromosomal DNA from six clones (lanes 2-7) obtained from this procedure compared with DNA from the H37Rv wild-type parent (lanes 1 and 8). In each clone the presence of a 3.7-kbp fragment hybridizing with an mmpL8 probe that recognizes a 2.1-kbp fragment on the wild-type chromosome suggests that all six have the expected 1.6-kbp insertion of the hygromycin resistance cassette. One of these mutants was selected for further analysis.Because of the chromosomal co-localization of mmpL8 and pks2 (Fig. 1B), we examined lipid extracts from the mmpL8::hyg mutant following metabolic labeling of the methyl-branched fatty acids of MTb H37Rv, MTb H37Rv mmpL8::hyg, and an MTb H37Rv pks2::hyg mutant with [1-14C]propionic acid. Bacteria-associated "apolar" (petroleum ether-extractable) and "polar" (chloroform:methanol-extractable) lipids were obtained from these strains and analyzed by TLC (Fig. 2A, lanes 1-8). SL-1, with an RF of 0.92, was mainly extracted in the apolar fraction, suggesting localization within the peripheral region of the cell wall. SL-1 was present in both H37Rv wild-type strains tested (the type strain from the American Type Tissue Collection and the sequenced strain from the Institut Pasteur) (Fig. 2A, lanes 1, 3, 5, and 7). As expected SL-1 was not produced by an MTb pks2 mutant (Fig. 2A, lanes 4 and 8), but surprisingly the MTb mmpL8::hyg mutant also failed to produce mature SL-1 (lanes 2 and 6). This mutant instead accumulated a more polar metabolite (RF 0.5) that incorporated propionate and appeared in the polar fraction (Fig. 2A, lane 6).Fig. 2Disruption of the M. tuberculosis mmpL8 gene interrupts SL-1 biosynthesis and results in accumulation of SL-N. A, autoradiogram of a TLC of [1-14C]propionic acid-labeled (lanes 1-8) and [35S]Na2SO4-labeled (lanes 9-12) lipids developed in chloroform:methanol: water (65:25:4). Lanes 1-4 are bacteria-associated "apolar lipids" (extracted with petroleum ether). Lanes 5-8 are bacteria-associated "polar lipids" (extracted with chloroform:methanol (2:1) after the petroleum ether extraction), and lanes 9-12 are bacteria-associated total lipids (total lipids extracted with chloroform:methanol (2:1) following the Folch method). Lipids are from H37Rv (Pasteur strain; lanes 1, 5, and 9), mmpL8::hyg mutant (lanes 2, 6, and 10), H37Rv (ATCC 27294; lanes 3, 7, and 11), and pks2::hyg mutant (lanes 4, 8, and 12). B, autoradiogram of a TLC of [1-14C]propionic acid-labeled bacteria-associated lipids (lanes 1-4) and lipids extracted from the culture supernatants (lanes 5-8). Lanes 1 and 5, apolar lipids from H37Rv (Pasteur); lanes 2 and 6, apolar lipids from mmpL8::hyg mutant; lanes 3 and 7, polar lipids from H37Rv (Pasteur); lanes 4 and 8, polar lipids from mmpL8::hyg mutant. The solvent is chloroform:methanol:H2O (65:25:4). C, neutral red assay of H37Rv (Pasteur) and mmpL8::hyg (mmpL8 KO).View Large Image Figure ViewerDownload (PPT)To establish whether this metabolite contained sulfate we labeled cells by growing them in the presence of [35S]Na2SO4 (Fig. 2A, lanes 9-12). In the wild-type strain the majority of the [35S]Na2SO4 was incorporated into SL-1 (Fig. 2A, lanes 9 and 11), confirming that this was the most abundant sulfolipid produced in MTb. In contrast, the mmpL8 mutant incorporated [35S]Na2SO4 into a molecule that co-migrated by TLC with the propionate-labeled metabolite (RF 0.5) (Fig. 2A, lane 10). These results suggest that inactivation of the mmpL8 gene of MTb interrupted the normal biosynthesis of SL-1 and led to the accumulation of a more polar molecule, designated SL-N, that contained both sulfate and methyl-branched fatty acids.SL-N Is Localized inside the Cell Envelope—We compared lipids associated with bacterial cells (Fig. 2B, lanes 1-4) with those found in the culture supernatant (Fig. 2B, lanes 5-8) for wild-type and mmpL8::hyg mutant strains. SL-1 was present in both the apolar and polar lipid extracts of wild-type culture supernatant (Fig. 2B, lanes 5 and 7), whereas SL-N was not found in the supernatant from the mmpL8 mutant (lanes 6 and 8). While some SL-1 was also found in association with the bacterial cells, SL-N was found exclusively in association with the bacterial cells. This result suggests that SL-1 normally occupies a peripheral location within the mycobacterial envelope and can be shed into the culture supernatant, whereas SL-N apparently occupies a more integral location and may be exclusively retained within the cytosol.In an attempt to address whether SL-N was peripherally associated with the cell membrane but was not efficiently shed into the medium, we examined the neutral red binding ability of the mmpL8::hyg mutant. The neutral red assay has been used extensively to distinguish between avirulent and virulent strains of MTb because of its specificity for labeling cells producing SL-1 (15Goren M.B. Brokl O. Schaefer W.B. Infect. Immun. 1974; 9: 142-149Crossref PubMed Google Scholar, 32Soto C.Y. Andreu N. Gibert I. Luquin M. J. Clin. Microbiol. 2002; 40: 3021-3024Crossref PubMed Scopus (22) Google Scholar, 34Morse W.C. Dail M.C. Olitzky I. Am. J. Public Health. 1953; 43: 36-39Crossref Google Scholar, 35Hughes D.E. Moss E.S. Hood M. Henson M. Am. J. Clin. Pathol. 1954; 24: 621-625Crossref PubMed Scopus (6) Google Scholar). The capacity to bind this molecule has been interpreted as indicating a surface-accessible location of the sulfatides, whose strongly acidic sulfate interacts ionically with the cationic dye (36Middlebrook G. Coleman
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