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Metabolism of Mycobacterium tuberculosis

  • Dany J. V. Beste
  • Johnjoe McFadden
Chapter

Abstract

Despite decades of research many aspects of the biology of Mycobacterium tuberculosis remain unclear and this is reflected in the antiquated tools available to treat and prevent tuberculosis. Consequently, this disease remains a serious public health problem responsible for 2–3 million deaths each year. Important discoveries linking M. tuberculosis metabolism and pathogenesis have renewed interest in the metabolic underpinning of the interaction between the pathogen and its host. Whereas, previous experimental studies tended to focus on the role of single genes, antigens or enzymes, the central paradigm of systems biology is that the role of any gene cannot be determined in isolation from its context. Therefore, systems approaches examine the role of genes and proteins embedded within a network of interactions. We here examine the application of this approach to studying metabolism of M. tuberculosis. Recent advances in high-throughput experimental technologies, such as functional genomics and metabolomics, provide datasets that can be analysed with computational tools such as flux balance analysis. These new approaches allow metabolism to be studied on a genome scale and have already been applied to gain insights into the metabolic pathways utilised by M. tuberculosis in vitro and identify potential drug targets. The information from these studies will fundamentally change our approach to tuberculosis research and lead to new targets for therapeutic drugs and vaccines.

Keywords

Metabolic Network Slow Growth Rate Metabolic Model Flux Balance Analysis Metabolic Flux Analysis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    McKinney JD, Honer zu Bentrup K, Munoz-Elias EJ, Miczak ACB, Chan W-T, Senson D, Sacchettini JC, Jacobs-WR J, Russell DG (2000) Persistance of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735–738PubMedCrossRefGoogle Scholar
  2. 2.
    Munoz-Elias EJ, McKinney JD (2005) Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med 11:638–644PubMedCrossRefGoogle Scholar
  3. 3.
    Fritz C, Maass S, Kreft A, Bange FC (2002) Dependence of Mycobacterium bovis BCG on anaerobic nitrate reductase for persistence is tissue specific. Infect Immun 70:286–291PubMedCrossRefGoogle Scholar
  4. 4.
    Miner MD, Chang JC, Pandey AK, Sassetti CM, Sherman DR (2009) Role of cholesterol in Mycobacterium tuberculosis infection. Indian J Exp Biol 47:407–411PubMedGoogle Scholar
  5. 5.
    Movahedzadeh F, Smith DA, Norman RA, Dinadayala P, Murray-Rust J, Russell DG, Kendall SL, Rison SC, McAlister MS, Bancroft GJ et al (2004) The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence. Mol Microbiol 51:1003–1014PubMedCrossRefGoogle Scholar
  6. 6.
    Singh A, Crossman DK, Mai D, Guidry L, Voskuil MI, Renfrow MB, Steyn AJ (2009) Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response. PLoS Pathog 5:e1000545PubMedCrossRefGoogle Scholar
  7. 7.
    Glickman MS, Cox JS, Jacobs WR Jr (2000) A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol Cell 5:717–727PubMedCrossRefGoogle Scholar
  8. 8.
    Keating LA, Wheeler PR, Mansoor H, Inwald JK, Dale J, Hewinson RG, Gordon SV (2005) The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth. Mol Microbiol 56:163–174PubMedCrossRefGoogle Scholar
  9. 9.
    Munoz-Elias EJ, Upton AM, Cherian J, McKinney JD (2006) Role of the methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular growth, and virulence. Mol Microbiol 60:1109–1122PubMedCrossRefGoogle Scholar
  10. 10.
    Gengenbacher M, Rao SP, Pethe K, Dick T (2010) Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability. Microbiology 156:81–87PubMedCrossRefGoogle Scholar
  11. 11.
    Baughn AD, Garforth SJ, Vilcheze C, Jacobs WR Jr (2009) An anaerobic-type alpha-ketoglutarate ferredoxin oxidoreductase completes the oxidative tricarboxylic acid cycle of Mycobacterium tuberculosis. PLoS Pathog 5:e1000662PubMedCrossRefGoogle Scholar
  12. 12.
    Beste D, Hooper T, Stewart GS, Bonde B, Avignone-Rossa C, Bushell M, Wheeler PR, Klamt S, Kierzek AM, McFadden JJ (2007) GSMN-TB: a web-based genome scale network model of Mycobacterium tuberculosis metabolism. Genome Biol 8:R89PubMedCrossRefGoogle Scholar
  13. 13.
    Beste DJ, Bonde B, Hawkins N, Ward JL, Beale MH, Noack S, Noh K, Kruger NJ, Ratcliffe RG, McFadden J (2011) 13C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation. PLoS Pathog 7:e1002091PubMedCrossRefGoogle Scholar
  14. 14.
    Tian J, Bryk R, Shi S, Erdjument-Bromage H, Tempst P, Nathan C (2005) Mycobacterium tuberculosis appears to lack alpha-ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes. Mol Microbiol 57:859–868PubMedCrossRefGoogle Scholar
  15. 15.
    Tian J, Bryk R, Itoh M, Suematsu M, Nathan C (2005) Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: identification of alpha-ketoglutarate decarboxylase. Proc Natl Acad Sci USA 102:10670–10675PubMedCrossRefGoogle Scholar
  16. 16.
    de Carvalho LP, Zhao H, Dickinson CE, Arango NM, Lima CD, Fischer SM, Ouerfelli O, Nathan C, Rhee KY (2010) Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase. Chem Biol 17:323–332PubMedCrossRefGoogle Scholar
  17. 17.
    Wagner T, Bellinzoni M, Wehenkel A, O’Hare HM, Alzari PM (2011) Functional plasticity and allosteric regulation of alpha-ketoglutarate decarboxylase in central mycobacterial metabolism. Chem Biol 18:1011–1020PubMedCrossRefGoogle Scholar
  18. 18.
    Watanabe S, Zimmermann M, Goodwin MB, Sauer U, Barry CE III, Boshoff HI (2011) Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis. PLoS Pathog 7:e1002287PubMedCrossRefGoogle Scholar
  19. 19.
    Kitano H (2007) Towards a theory of biological robustness. Mol Syst Biol 3:137PubMedCrossRefGoogle Scholar
  20. 20.
    Hoskisson PA, Hobbs G (2005) Continuous culture–making a comeback? Microbiology 151:3153–3159PubMedCrossRefGoogle Scholar
  21. 21.
    Boer VM, de Winde JH, Pronk JT, Piper MD (2003) The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J Biol Chem 278:3265–3274PubMedCrossRefGoogle Scholar
  22. 22.
    Boer VM, Tai SL, Vuralhan Z, Arifin Y, Walsh MC, Piper MD, de Winde JH, Pronk JT, Daran JM (2007) Transcriptional responses of Saccharomyces cerevisiae to preferred and nonpreferred nitrogen sources in glucose-limited chemostat cultures. FEMS Yeast Res 7:604–620PubMedCrossRefGoogle Scholar
  23. 23.
    Hayes A, Zhang N, Wu J, Butler PR, Hauser NC, Hoheisel JD, Lim FL, Sharrocks AD, Oliver SG (2002) Hybridization array technology coupled with chemostat culture: tools to interrogate gene expression in Saccharomyces cerevisiae. Methods 26:281–290PubMedCrossRefGoogle Scholar
  24. 24.
    James BW, Williams A, Marsh PD (2000) The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium. J Appl Microbiol 88:669–677PubMedCrossRefGoogle Scholar
  25. 25.
    Bacon J, James BW, Wernisch L, Williams A, Morley KA, Hatch GJ, Mangan JA, Hinds J, Stoker NG, Butcher PD et al (2004) The influence of reduced oxygen availability on pathogenicity and gene expression in Mycobacterium tuberculosis. Tuberculosis (Edinb) 84:205–217CrossRefGoogle Scholar
  26. 26.
    Bacon J, Dover LG, Hatch KA, Zhang Y, Gomes JM, Kendall S, Wernisch L, Stoker NG, Butcher PD, Besra GS et al (2007) Lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron-limitation in continuous culture: identification of a novel wax ester. Microbiology 153:1435–1444PubMedCrossRefGoogle Scholar
  27. 27.
    Jenkins C, Bacon J, Allnutt J, Hatch KA, Bose A, O’Sullivan DM, Arnold C, Gillespie SH, McHugh TD (2009) Enhanced heterogeneity of rpoB in Mycobacterium tuberculosis found at low pH. J Antimicrob Chemother 63:1118–1120PubMedCrossRefGoogle Scholar
  28. 28.
    Daran-Lapujade P, Daran JM, Kotter P, Petit T, Piper MD, Pronk JT (2003) Comparative genotyping of the Saccharomyces cerevisiae laboratory strains S288C and CEN.PK113-7D using oligonucleotide microarrays. FEMS Yeast Res 4:259–269PubMedCrossRefGoogle Scholar
  29. 29.
    Beste DJ, Peters J, Hooper T, Avignone-Rossa C, Bushell ME, McFadden J (2005) Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism. J Bacteriol 187:1677–1684PubMedCrossRefGoogle Scholar
  30. 30.
    Beste DJ, Espasa M, Bonde B, Kierzek AM, Stewart GR, McFadden J (2009) The genetic requirements for fast and slow growth in mycobacteria. PLoS One 4:e5349PubMedCrossRefGoogle Scholar
  31. 31.
    Beste DJ, Laing E, Bonde B, Avignone-Rossa C, Bushell ME, McFadden JJ (2007) Transcriptomic analysis identifies growth rate modulation as a component of the adaptation of mycobacteria to survival inside the macrophage. J Bacteriol 189:3969–3976PubMedCrossRefGoogle Scholar
  32. 32.
    Bumann D (2009) System-level analysis of Salmonella metabolism during infection. Curr Opin Microbiol 12:559–567PubMedCrossRefGoogle Scholar
  33. 33.
    Feist AM, Herrgard MJ, Thiele I, Reed JL, Palsson BO (2009) Reconstruction of biochemical networks in microorganisms. Nat Rev Microbiol 7:129–143PubMedGoogle Scholar
  34. 34.
    Durot M, Bourguignon PY, Schachter V (2009) Genome-scale models of bacterial metabolism: reconstruction and applications. FEMS Microbiol Rev 33:164–190PubMedCrossRefGoogle Scholar
  35. 35.
    Bettenbrock K, Fischer S, Kremling A, Jahreis K, Sauter T, Gilles ED (2006) A quantitative approach to catabolite repression in Escherichia coli. J Biol Chem 281:2578–2584PubMedCrossRefGoogle Scholar
  36. 36.
    AbuOun M, Suthers PF, Jones GI, Carter BR, Saunders MP, Maranas CD, Woodward MJ, Anjum MF (2009) Genome scale reconstruction of a Salmonella metabolic model: comparison of similarity and differences with a commensal Escherichia coli strain. J Biol Chem 284:29480–29488PubMedCrossRefGoogle Scholar
  37. 37.
    Duarte NC, Becker SA, Jamshidi N, Thiele I, Mo ML, Vo TD, Srivas R, Palsson BO (2007) Global reconstruction of the human metabolic network based on genomic and bibliomic data. Proc Natl Acad Sci USA 104:1777–1782PubMedCrossRefGoogle Scholar
  38. 38.
    Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007) A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Mol Syst Biol 3:121PubMedCrossRefGoogle Scholar
  39. 39.
    Mo ML, Jamshidi N, Palsson BO (2007) A genome-scale, constraint-based approach to systems biology of human metabolism. Mol Biosyst 3:598–603PubMedCrossRefGoogle Scholar
  40. 40.
    Oberhardt MA, Puchalka J, Fryer KE, Martins dos Santos VA, Papin JA (2008) Genome-scale metabolic network analysis of the opportunistic pathogen Pseudomonas aeruginosa PAO1. J Bacteriol 190:2790–2803PubMedCrossRefGoogle Scholar
  41. 41.
    Thiele I, Jamshidi N, Fleming RM, Palsson BO (2009) Genome-scale reconstruction of Escherichia coli’s transcriptional and translational machinery: a knowledge base, its mathematical formulation, and its functional characterization. PLoS Comput Biol 5:e1000312PubMedCrossRefGoogle Scholar
  42. 42.
    Kim HU, Kim TY, Lee SY (2008) Metabolic flux analysis and metabolic engineering of microorganisms. Mol Biosyst 4:113–120PubMedCrossRefGoogle Scholar
  43. 43.
    Wiechert W (2001) 13C metabolic flux analysis. Metab Eng 3:195–206PubMedCrossRefGoogle Scholar
  44. 44.
    Boyle NR, Morgan JA (2009) Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii. BMC Syst Biol 3:4PubMedCrossRefGoogle Scholar
  45. 45.
    Raman K, Chandra N (2009) Flux balance analysis of biological systems: applications and challenges. Brief Bioinform 10:435–449PubMedCrossRefGoogle Scholar
  46. 46.
    Varma A, Palsson BO (1994) Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110. Appl Environ Microbiol 60:3724–3731PubMedGoogle Scholar
  47. 47.
    Antoniewicz MR, Kraynie DF, Laffend LA, Gonzalez-Lergier J, Kelleher JK, Stephanopoulos G (2007) Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol. Metab Eng 9:277–292PubMedCrossRefGoogle Scholar
  48. 48.
    Kayser A, Weber J, Hecht V, Rinas U (2005) Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state. Microbiology 151:693–706PubMedCrossRefGoogle Scholar
  49. 49.
    Peng L, Arauzo-Bravo MJ, Shimizu K (2004) Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements. FEMS Microbiol Lett 235:17–23PubMedCrossRefGoogle Scholar
  50. 50.
    Raman K, Rajagopalan P, Chandra N (2005) Flux balance analysis of mycolic acid pathway: targets for anti-tubercular drugs. PLoS Comput Biol 1:e46PubMedCrossRefGoogle Scholar
  51. 51.
    Schuetz R, Kuepfer L, Sauer U (2007) Systematic evaluation of objective functions for predicting intracellular fluxes in Escherichia coli. Mol Syst Biol 3:119PubMedCrossRefGoogle Scholar
  52. 52.
    Jamshidi N, Palsson BO (2007) Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets. BMC Syst Biol 1:26PubMedCrossRefGoogle Scholar
  53. 53.
    Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84PubMedCrossRefGoogle Scholar
  54. 54.
    Feist AM, Palsson BO (2008) The growing scope of applications of genome-scale metabolic reconstructions using Escherichia coli. Nat Biotechnol 26:659–667PubMedCrossRefGoogle Scholar
  55. 55.
    Sassetti CM, Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci USA 100:12989–12994PubMedCrossRefGoogle Scholar
  56. 56.
    Sohaskey CD, Wayne LG (2003) Role of narK2X and narGHJI in hypoxic upregulation of nitrate reduction by Mycobacterium tuberculosis. J Bacteriol 185:7247–7256PubMedCrossRefGoogle Scholar
  57. 57.
    Bordbar A, Lewis NE, Schellenberger J, Palsson BO, Jamshidi N (2010) Insight into human alveolar macrophage and M. tuberculosis interactions via metabolic reconstructions. Mol Syst Biol 6:422PubMedCrossRefGoogle Scholar
  58. 58.
    Weber I, Fritz C, Ruttkowski S, Kreft A, Bange FC (2000) Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Mol Microbiol 35:1017–1025PubMedCrossRefGoogle Scholar
  59. 59.
    Oberhardt MA, Palsson BO, Papin JA (2009) Applications of genome-scale metabolic reconstructions. Mol Syst Biol 5:320PubMedCrossRefGoogle Scholar
  60. 60.
    Covert MW, Knight EM, Reed JL, Herrgard MJ, Palsson BO (2004) Integrating high-throughput and computational data elucidates bacterial networks. Nature 429:92–96PubMedCrossRefGoogle Scholar
  61. 61.
    Cappelli G, Volpe E, Grassi M, Liseo B, Colizzi V, Mariani F (2006) Profiling of Mycobacterium tuberculosis gene expression during human macrophage infection: upregulation of the alternative sigma factor G, a group of transcriptional regulators, and proteins with unknown function. Res Microbiol 157:445–455PubMedCrossRefGoogle Scholar
  62. 62.
    Raju B, Hoshino Y, Belitskaya-Levy I, Dawson R, Ress S, Gold JA, Condos R, Pine R, Brown S, Nolan A et al (2008) Gene expression profiles of bronchoalveolar cells in pulmonary TB. Tuberculosis (Edinb) 88:39–51CrossRefGoogle Scholar
  63. 63.
    Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, Monahan IM, Dolganov G, Efron B, Butcher PD, Nathan C et al (2003) Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med 198:693–704PubMedCrossRefGoogle Scholar
  64. 64.
    Tailleux L, Waddell SJ, Pelizzola M, Mortellaro A, Withers M, Tanne A, Castagnoli PR, Gicquel B, Stoker NG, Butcher PD et al (2008) Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages. PLoS One 3:e1403PubMedCrossRefGoogle Scholar
  65. 65.
    Talaat AM, Lyons R, Howard ST, Johnston SA (2004) The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc Natl Acad Sci USA 101:4602–4607PubMedCrossRefGoogle Scholar
  66. 66.
    Salmon K, Hung SP, Mekjian K, Baldi P, Hatfield GW, Gunsalus RP (2003) Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR. J Biol Chem 278:29837–29855PubMedCrossRefGoogle Scholar
  67. 67.
    Kacser H, Burns JA (1995) The control of flux. Biochem Soc Trans 23:341–366PubMedGoogle Scholar
  68. 68.
    Heinrich R, Rapoport TA (1974) A linear steady-state treatment of enzymatic chains. Critique of the crossover theorem and a general procedure to identify interaction sites with an effector. Eur J Biochem 42:97–105PubMedCrossRefGoogle Scholar
  69. 69.
    Patil KR, Nielsen J (2005) Uncovering transcriptional regulation of metabolism by using metabolic network topology. Proc Natl Acad Sci USA 102:2685–2689PubMedCrossRefGoogle Scholar
  70. 70.
    Shlomi T, Cabili MN, Herrgard MJ, Palsson BO, Ruppin E (2008) Network-based prediction of human tissue-specific metabolism. Nat Biotechnol 26:1003–1010PubMedCrossRefGoogle Scholar
  71. 71.
    Colijn C, Brandes A, Zucker J, Lun DS, Weiner B, Farhat MR, Cheng TY, Moody DB, Murray M, Galagan JE (2009) Interpreting expression data with metabolic flux models: predicting Mycobacterium tuberculosis mycolic acid production. PLoS Comput Biol 5:e1000489PubMedCrossRefGoogle Scholar
  72. 72.
    Boshoff HI, Myers TG, Copp BR, McNeil MR, Wilson MA, Barry CE III (2004) The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. J Biol Chem 279:40174–40184PubMedCrossRefGoogle Scholar
  73. 73.
    Shi L, Sohaskey CD, Pfeiffer C, Datta P, Parks M, McFadden J, North RJ, Gennaro ML (2010) Carbon flux rerouting during Mycobacterium tuberculosis growth arrest. Mol Microbiol 78:1199–1215PubMedCrossRefGoogle Scholar
  74. 74.
    Bonde BK, Beste D, Laing E, Kierzek A, McFadden J (2011) Differential Producibility Analysis (DPA) of transcriptomic data with metabolic networks: deconstructing the metabolic response of M. tuberculosis. PLoS Comput Biol 7:e1002060PubMedCrossRefGoogle Scholar
  75. 75.
    Garton NJ, Waddell SJ, Sherratt AL, Lee SM, Smith RJ, Senner C, Hinds J, Rajakumar K, Adegbola RA, Besra GS et al (2008) Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med 5:e75PubMedCrossRefGoogle Scholar
  76. 76.
    de Carvalho LP, Fischer SM, Marrero J, Nathan C, Ehrt S, Rhee KY (2010) Metabolomics of Mycobacterium tuberculosis reveals compartmentalized co-catabolism of carbon substrates. Chem Biol 17:1122–1131PubMedCrossRefGoogle Scholar
  77. 77.
    Thomas ST, Vanderven BC, Sherman DR, Russell DG, Sampson NS (2011) Pathway profiling in Mycobacterium tuberculosis: elucidation of cholesterol-derived catabolite and enzymes that catalyze its metabolism. J Biol Chem 286:43668–43678PubMedCrossRefGoogle Scholar
  78. 78.
    Marrero J, Rhee KY, Schnappinger D, Pethe K, Ehrt S (2010) Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection. Proc Natl Acad Sci USA 107:9819–9824PubMedCrossRefGoogle Scholar
  79. 79.
    Zamboni N, Fendt SM, Ruhl M, Sauer U (2009) (13)C-based metabolic flux analysis. Nat Protoc 4:878–892PubMedCrossRefGoogle Scholar
  80. 80.
    Munoz-Elias EJ, McKinney JD (2006) Carbon metabolism of intracellular bacteria. Cell Microbiol 8:10–22PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Faculty of Health and Medical SciencesUniversity of SurreyGuildfordUK

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