Abstract
Macrophages fulfill most of their microbicidal duties in their phagosomes following uptake of microbes. However, some microbes, such as Mycobacterium tuberculosis, have evolved mechanisms to subvert the normal maturation process of their phagocytic compartment to limit the hostility of this environment. The experimental analysis of this process and its subsequent impact on bacterial fitness is technically demanding and has required the development of a broad range of readouts to correlate function and outcome. In this chapter we detail two technically divergent platforms to measure the environment within the phagosomal compartment that contains Mtb in the short term, and more long-term readouts of bacterial fitness and Mtb’s reaction to host-derived stresses. The readouts are all fluorescence-based and are adaptable to measurement by a range of platforms, including spectrofluorometry, confocal microscopy, and flow cytometry.
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References
Draper P, Hart PD, Young MR (1979) Effects of anionic inhibitors of phagosome-lysosome fusion in cultured macrophages when the ingested organism is Mycobacterium lepraemurium. Infect Immun 24:558–561
Hart PD, Armstrong JA (1974) Strain virulence and the lysosomal response in macrophages infected with Mycobacterium tuberculosis. Infect Immun 10:742–746
Hart PD, Young MR (1991) Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast. J Exp Med 174:881–889
Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins HL, Fok AK, Allen RD, Gluck SL, Heuser J, Russell DG (1994) Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263:678–681
Pethe K, Swenson DL, Alonso S, Anderson J, Wang C, Russell DG (2004) Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation. Proc Natl Acad Sci U S A 101:13642–13647
Clemens DL, Horwitz MA (1995) Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med 181:257–270
Clemens DL, Horwitz MA (1996) The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin. J Exp Med 184:1349–1355
Russell DG, Dant J, Sturgill-Koszycki S (1996) Mycobacterium avium- and Mycobacterium tuberculosis-containing vacuoles are dynamic, fusion-competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma. J Immunol 156:4764–4773
Sturgill-Koszycki S, Schaible UE, Russell DG (1996) Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J 15:6960–6968
Via LE, Fratti RA, McFalone M, Pagan-Ramos E, Deretic D, Deretic V (1998) Effects of cytokines on mycobacterial phagosome maturation. J Cell Sci 111(Pt 7):897–905
Schaible UE, Sturgill-Koszycki S, Schlesinger PH, Russell DG (1998) Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. J Immunol 160:1290–1296
Abramovitch RB, Rohde KH, Hsu FF, Russell DG (2011) aprABC: a Mycobacterium tuberculosis complex-specific locus that modulates pH-driven adaptation to the macrophage phagosome. Mol Microbiol 80:678–694
Rohde KH, Abramovitch RB, Russell DG (2007) Mycobacterium tuberculosis invasion of macrophages: linking bacterial gene expression to environmental cues. Cell Host Microbe 2:352–364
Rohde KH, Veiga DF, Caldwell S, Balazsi G, Russell DG (2012) Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection. PLoS Pathog 8, e1002769
Tan S, Sukumar N, Abramovitch RB, Parish T, Russell DG (2013) Mycobacterium tuberculosis responds to chloride and pH as synergistic cues to the immune status of its host cell. PLoS Pathog 9, e1003282
Sukumar N, Tan S, Aldridge BB, Russell DG (2014) Exploitation of Mycobacterium tuberculosis reporter strains to probe the impact of vaccination at sites of infection. PLoS Pathog 10, e1004394
Reyes-Lamothe R, Possoz C, Danilova O, Sherratt DJ (2008) Independent positioning and action of Escherichia coli replisomes in live cells. Cell 133:90–102
Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, Wiles S, Robertson BD, Ripoll J, Ward TH, Bancroft GJ, Schaible UE, Parish T (2010) Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. PLoS One 5, e9823
Yates RM, Russell DG (2008) Real-time spectrofluorometric assays for the lumenal environment of the maturing phagosome. Methods Mol Biol 445:311–325
Yates RM, Russell DG (2005) Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4. Immunity 23:409–417
Acknowledgments
S.T. and D.G.R. are supported by the following awards from the National Institutes of Health, USA; AI114952 (S.T.) and AI067027, AI118582, and HL055936 (D.G.R.). R.M.Y. is supported by awards from the Canadian Institutes of Health Research and Natural Sciences and the Engineering Research Council of Canada.
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Tan, S., Yates, R.M., Russell, D.G. (2017). Mycobacterium tuberculosis: Readouts of Bacterial Fitness and the Environment Within the Phagosome. In: Botelho, R. (eds) Phagocytosis and Phagosomes. Methods in Molecular Biology, vol 1519. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6581-6_23
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DOI: https://doi.org/10.1007/978-1-4939-6581-6_23
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