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Testing Chemical and Genetic Modulators in Mycobacterium tuberculosis Infected Cells Using Phenotypic Assays

  • Vincent Delorme
  • Ok-Ryul Song
  • Alain Baulard
  • Priscille BrodinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)

Abstract

Mycobacterium tuberculosis is able to colonize host cells, and it is now well admitted that the intracellular stage of the bacteria contributes to tuberculosis pathogenesis as well as to making it a persistent infection. There is still limited understanding on how the tubercle bacillus colonizes the cell and what are the factors impacting on its intracellular persistence. Recent advances in imaging technique allow rapid quantification of biological objects in complex environments. Furthermore, M. tuberculosis is a microorganism that is particularly genetically tractable and that tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of M. tuberculosis expressing fluorescent proteins can be easily quantified by the use of confocal microscopy. Here we describe high-content/high-throughput imaging methods that enable tracking the bacillus inside host settings, taking into account the heterogeneity of colonization.

Key words

Mycobacterium tuberculosis High-content/high-throughput screening Chemogenomics Drug discovery siRNA library Automated confocal microscopy Image-based analysis 

References

  1. 1.
    Soldati T, Neyrolles O (2012) Mycobacteria and the intraphagosomal environment: take it with a pinch of salt(s)! Traffic 13(8):1042–1052CrossRefPubMedGoogle Scholar
  2. 2.
    Christophe T, Jackson M, Jeon HK, Fenistein D, Contreras-Dominguez M, Kim J, Genovesio A, Carralot JP, Ewann F, Kim EH, Lee SY, Kang S, Seo MJ, Park EJ, Skovierova H, Pham H, Riccardi G, Nam JY, Marsollier L, Kempf M, Joly-Guillou ML, Oh T, Shin WK, No Z, Nehrbass U, Brosch R, Cole ST, Brodin P (2009) High content screening identifies decaprenyl-phosphoribose 2′ epimerase as a target for intracellular antimycobacterial inhibitors. PLoS Pathog 5(10):e1000645CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Christophe T, Ewann F, Jeon HK, Cechetto J, Brodin P (2010) High-content imaging of Mycobacterium tuberculosis-infected macrophages: an in vitro model for tuberculosis drug discovery. Future Med Chem 2(8):1283–1293CrossRefPubMedGoogle Scholar
  4. 4.
    Queval CJ, Song OR, Delorme V, Iantomasi R, Veyron-Churlet R, Deboosere N, Landry V, Baulard A, Brodin P (2014) A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening. J Vis Exp 83:e51114Google Scholar
  5. 5.
    Stanley SA, Barczak AK, Silvis MR, Luo SS, Sogi K, Vokes M, Bray MA, Carpenter AE, Moore CB, Siddiqi N, Rubin EJ, Hung DT (2014) Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog 10(2):e1003946CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    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(3):e9823CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Raschke WC, Baird S, Ralph P, Nakoinz I (1978) Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 15(1):261–267, 0092-8674(78)90101-0 [pii]CrossRefPubMedGoogle Scholar
  8. 8.
    Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K (1980) Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 26(2):171–176CrossRefPubMedGoogle Scholar
  9. 9.
    Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51(5):1417–1423PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Vincent Delorme
    • 1
  • Ok-Ryul Song
    • 1
  • Alain Baulard
    • 1
  • Priscille Brodin
    • 1
    Email author
  1. 1.Institut Pasteur de LilleUniversity of Lille Inserm U1019, CNRS UMR8204, Institut Pasteur de LilleLille CedexFrance

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