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Transport Assays and Permeability in Pathogenic Mycobacteria

  • Marie-Antoinette Lanéelle
  • Mamadou Daffé
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 465)

Abstract

Mycobacteria produce an effective permeability layer that consists of a mycolic acid–containing cell wall. This protection confers a natural resistance to many chemical agents and results in a low permeability toward both hydrophilic and lipophilic agents. The permeability of cells is classically measured using methods that generally need cell suspensions and are hazardous with pathogens (e.g., nutrient and antibiotic uptake). A major problem encountered with mycobacteria is their propensity to form aggregates; the addition of detergent to the cell suspension is not recommended as this disorganizes the cell envelope, rendering it more permeable to antibiotics. To circumvent this problem, growing cells are uniformly labeled with [3H]-uracil, allowing a quantification of the aliquots; then, the uptake of [14C]-labeled probes is followed during the first minutes. To avoid the generation of aerosols associated with the commonly used filtration methods, centrifugation through an oil mixture is the preferred alternative technique for use with Mycobacterium tuberculosis.

Keywords

chenodeoxycholate uptake glycerol uptake Mycobacterium tuberculosis permeability barrier transport assays 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Molecular Mechanisms of Mycobacterial InfectionsInstitut of Pharmacology and Structural Biology, UMR 5089 of the National Centre for Scientific Research (CNRS) and University Paul Sabatier (Toulouse III)Toulouse IIIFrance
  2. 2.Department of ‘Molecular Mechanisms of Mycobacterial Infections’Institut de Pharmacologie et Biologie Structurale du Centre National de la Recherche Scientifique and Université Paul SabatierToulouse cedex 04France

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