title: Breaking down the wall: fractionation of mycobacteria.
abstract: Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.
authors.affiliation: Institut für Medizinische Mikrobiologie, Gloriastrasse 32, Universität Zürich CH-8006 Zürich, Switzerland.
authors.forename: Mandana
authors.initials: M
authors.lastname: Rezwan
authors.forename: Marie-Antoinette
authors.initials: MA
authors.lastname: Lanéelle
authors.forename: Peter
authors.initials: P
authors.lastname: Sander
authors.forename: Mamadou
authors.initials: M
authors.lastname: Daffé
mesh_terms: D002241:Carbohydrates / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods*; D002473:Cell Wall / Q000737:chemistry / Q000201:enzymology; D008563:Membrane Lipids / Q000032:analysis; D009097:Multienzyme Complexes / Q000032:analysis; D009161:Mycobacterium / Q000737:chemistry* / Q000201:enzymology; D009171:Mycolic Acids / Q000032:analysis; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis; D013010:Sonication; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology
chemical_list: D002241:Carbohydrates; D008563:Membrane Lipids; D009097:Multienzyme Complexes; D009171:Mycolic Acids; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases