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Breaking down the wall: fractionation of mycobacteria


Rezwan, M; Lanéelle, M; Sander, P; Daffé, M (2007). Breaking down the wall: fractionation of mycobacteria. Journal of Microbiological Methods, 68(1):32-39.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Microbiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2007
Deposited On:26 Mar 2009 09:44
Last Modified:05 Apr 2016 12:26
Publisher:Elsevier
ISSN:0167-7012
Publisher DOI:10.1016/j.mimet.2006.05.016
PubMed ID:16839634
Permanent URL: http://doi.org/10.5167/uzh-3147

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