A NEW INSIGHT INTO THE MYCOBACTERIAL CELL-ENVELOPE ARCHITECTURE BY THE LOCALIZATION OF ANTIGENS IN ULTRATHIN SECTIONS

In an attempt to have a better insight into the mycobacterial cell envelope architecture, various subcellular fractions of Mycobacterium avium were prepared and characterized chemically and ultrastructurally. The various fractions corresponding to the mycobacterial "capsular material", outer layer, cell wall skeleton, cytoplasmic membrane, and cytosol as well as intact bacteria were then used to raise antisera in rabbits. The antisera so raised were then used to immunolabel the intact bacteria prior to embedding in epon. In parallel studies, bacteria were processed by a novel gelatin-uranyl acetate-low temperature Lowicryl HM20 embedding which preserved mycobacterial antigens, permitting to immunolabel antigens on ultrathin sections. Immunolabelling of epon-embedded intact bacteria showed that in the tripartite structure of the bacterial cell envelope, the middle electron-transparent layer acted as a barrier, not permitting the antibodies to penetrate into deeper structures. Immunolabelling of ultrathin sections showed that mycobacteria were surrounded by a "capsule" containing specific surface antigens with a glycocalyx-like topography, and that the intermediate electron transparent layer which separated the surface amphiphils from the inner arabinogalactan-peptidoglycan layer, was a virtual no man's land as it only seldom contained a single gold particle irrespective of the various antisera used. Furthermore, location of various layers in the cell envelope of M. avium using antisera raised against the subcellular fractions prepared was in agreement with chemical and ultrastructural data. A cell envelope model compatible with chemical, ultrastructural and immunolabelling data is proposed and its validity discussed.