Disposition of exposed antigens on the faces of isolated Mycoplasma gallisepticum membranes

O Vinther, E A Freundt
1980 Journal of Bacteriology  
The transverse disposition of exposed protein antigens on the two faces of isolated Mycoplasma gallisepticum membranes have been investigated by using indirect immunoferritin labeling to accomplish visualization of the antigens at the ultrastructural level. Comparison between the labeling patterns obtained with unabsorbed specific mycoplasma antiserum and antiserum from which antibodies directed against outer side determinants had been removed revealed that the majority of protein antigens were
more » ... the same on the opposed membrane faces or at least displayed extensive interside cross-reactivity. The relatively scarce tagging of isolated Acholeplasma laidlawii membranes, contrary to membranes on intact organisms observed in this investigation, precluded conclusions regarding the disposition of membrane antigens of this species. The advantages and limitations of the employed method in disposition studies and the factors influencing the transverse distribution of membrane proteins in mycoplasmas are discussed. An asymmetric transverse distribution of mycoplasma membrane proteins with more proteins facing the cytoplasmic side of the membrane than exposed on the outer face has been inferred from several studies (24, 9, 10) . The membranes used as models in these investigations of membrane architecture have been derived most often from Acholeplasma laidlawii and to some extent from Mycoplasma hominis (2, 3) and Mycoplasma pneumoniae (10). The methods, which have been used to reveal dispositional asymmetry ofthe membrane proteins, have been based on radioactive labeling or proteolytic digestion of exposed proteins or on immunoelectrophoretic assay of detergent-solubilized membranes (9). The predominance of proteins on the cytoplasmic side of the lipid bilayer is illustrated also by the higher number of intramembranous particles on the convex fracture faces than on the concave faces showing up in freeze-etching experiments (15). In the present paper, the results of an electron microscopic study of the transverse disposition of membrane proteins of Mycoplasma gallisepticum are presented, applying indirect immunoferritin labeling of membrane antigens (18) as a means of visualizing the proteins. M. gallisepticum membranes show a higher density and a greater protein-to-lipid ratio than other mycoplasmas (13, 17), possibly indicating a different protein organization. For comparison, membranes of A. laidlawii were included in the investigation. MATERIALS AND METHODS Organisms. The type strains of M. gallisepticum PG 31 and of A. laidlawii PG 8 were used. Culture conditions. The organisms were grown in equal amounts of liquid B medium (8) for the production of membranes and for the preparation of immunization antigens and antiserum absorption antigens. Cells were harvested at the late exponential growth phase in yields of 107 to 108 colony-forming units per ml. Growth on solid B medium agar took place as previously described (18) . Preparation of membranes. Membranes of the two organisms were isolated by the methods of Rottem et al. (17) , i.e., by osmotic lysis of a washed cell sediment of A. laidlawii in distilled water and by osmotic lysis of glycerol-loaded M. gallisepticum organisms. The collected membranes were washed in deionized water. Membranes were suspended in dilute fl-buffer (17) at a concentration of 0.25 mg/ml of protein (Lowry) and stored at -70°C until used. Indirect immunoferritin labeling. Indirect immunological labeling of the mycoplasma membranes was carried out by a previously published procedure (18). Membranes were fixed with 0.3% glutaraldehyde for 1 h at room temperature before agar embedding and reaction with the first-step antibody, which was specific rabbit-antiserum diluted 1:100 (1:200 in some experiments) or the same antiserum absorbed with a preparation of intact cells. Control labelings in which preimmune rabbit serum or buffer was substituted for hyperimmune serum were always carried out in parallel. The ferritin label consisted of ferritin-conjugated gamma globulin fraction of goat anti-rabbit immunoglobulin G (IgG) (Miles-Yeda, Ltd, Israel) and was utilized in the dilution 1:60. Ferritin labeling of agargrown mycoplasma colonies prefixed with 0.3% glutaraldehyde also proceeded as previously described. Electron microscopy. Ultrathin sections of Vestopal-W-embedded mycoplasma cells or membranes, fixed with 3% glutaraldehyde and 1% OS04 fixatives after tagging with ferritin (18), were examined in a JEOL JEM 100B electron microscope either unstained or after section staining with lead citrate alone or lead 683 on May 9, 2020 by guest
doi:10.1128/jb.142.2.683-688.1980 fatcat:ifxxnvwkfvbcndwvbgk6tj4cr4