Immunoblotting and ligand blotting of envelope structures of Staphylococcus aureus grown under iron insufficiency and in a sub-minimal inhibitory concentration of penicillin G.
Strip immunoblotting with specific, hyperimmune antisera and normal human sera (NHS), in conjunction with lectin and avidin ligand blotting and surface iodination, was used to investigate cell envelope components of phenotypes of Staphylococcus aureus NCTC 6571. Cocci were grown with a relatively slow doubling time (48 min) under iron sufficiency (Fe+) and iron insufficiency (Fe-), and with or without a sub-MIC of penicillin G (pen G), to approximate to in vivo conditions. Fe+ phenotypes demonstrated extra bands with prominent antigens at 48, 52 and 54 kD. Iron depletion or pen G simplified profiles, notably in the mid-range. In this particular S. aureus strain, protein A was ascribed a molecular mass of 35.5 kD and its detection was not affected by iron or pen G conditions. NHS reacted poorly with the Fe+ phenotype, supplying indirect evidence that this phenotype may not be common in vivo. Lectin blotting demonstrated the presence of glycosylated residues. Lectin affinities were not affected by the pen G treatment but in the Fe- phenotypes only, a 30 kD fucose-containing structure was seen. Avidin blotting visualised a major 86 kD binding site in all phenotypes which was not detected in immunoblotting. Whole cell radioiodination revealed that five major proteins of 21, 35.5, 48, 52 and 68 kD were surface-associated but their immunoreactivity depended upon the phenotype and source of sera.
Domingue
P A
PA
Microbiology Research Group, Pharmaceutical Sciences Institute, Aston University, Birmingham, Great Britain.
Lambert
P A
PA
Brown
M R
MR
D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D008297:Male; D010400:Penicillin G / Q000494:pharmacology*; D010641:Phenotype; D011817:Rabbits; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000187:drug effects / Q000254:growth & development / Q000276:immunology / Q000648:ultrastructure
D000942:Antigens, Bacterial; D007106:Immune Sera; D008024:Ligands; D007501:Iron; D010400:Penicillin G