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Staphylococcal membrance proteins are potent initiators of delayed hypersensitivity following multiple subcutaneous injections of viable organisms. When the membranes are separated by exclusion chromatography they separate into three distinct fractions, one of which was responsible for the elicitation of footpad (FP) reactivity in sensitized mice. The active immunogen was characterized as a glycoprotein having a molecular weight of approximately 15,600 Daltons, with the peptide and carbohydrate moieties linked by covalent bonding. In vitro spleen cell stimulation and macrophage migration inhibition studies revealed that the active FP fraction was also the immunogen involved in these responses. The immunogenic fraction also had mitogenic properties as evidenced by the stimulation of non-sensitized spleen cells. These data characterize a glycoprotein present in Staphylococcus aureus cell membrane which is both immunogenic and mitogenic and is the principal immunogen responsible for the early delayed hypersensitivity response.

The development of delayed hypersensitivity (DH) to Staphylococcus aureus in Swiss mice was evaluated by the footpad (FP) assay. In order to determine which component of the bacteria was responsible for the in vivo immune reactivity, purified Staphylococcal cell wall, cell membrane, protein A, lipoteichoic acid, teichoic acid, as well as lipid-free membrane proteins were isolated. The immune responses of mice receiving one to eight S. aureus injections indicated that the first DH peak, following three injections, was primarily dependent upon protein antigens associated with the bacterial membrane. Increased bacterial injections gave rise to a second DH peak following seven injections which was dependent upon multiple bacterial components including cell wall, protein A, and membrane proteins.

Isolated Staphylococcal cellular components were used to evaluate the in vitro reactivity of lymphocytes from mice with delayed hypersensitivity to Staphylococcus aureus. Macrophage migration inhibition studies showed that splenic lymphocytes from mice sensitized with three injections of S. aureus inhibited macrophage migration when stimulated with S. aureus sonicate antigen (SASA), cell membrane (CM), and purified membrane protein (PMP). Continued injections (seven) resulted in migration inhibition when the sensitized cells were reacted with SASA, CM, PMP, cell wall (CW), and protein A (PA). Lymphocyte stimulation studies following three injections further illustrated the role of membrane proteins in the early phase of mouse reactivity. Splenic lymphocytes were maximally stimulated by SASA, CM, and PMP. Lipoteichoic acid (LTA), teichoic acid (TA), and CW also were stimulatory but to a much lesser degree. Mice receiving seven S. aureus injections had a high basal stimulatory response which overshadowed the responses to the isolated staphylococcal components. All of the staphylococcal components except LTA were mitogenic for splenic B lymphocytes. The mitogenicity was dependent upon the presence of macrophages. Only SASA, CM, and PMP were mitogenic for non-enriched splenic lymphocytes.