Immunological studies on staphylococcal alpha toxin and its fragments

by Sawicki, Maria

Abstract (Summary)
The growing prevalence of antibiotic resistant Staphyhcocczs aweus strains, an important comrnunity acquired and nosocornial pathogen for compromised hosts, threatens the effectiveness of current strategies and demonstrates the need for other means of control and prevention. Designing vaccines against bacterial toxins is a challenge - a protective immune response must be induced in the absence of toxic biological activity. Previous development of anti toxic immunity involved indiscriminate chernical or proteolytic inactivation of alpha toxin, a major pathogenicity factor of Stuphy~ococcus aurem In this study we used isoelecaic focusing to isolate the 28 kD naturally occumng fragment of alpha toxin that was the product of auto-digestion by ' staphylococcal proteases. Whiie the fiagment is neither hemolytic nor lethal, it still protects rabbit erythrocytes in vitro fkom alpha toxin. This fragment is immunologically reactive with both monoclonal and polyclonal antibodies to the hemolytic and lethal domains of alpha toxin. Thus the purified fragment can potentially be useful as a protective immunogen in Stuphy~oc0ccu.s aurerrs vaccine studies. Many questions remain unanswered about events occumingbetween membrane receptor binding, oligomerization and pore formation. In young cells alpha toxin binds to Band 3, aggregates into an oligomenc pore and causes leakage of ions from the cell. Old cells with degraded Band 3 exhibit a monomenc pattern of lysis. Anti alpha toxin Monoclonal Antibodies (MAE3s) were used to dissect these events. Al1 of the MABs reacted with the toxin on ELISA and on Western blots and neutralized hemolytic activity on rabbit erythrocytes. One MAB (1G6) failed to block the toxin afier it was bound to the membrane. The kinetic hemolytic assay was adapted to masure the rate of hemolysis inhibition. When we compared the capacity of MABs to neutralize toxin on young and old erythrocytes only one MAB (3C6) that blocked bound toxin on young cells failed to do so on aged cells. As the remaining MABs were equally effective on both populations of erythrocytes they blocked the monomer, while the other MAB blocked the membrane bound oligomer.
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Source Type:Master's Thesis



Date of Publication:01/01/1999

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