Actinobacillus actinomycetemcomitans, a major pathogen in certain human periodontal diseases, elaborates a potent leukotoxin. This polypeptide kills human neutrophils, monocytes and T lymphocytes and may impair host antibacterial resistance by destroying or perturbing these cells in infected tissues. Results of current studies indicate that the toxin disrupts and permeability barrier of the plasma membrane of susceptible cells. This results in a rapid influx of Ca2+ (and probably other ions) and water into the cells and ultimately leads to osmotic lysis. In the proposed study, we shall more completely define the lytic mechanism of action of the leukotoxin.
In Aim #1, experiments will explore whether toxin-induced membrane disturbances are a consequence of the formation of """"""""foreign"""""""", ion- permeant pores in the plasmalemma or whether the toxin perturbs existing channels or carrier systems in the membrane.
In Aim #2, experiments will delineate whether Ca2+ plays a functional role in the leukocidal reaction. the sustained rise in cytoplasmic free Ca2+ seen during intoxication may be important because high levels of this electrolyte can have deleterious effects on numerous cellular functions. Preliminary data indicate that the leukotoxic reaction is inhibited by omitting Ca2+ from the cell culture medium.
Aim #3 relates to the role of phospholipase A2 in the leukocidal process: the toxin is a powerful activator of this enzyme and we have found that anti-phospholipase drugs inhibit leukotoxicity. Phospholipase A2 hydrolyzes membrane phospholipids and gives rise to products and metabolites with membranolytic properties. Experiments are proposed to study the mechanism and consequences of leukotoxin stimulation of phospholipase A2. Finally in Aim #4, a strategy is presented to identify the specific domain(s) on the toxin responsible for disrupting the plasma membrane and activating phospholipase A2. Recombinant DNA techniques will be employed to express toxin-derived peptides which will be analyzed for biological activities. Fundamental insights into the pathobiology of the Actinobacillus leukotoxin will bring us closer to a molecular definition of its role in human periodontal infections. In addition, detailed studies on the action of the Actinobacillus leukotoxin will contribute to a better understanding of the biology of other membrane-active bacterial cytolysins.
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