We have discovered that alpha1-antitrypsin (alpha1-AT), a major plasma serine proteinase inhibitor, is a potent inhibitor of a group of bacterial hemolytic toxins (hemolysins) by a mechanism that appears to be distinct from its antiprotease function. We propose that alpha1-AT may protect the lung and other tissues from injury by inhibiting toxin- mediated cellular injury during infections, in addition to functioning as an inhibitor of leukocyte elastase. Although alpha1-AT has been extensively characterized by biochemical, molecular, and clinical studies as the major plasma inhibitor of leukocyte elastase, its ability to inhibit non-protease bacterial hemolysins has been completely unexplored. The long term goal of our research is to understand the role of alpha1-AT in host defense against acute lung injury from bacterial infections. The goal of this proposal is to explore the biological significance and biochemical mechanisms of this newly discovered """"""""antihemolysin"""""""" activity of alpha1-AT. Within the scope of this proposal we will focus on the antihemolysin activity of alpha1-AT against one particular toxin, the S. pneumoniae toxin pneumolysin. Specifically, we propose: 1. To establish that alpha1-AT is a biologically significant inhibitor of pneumolysin. a. Is alpha1-AT a major plasma inhibitor of pneumolysin in normal subjects? b. Do patients with various degrees of alpha1-AT deficiency have proportionately reduced plasma antihemolysin activity? c. Is alpha1-AT a major inhibitor of pneumolysin in bronchoalveolar fluid? d. Do other mammalian alpha1-ATs that lack significant antielastase activity have antihemolysin activity? e. Are experimentally alpha1-AT-depleted rats more susceptible to pneumolysin toxicity? 2. To investigate the biochemical mechanisms of the inhibition of pneumolysin by alpha1-AT. a. Does alpha1-AT prevent binding of pneumolysin to cell membranes? b. What is the stoichiometry of pneumolysin binding to alpha1-AT? c. Does the inhibition of pneumolysin by alpha1-AT involve a thiol interaction? d. Is glycosylation required for alpha1-AT antihemolysin activity? Future investigations will extend these observations to the other thiol- activated toxins, study the role that bacterial toxins play in the pathogenesis of emphysema in normal and alpha1-AT deficient patients, and explore the possibility that alpha1-AT augmentation can meliorate pulmonary tissue destruction from bacterial pneumonitis.
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