Intracellular bacteria are responsible for a wide range of chronic and lethal disease in man including tuberculosis and undulant fever (brucellosis), which are caused by Mycobacterium tuberculosis and Brucella abortus, respectively. These bacteria are difficult for the immune system to kill and require lung antibiotic treatment because they hide insight the host's macrophages. For many of these infections, even after disease symptoms have been eliminated, residual live bacteria are often left in macrophage conglomerations known as granulomas. This residual infection is normally harmless, but is responsible for recurrence of infection, which often results in death for immuno-comprised individuals such as those with AIDS. The purpose of the proposed study is to explore a new mechanism for treating intracellular pathogenic diseases by selective lysis of infected macrophages by helical amphipathic peptides. The novel peptides in this work contain large percentages of unnatural alpha, alpha-distributed amino acids, which makes them highly helical even when they are relatively short (10 amino acids). The main hypothesis is that these peptides selectively destroy macrophages infected with intracellular pathogens, releasing the pathogens from their protective niche, thus making them more vulnerable to added antibiotics and the immune response. The mechanism and scope of this novel indirect antimicrobial activity as well as the peptide structural requirements responsible for the observed selectivity will be examined according the following Specific Aims: (1) To probe the mechanism of peptide induced lysis of susceptible macrophage in vitro. Microscopy experiments using labeled bacteria and peptides will determine optimal concentration for selective lysis of infected macrophages and at what concentration peptides bind to and lyse the macrophages. (2) Identification of the most active and selective peptide or peptide mimetic for in vitro and in vivo activity. Starting with the most active peptides currently known the peptide structure and size will be varied to determine what is required for optimal activity and selectivity in lysing of infected macrophages. (3) Optimization of multiple peptide doses and possible synergistic effects of peptides with antibiotics against Brucella abortus and Mycobacterium tuberculosis ex vivo and in vivo. Multiple peptides doses with and without added antibiotics will be studied ex vivo using infected granulomas and in vivo using established mouse model systems.
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