Drug-resistant infectious agents are an increasingly important medical problem. Infectious agents employ a variety of strategies to avoid the effects of antimicrobial therapy. One of these is to seek refuge in the cytoplasm of phagocytes. organisms that employ this strategy (e.eg.Mycobacteria, Legionella pneumophila), cause life threatening infections, especially in immunosuppressed individuals. The intracellular environment aids the survival of these bacteria by protecting them against anti-infective agents, many of which (e.g., penicillin G), are charged water soluble compounds that do not readily penetrate the membranes of animal cells. We have discovered another mechanism by which eucaryotic cells protect microbial pathogens from antiinfective agents. We have shown that mononuclear phagocytes possess membrane transporters that promote the secretion of these substances from their cytoplasm. These transporters recognize a number of anionic organic drugs that are membrane permeant (e.g., fluoroquinolone antibiotics such as Ciprofloxacin). By removing these drugs from the cytoplasm these transporters reduce their efficacy against intracellular bacteria. Since these transporters are found in many types of cells (e.g., fibroblasts, epithelial cells), they probably reduce the efficacy of antibiotics against organisms growing within these cells as well. We have found that Gemfibrozil, a drug used clinically to lower blood lipoproteins in man, is a highly effective inhibitor of organic anion transport in mononuclear phagocytes, and that by increasing intracellular accumulation/retention of fluoroquinolone antibiotics it markedly enhances the efficacy of these drugs against Listeria monocytogenes growing intracellulary in these cells in vitro. The overall goal of the research described in this application is to enhance the efficacy of phagocytic leukocytes in defending the host against microbial pathogens by enhancing the ability of these cells to accumulate and retain antibiotics intracellularly, and thereby to target these anti- infective agents to the intracellular compartments within which pathogens reside. To this end this research has four complementary specific aims. First, to determine whether the organic anion transport pathway we have discovered in mouse macrophages affects the efficacy of therapy with fluoroquinolone and beta lactam antibiotics against facultative intracellular bacterial pathogens such as Listeria monocytogenes, Legionella pneumophila, and mycobacteria in vivo in mice. Second, to determine whether human PMNs, monocytes and macrophages express similar organic anion transporters, and whether inhibitors of these organic anion transporters enhance the efficacy of fluoroquinolone and beta-lactam antibiotics against facultative intracellular bacterial such as Listeria monocytogenes, Legionella pneumophila, and Mycobacteria in vitro. Third, to determine whether inhibitors of monocyte/macrophage organic anion transporters promote the secretion of other anti-infective agents, as well as endogenous substances, from macrophages. Fourth, to molecularly identify and clone the macrophage membrane protein(s) that mediates the secretion of organic anions from these cells.
