DELIVERY OF THE NON-MEMBRANE-PERMEATIVE ANTIBIOTIC GENTAMICIN INTO MAMMALIAN-CELLS BY USING SHIGELLA-FLEXNERI MEMBRANE-VESICLES

We developed a model to test whether non-membrane-permeative therapeut ic agents such as gentamicin could be delivered into mammalian cells b y means of bacterial membrane vesicles. Many gram-negative bacteria bl eb off membrane vesicles (MVs) during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 199 5), which can be entrapped within the MVs. Gentamicin-induced MVs (g-M Ws) were isolated from Shigella flexneri and contained 85 +/- 2 ng of gentamicin per mu g of MV protein. Immunogold electron microscopic lab eling of thin sections with antibodies specific to S. flexneri lipopol ysaccharide (LPS) demonstrated the adherence and subsequent engulfment of MVs by the human Henle 407 intestinal epithelial cell line. Furthe r incubation of g-MVs with S. flexneri-infected Henle cells revealed t hat the g-MVs penetrated throughout the infected cells and reduced the intracellular pathogen by similar to 1.5 log(10) CFU in the first hou r of incubation. Antibiotic was detected in the cytoplasms of host cel ls, indicating the intracellular placement of the drug following the p enetration of g-MVs. Soluble antibiotic, added as a fluid to the tissu e culture growth medium, had no effect on intracellular bacterial grow th, confirming the impermeability of the cell membranes of the tissue to gentamicin. Western blot analysis of MVs with S. flexneri Ipa-speci fic antibodies demonstrated that the invasion protein antigens IpaB, I paC, and IpaD were present in MVs. Being bilayered, with outer faces c omposed of LPS and Ipa proteins, these MVs were readily engulfed by th e otherwise impermeable membranes and eventually liberated their conte nts into the cytoplasmic substance of the host tissue.