GROWTH OF FRANCISELLA-TULARENSIS LVS IN MACROPHAGES - THE ACIDIC INTRACELLULAR COMPARTMENT PROVIDES ESSENTIAL IRON REQUIRED FOR GROWTH

Murine macrophages supported exponential intracellular growth of Franc isella tularensis LVS in vitro with a doubling time of 4 to 6 h, LVS w as internalized and remained in a vacuolar compartment throughout its growth cycle, The importance of endosome acidification to intracellula r growth of this bacterium was assessed by treatment of LVS-infected m acrophages with several different lysosomotropic agents (chloroquine, NH4Cl, and ouabain), Regardless of the agent used or its mechanism of action, macrophages treated with agents that blocked endosome acidific ation no longer supported replication of LVS, Over several experiments for each lysosomotropic agent, the number of CFU of LVS recovered fro m treated macrophage cultures was equivalent to the input inoculum (ap proximately 10(4) CFU) at 72 h, In contrast, over 10(8) CFU was consis tently recovered from untreated cultures, Pretreatment of macrophages with these endosome acidification inhibitors did not alter their inges tion of bacteria. Further, the effects of the inhibitors were complete ly reversible: inhibitor-pretreated LVS-infected macrophages washed fr ee of the agent and cultured in medium fully supported LVS growth over 72 h, Endosome acidification is an important cellular event essential for release of iron from transferrin, The growth-inhibitory effects o f both chloroquine and NH4Cl were completely reversed by addition of f erric PPi, a transferrin-independent iron source, at a neutral pH but not by addition of excess holotransferrin, Thus, intracellular localiz ation in an acidic vesicle which facilitates the availability of iron essential for Francisella growth is a survival tactic of this bacteriu m, and iron depletion is one mechanism that macrophages use to inhibit its growth.