Growth of Francisella tularensis LVS in macrophages: the acidic intracellular compartment provides essential iron required for growth

Abstract

Murine macrophages supported exponential intracellular growth of Francisella tularensis LVS in vitro with a doubling time of 4 to 6 h. LVS was internalized and remained in a vacuolar compartment throughout its growth cycle. The importance of endosome acidification to intracellular growth of this bacterium was assessed by treatment of LVS-infected macrophages with several different lysosomotropic agents (chloroquine, NH 4 Cl, and ouabain). Regardless of the agent used or its mechanism of action, macrophages treated with agents that blocked endosome acidification no longer supported replication of LVS. Over several experiments for each lysosomotropic agent, the number of CFU of LVS recovered from treated macrophage cultures was equivalent to the input inoculum (approximately 10 4 CFU) at 72 h. In contrast, over 10 8 CFU was consistently recovered from untreated cultures. Pretreatment of macrophages with these endosome acidification inhibitors did not alter their ingestion of bacteria. Further, the effects of the inhibitors were completely reversible: inhibitorpretreated LVS-infected macrophages washed free 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 of both chloroquine and NH 4 Cl were completely reversed by addition of ferric PP i , a transferrin-independent iron source, at a neutral pH but not by addition of excess holotransferrin. Thus, intracellular localization in an acidic vesicle which facilitates the availability of iron essential for Francisella growth is a survival tactic of this bacterium, and iron depletion is one mechanism that macrophages use to inhibit its growth.

Recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century

“…2); and (iv) survival and multiplication in [5,9,10]. There are many arguments suggesting that the low pH environment within phagosomal compartments of the cell is critical for many intracellular pathogens to access cellular iron for growth [11,12]. CQ treatment of different cells, including macrophages, could inhibit the growth of several of these intracellular bacteria by neutralising the phagolysosomal pH (Table 1; Figs.…”

“…palearctica are facultative intracellular bacteria responsible for tularaemia. It has been demonstrated that F. tularensis finds a successful niche for replication in an acidified vacuole where iron is concentrated [12]. Growth of F. tularensis in murine macrophages has been shown to be dramatically inhibited in vitro by CQ in a dose-dependent manner [12].…”

“…It has been demonstrated that F. tularensis finds a successful niche for replication in an acidified vacuole where iron is concentrated [12]. Growth of F. tularensis in murine macrophages has been shown to be dramatically inhibited in vitro by CQ in a dose-dependent manner [12]. Intracellular localisation in an acidic vesicle, which facilitates the availability of iron essential for Francisella growth, is a survival tactic of this bacterium and iron depletion is one mechanism that macrophages use to inhibit its growth [12].…”

Francisella tularensisvaccines

“…Iron acquisition systems also appear to play a key role in promoting the growth of F. tularensis in macrophages (Fortier et al, 1995). Initial studies suggested that acidification of phagosomes was required for the acquisition of iron by F. tularensis (Fortier et al, 1995), but a more recent study has shown that the bacterium blocks phagosome acidification (Clemens et al, 2004). The findings outlined above would indicate that the bacterium must also be able to acquire iron after escape into the cytosol.…”

Uptake of serum-opsonized Francisella tularensis by macrophages can be mediated by class A scavenger receptors

“…F. tularensis is a faculative intracellular pathogen because it can be cultured in cell-free growth medium as well as inside host cells (Ellis et al ., 2002;Elkins et al ., 2003), but there are no data demonstrating extracellular replication during human or animal infection (Conlan and North, 1992;Fortier et al ., 1994). Thus, F. tularensis may be an obligate intracellular pathogen in vivo , where it has been found to survive and replicate within macrophages, among other cell types (Anthony et al ., 1991;Conlan and North, 1992;Fortier et al ., 1995). Humans can be infected by inhaling as few as 10 F. tularensis , making this one of the most infectious pathogens known (Dennis et al ., 2001;Ellis et al ., 2002).…”