Single confocal planes for merged fluorescence channels are shown. B. RAW264.7 cells were infected with live or formalin-inactivated Francisella (dead) for two and twenty-four hours. Immunoblotting

of solubilized proteins was done with mouse anti-TfR1 and mouse MEK162 mw anti-GAPDH as control. Visualization was by chemiluminescence. C. mRNA levels for TfR1 in RAW264.7 macrophages were determined after 2 or 24 h of infection with Francisella by quantitative light cycler PCR; levels are normalized to GAPDH-mRNA levels. Means of n = 6 experiments +/- 1 standard error of mean (SEM) are shown. Increased level of transferrin receptor in infected cells can increase the labile iron pool An increased TfR1 expression could translate into enhanced

transferrin-mediated delivery of iron into the host cell and increased iron availability for Francisella. For Francisella, this could be accomplished by transferrin directly binding to the bacterial cell surface via a transferrin-binding protein, as has been described for other, mostly extracellular bacteria [20]. Search of the Francisella genome did not reveal any homologue to transferrin-binding proteins ((S.Daefler, unpublished observation). We could also experimentally verify that apo-transferrin and holotransferrin do not bind to Francisella (data not shown). We therefore asked if the increased expression of TfR1 correlates with an increase of iron delivery to the host cell. In most cells, uptake of transferrin-bound iron leads to

fast delivery find more into the cytosolic labile iron pool, which can be operationally defined as the cell chelatable pool that includes Fe2+ and Fe3+ associated with ligands such as organic anions, polypeptides, or surface membrane components [29]. The labile iron pool (LIP) composes the metabolically active ID-8 and regulatory forms of iron [[29, 30], Breuer et al., 2007, Int J Biochem Cell Biol]. A sensitive way to measure the labile iron pool without cell disruption is the use of a membrane permeable fluorescent probe such as calcein. Calcein rapidly forms a complex with iron in a 1:1 stoichiometry. This results in quenching of the green fluorescence of calcein. When cells are loaded so that there is a minor excess of free fluorescent calcein, an increase in the LIP will result in a decrease of the fluorescence signal [31], whereas the total cell-associated LIP can be determined after dequenching of the fluorescence signal with a cell-permeant Fe-chelator [29]. Macrophages were infected with Francisella for two and twenty-four hours or left uninfected as control. After loading with calcein, cells were exposed to holotransferrin as delivery vehicle for iron while the fluorescence signal was measured. In macrophages infected with Francisella, there is a rapid iron uptake as determined by the slope of the fluorescence quenching, which is steeper than in the control Epigenetics inhibitor sample (uninfected cells) (Figure 4A, 4B, and 4D; p = 0.