91) or Francisella (p = 0.89) between non-transfected and transfected macrophages (Figure 1C and 1D). This suggests that expression of TfR1 does not affect bacterial entry processes. Francisella, however, failed to proliferate in macrophages in which expression of the transferrin receptor was suppressed (Figure 1C; p = 0.005). The amount of Francisella recovered after 24 h most likely represents growth in macrophages which
could not be transfected with siRNA. In contrast, intracellular proliferation of S. typhimurium was not affected by the lack of TfR1 (Figure 1D; p = 0.89). Addition of lactoferrin – chelated iron (Fe content >0.15% w/w, final lactoferrin concentration of 0.01 mg/ml) as external iron source to macrophages with suppressed TfR1 rescued the proliferation of Francisella at intermediate levels (data not shown). Spatial Evofosfamide clinical trial relationship of transferrin receptor and Francisella-containing vacuole Some intracellular pathogens have devised ways to attract transferrin receptors to the intracellular vesicles they reside in . When Salmonella enters
macrophages, it localizes to an early endosome that is characterized by EEA1 Staurosporine mw and recruitment of the transferrin receptor (TfR1). As the Salmonella-containing vacuole matures and acquires markers of late endosomes (Rab7, Rab9), it also loses TfR1 [25, 26]. Francisella differs from Salmonella by escaping early during infection from its endosomal environment. Since little is known about TfR1 in macrophages BIBW2992 manufacturer infected with Francisella, we investigated the role of the transferrin receptor during infection and Phosphatidylinositol diacylglycerol-lyase its relation to the maturation of the Francisella-containing vacuole (FCV). Murine macrophages (RAW264.7) were infected with Francisella LVS that constitutively expressed Gfp. At defined
time intervals, infected cells were fixed and prepared for immunostaining. This demonstrated that early during entry (15 and 30 minutes after infection), there is significant co-localization of FCV and TfR1 (Figure 2A and 2E). As Francisella is trafficking away from the cell membrane during the time course of the infection, the co-localization with TfR1 is lost (Figure 2B and 2E; p = 0.88 for comparison of 15 and 30 minutes timepoints, p = 0.006 for 30 and 45 minute timepoints, and p = 0.61 for 45 and 60 minute timepoints (Student’s t-test). Figure 2 Transferrin receptor TfR1 and Rab5, but not Rab7, co-localize with Francisella. Macrophages (RAW264.7) were infected with Francisella that constitutively expressed green fluorescence protein (Gfp). At defined time intervals of infection, cells were fixed and stained with goat anti-TfR1 (A, B), with rabbit anti-Rab5 (C), or goat anti-Rab7 (D), followed by reaction with goat-anti-rabbit or rabbit-anti-goat IgG conjugated to Alexa594 (red fluorescence). Representative confocal images for thirty minutes of infection from twenty z-stacks acquired at 0.2 μm intervals are shown for each fluorescence channel, which were then merged using Volocity 4.