On the selleck inhibitor bacterial side, many operons responsible for iron acquisition and scavenging have been described. However, much less is known how the host cell modulates its iron homeostasis and how pathogens might actively influence such homeostasis. Results Transferrin receptor is required for Francisella intracellular proliferation but not for Salmonella In order to determine if expression of TfR1 is required for proliferation of Francisella and Salmonella inside macrophages, siRNA was used to silence the expression of TfR1 in murine macrophages (RAW264.7). Expression
of the transferrin receptor was suppressed significantly 48 h after transfection with siRNA as measured by fluorescence microscopy and immunoblotting (Figure 1A and 1B). Our transfection efficiency for siRNA was 63% (+/- 7%), which was determined by counting cells, which had taken up siRNA labeled with the red fluorescence dye Alexa Fluor 555 (Figure 1A). Transfected
cells appear to have an almost complete reduction of TfR1 (Figure 1A). Thus, the residual expression of transferrin receptors seen by immunoblot (Figure 1B) is most likely due to non-transfected cells. Figure 1 Francisella , but not Salmonella requires TfR1 for proliferation inside macrophages. A. RAW264.7 macrophages were transfected with siRNA (coupled to Alexa Fluor 555, red fluorescence) specific for TfR1 or as selleck products control with random siRNA (no red fluorescence). After 48 h cells were fixed and processed for immunofluorescence with a mouse anti-TfR1 AMPK activator antibody followed by an Alexa488 conjugated goat-anti-mouse IgG (green fluorescence). Overlay of both fluorescence channels is shown. B. Proteins were solubilized from transfected and infected cells as above, separated on a 9% SDS-PAGE, transferred to Westran membranes, and immunoblotted with antiserum to TfR1. Visualization was by chemiluminescence
C. RAW264.7 macrophages were transfected with TfR1-siRNA or with random siRNA (control). 48 h cells after transfection cells were infected with Francisella for 2 h or 24 h. The number of intracellular bacteria was obtained by plating a lysate of the host cells on chocolate agar plates for colony-forming units (cfus). Means of triplicate experiments +/- 1 Org 27569 standard error of mean are shown. D. RAW264.7 cells were treated as in C and then infected with Salmonella for 2 h or 24 h. The number of intracellular bacteria was determined as in C. Means of triplicate experiments +/- 1 standard error of mean are shown. Macrophages (RAW264.7) transfected with TfR1-siRNA were infected with Francisella tularensis subspecies holarctica vaccine strain (F. tularensis LVS) or wild-type Salmonella typhimurium (ATTC 14208). F. tularensis LVS has been developed from fully virulent type B Francisella strains. It is attenuated in humans, but virulent in a mouse model [24].