Our assessment of the labile iron pool after infection with Salmonella after 24 h shows a decrease (Figure 5) and agrees with the findings reported by Nairz [28]. Conclusions Iron acquisition and utilization by microbes is of critical importance for bacterial pathogenesis. Defects in the bacterium’s ability to efficiently scavenge iron and use it in its metabolism usually lead to avirulence.

However, little is known how bacteria might modulate the iron handling properties of their host cells. We identified two distinct iron-handling scenarios for two different bacterial pathogens. Francisella tularensis drives an active iron acquisition program via the TfR1 pathway program with induction of ferrireductase (Steap3), iron membrane transporter Dmt1, and iron regulatory proteins IRP1 and IRP2, which is associated with a sustained increase of the labile iron pool inside the macrophage. OSI-906 research buy Expression of TfR1 is critical for Francisella’s intracellular proliferation. This contrasts with infection of macrophages by wild-type Salmonella typhimurium, which does not require expression of TfR1 for successful intracellular survival. Macrophages infected with Salmonella lack significant

induction of Dmt1, Steap3, and IRP1, and maintain their labile iron this website pool at normal levels. Methods Bacterial strains, cell lines, growth conditions, and plasmids Francisella tularensis subspecies holarctica vaccine strain (F. tularensis LVS, army lot 11) was generously provided to us by Dr. Karen Elkins (FDA). F. tularensis LVS see more was transformed with plasmid pFNLTP6 gro-gfp to produce a Francisella strain constitutively expressing green fluorescent protein (SD833). Wild-type Salmonella strain ATCC 14028 was used. Salmonella mutant strains spiC::kan (EG10128) and spiA::kan (EG5793) are isogenic derivatives

[32]. Francisella was grown on chocolate II agar enriched with IsoVitaleX (BD Biosciences, San Jose, CA) for 40-48 hrs at 37°C. For liquid medium, we used Mueller-Hinton broth supplemented with IsoVitaleX. Salmonella strains and E.coli XL-1 were grown at 37°C with shaking in LB broth without glucose or on LB plates [53]. When indicated antibiotics were present (in μg/ml) at: kanamycin, 50; chloramphenicol, 50; for Francisella, kanamycin was used at 10 μg/ml. RAW264.7 murine macrophages were obtained from ATCC (TIB-71). Dulbecco’s Modification of Eagle’s Medium (DMEM; Cellgro) was supplemented with 10% fetal bovine serum (Hyclone, not heat-inactivated) and penicillin (100 I.U./ml) and streptomycin (100 μg/ml). When cells were used for Francisella infection assays, no antibiotics were added 24 h prior to infection. Cells were grown at 37°C and 5%CO2. A shuttle plasmid which encodes Gfp under the control of the groE promoter (pFNLTP6 gro-gfp) was kindly provided to us by Dr. Zahrt [54]. It carries a kanamycin antibiotic resistance marker. Infection Assay Several colonies of F.