Role of the hepcidin-ferroportin axis in controlling the iron content of the cytosol and Salmonella-containing vacuoles in infected macrophages

Abstract

Abstract Iron plays a dual-role in bacterial infection: First, it is a critical micronutrient required for the proliferation of infecting bacteria and second, it acts as a cofactor in the generation of bactericidal free radicals. Macrophages provide a major source of serum iron by releasing cytoplasmic iron via the membrane bound iron export protein, Ferroportin (FPN), degradation of which is triggered by hepcidin produced by hepatocytes upon bacterial infection. Salmonella typhimurium is an intracellular pathogen capable of invading macrophages and proliferating in the membranous Salmonella-Containing Vacuole (SCV). In this study, we first demonstrate that FPN is localized on the SCV and plays a role in iron transport into the SCV. To measure iron content in the SCV, a biosensor was constructed by fusing the iron responsive iroB promoter of Salmonella to a mutant GFP with a short half-life (gfpOVA). Using this construct, we estimated the iron levels in macrophages in animals as well as in in vitro cultured macrophages in the presence and absence of hepcidin. In contrast to the generally accepted opinion, the iron level in the SCV in the presence of the iron transporter (FPN) was higher than in its absence (+hepcidin). In general, host defense against pathogens relies on the generation of Reactive Oxygen Species (ROS) in phagocytic cells, especially during the early stage of infection. Thus, we examined for the generation of bactericidal ROS in the SCV using another biosensor composed of the ROS-responsive katG promoter of Salmonella fused to gfpOVA. To our surprise, ROS generation in the SCV was higher in the presence of FPN than in its absence. The relatively high level of iron in the SCV increased the generation of bactericidal ROS, which in turn decreased the number of intra-macrophage Salmonella and extended infected animal survival. Thus, this study reveals the mechanism via which a block in the hepcidin-FPN circuit controls intra-macrophage Salmonella infection.