Anaplasma phagocytophilum Subversion of Host Hepcidin-Ferroportin Iron Nutritional Immunity

Abstract

Obligatory intracellular bacterium Anaplasma phagocytophilum (Aph) causes an emerging infectious disease called human granulocytic anaplasmosis. Within host cells, Aph proliferates in a membrane-bound vacuole (Aph-vacuole), to which all required nutrients must be routed, including essential iron. Here, using a fluorescent labile iron-binding dye, we found that Aph-vacuoles are enriched with labile iron. Further, ferroportin (Fpn), the only known iron exporter in the plasma membrane of host cells, was increasingly localized to Aph-vacuole post-invasion. Plasma membrane Fpn is tightly regulated by the hormone hepcidin, which binds to Fpn and triggers its ubiquitination and internalization. We found Fpn-GFP mutants deficient in ubiquitination (domain deletion or Y64H) had reduced or absent localization to Aph-vacuoles. Fpn-GFP (D131V) and Fpn-GFP (D039A) mutants that are deficient in iron binding and transport could still localize to Aph-vacuoles, but significantly reduced Aph growth and labile iron in Aph-vacuoles. Aph infection upregulated host cell expression of the hepcidin mRNA and protein. Furthermore, the gene encoding hepcidin is upregulated by inflammation, and we found Aph induced strong IL-6, IL-1{beta}, and TNF- mRNA expression in the host cells. Altogether, these results suggest that Aph induces production of proinflammatory cytokines for autocrine or paracrine hepcidin secretion to trigger the Fpn internalization, which can then be diverted to Aph-vacuoles in a ubiquitination-dependent manner as a mechanism of iron acquisition for thebacteria. This finding illuminates pathogen manipulation of cellular iron export mechanisms, and subversion of host hepcidin-Fpn iron nutritional immunity against pathogens. IMPORTANCEIron is an essential element for both humans and microorganisms, serving as a cofactor in key metabolic processes. Obligatory intracellular bacterium Anaplasma phagocytophilum infects and proliferates within a membrane-bound vacuole (Aph-vacuoles) of neutrophils and endothelial cells, competitively acquiring intracellular iron from the host. Upon exploration of cytoplasmic labile free iron levels and distribution in the A. phagocytophilum-infected and uninfected host cells, we uncovered the unique ability of this bacterium to enrich intravacuolar iron. Ferroportin is the only cellular iron exporter of the host cells, which is regulated by the hepatic hormone hepcidin. Herein, we investigate the role of ferroportin and endogenous hepcidin locally produced by the host cells for iron enrichment in Aph-vacuoles. Ultimately, this study provides new insights into the novel mechanisms of microbial manipulation of host cells to acquire the essential micronutrient iron and overcoming host nutritional immunity, which may facilitate more effective treatment and prevention.