Anaplasma phagocytophilum subversion of host hepcidin-ferroportin iron nutritional immunity

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 in live image analysis, we found that Aph -vacuoles are enriched with labile iron. Furthermore, ferroportin (Fpn), the only known iron exporter on the plasma membrane of host cells, increasingly encircled the Aph -vacuole post-invasion in a ring-like pattern. Fpn is regulated by the hormone hepcidin, which binds to Fpn and triggers its ubiquitination and internalization, thereby, inhibits iron availability to blood-borne pathogens. Fpn-GFP mutants deficient in ubiquitination (domain deletion or Y64H) did not encircle Aph- vacuoles. Fpn-GFP (D181V) and Fpn-GFP (D39A) mutants that are deficient in iron binding and transport could encircle Aph- vacuoles, but unlike Fpn-GFP (wild-type) did not increase labile iron in Aph -vacuoles or Aph growth. The gene encoding hepcidin is upregulated by inflammation. We found Aph infection induced the expression of the hepcidin mRNA and protein, and strong IL-6, IL-1β, and TNF-α mRNA expression by host cells. Altogether, these results suggest that Aph induces the production of proinflammatory cytokines for the upregulation of hepcidin which triggers the Fpn endocytosis and encasement of Aph- ­vacuoles in a ubiquitination-dependent manner. Fpn internalization raises host cell cytoplasmic iron and may export iron into the vacuoles to enrich intravacuolar iron for the benefit of bacteria. This finding demonstrates pathogen manipulation of cellular iron export mechanisms and subversion of host hepcidin-Fpn iron nutritional immunity against pathogens. IMPORTANCE Iron 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 host cells, which is regulated by the hepatic hormone hepcidin. Herein, we investigate the role of ferroportin and endogenous hepcidin locally produced by infected 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.