{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:52:15Z","timestamp":1785430335869,"version":"3.56.0"},"posted":{"date-parts":[[2026,3,4]]},"group-title":"Molecular Biology","reference-count":74,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T00:00:00Z","timestamp":1772582400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R01AI151065"],"award-info":[{"award-number":["R01AI151065"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Obligatory intracellular bacterium\n                  <jats:italic>Anaplasma phagocytophilum<\/jats:italic>\n                  (\n                  <jats:italic>Aph<\/jats:italic>\n                  ) causes an emerging infectious disease called human granulocytic anaplasmosis. Within host cells,\n                  <jats:italic>Aph<\/jats:italic>\n                  proliferates in a membrane-bound vacuole (\n                  <jats:italic>Aph<\/jats:italic>\n                  -vacuole), to which all required nutrients must be routed, including essential iron. Here, using a fluorescent labile iron-binding dye, we found that\n                  <jats:italic>Aph<\/jats:italic>\n                  -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\n                  <jats:italic>Aph<\/jats:italic>\n                  -vacuole post-invasion.\n                <\/jats:p>\n                <jats:p>\n                  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\n                  <jats:italic>Aph-<\/jats:italic>\n                  vacuoles. Fpn-GFP (D131V) and Fpn-GFP (D039A) mutants that are deficient in iron binding and transport could still localize to\n                  <jats:italic>Aph-<\/jats:italic>\n                  vacuoles, but significantly reduced\n                  <jats:italic>Aph<\/jats:italic>\n                  growth and labile iron in\n                  <jats:italic>Aph<\/jats:italic>\n                  -vacuoles.\n                  <jats:italic>Aph<\/jats:italic>\n                  infection upregulated host cell expression of the hepcidin mRNA and protein. Furthermore, the gene encoding hepcidin is upregulated by inflammation, and we found\n                  <jats:italic>Aph<\/jats:italic>\n                  induced strong IL-6, IL-1\u03b2, and TNF-\u03b1 mRNA expression in the host cells. Altogether, these results suggest that\n                  <jats:italic>Aph<\/jats:italic>\n                  induces production of proinflammatory cytokines for autocrine or paracrine hepcidin secretion to trigger the Fpn internalization, which can then be diverted to\n                  <jats:italic>Aph<\/jats:italic>\n                  -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.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    Iron is an essential element for both humans and microorganisms, serving as a cofactor in key metabolic processes. Obligatory intracellular bacterium\n                    <jats:italic>Anaplasma phagocytophilum<\/jats:italic>\n                    infects and proliferates within a membrane-bound vacuole (\n                    <jats:italic>Aph-<\/jats:italic>\n                    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\n                    <jats:italic>A. phagocytophilum<\/jats:italic>\n                    -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\n                    <jats:italic>Aph-<\/jats:italic>\n                    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.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.02.709072","type":"posted-content","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T23:10:23Z","timestamp":1772665823000},"source":"Crossref","is-referenced-by-count":0,"title":["<i>Anaplasma phagocytophilum<\/i>\n                  Subversion of Host Hepcidin-Ferroportin Iron Nutritional Immunity"],"prefix":"10.64898","author":[{"given":"Stephen L.","family":"Denton","sequence":"first","affiliation":[{"name":"Department of Veterinary Biosciences, College of Veterinary Medicine, Infectious Diseases Institute, The Ohio State University, Columbus, OH"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9964-5460","authenticated-orcid":false,"given":"Mingqun","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, College of Veterinary Medicine, Infectious Diseases Institute, The Ohio State University, Columbus, OH"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeta","family":"Nemeth","sequence":"additional","affiliation":[{"name":"David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuko","family":"Rikihisa","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, College of Veterinary Medicine, Infectious Diseases Institute, The Ohio State University, Columbus, OH"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026030705400572000_2026.03.02.709072v1.1","doi-asserted-by":"publisher","DOI":"10.15585\/mmwr.rr6501e1"},{"key":"2026030705400572000_2026.03.02.709072v1.2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM199601253340401"},{"key":"2026030705400572000_2026.03.02.709072v1.3","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2016.2884"},{"key":"2026030705400572000_2026.03.02.709072v1.4","doi-asserted-by":"crossref","first-page":"e0012377","DOI":"10.1371\/journal.pntd.0012377","article-title":"Human monocytotropic ehrlichiosis-A systematic review and analysis of the literature","volume":"18","year":"2024","journal-title":"PLoS Negl Trop Dis"},{"key":"2026030705400572000_2026.03.02.709072v1.5","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.32.3.589-595.1994"},{"key":"2026030705400572000_2026.03.02.709072v1.6","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM198704023161406"},{"key":"2026030705400572000_2026.03.02.709072v1.7","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.29.12.2741-2745.1991"},{"key":"2026030705400572000_2026.03.02.709072v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2013.04.010"},{"key":"2026030705400572000_2026.03.02.709072v1.9","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.58.030603.123811"},{"key":"2026030705400572000_2026.03.02.709072v1.10","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2007-12-126854"},{"key":"2026030705400572000_2026.03.02.709072v1.11","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1000949"},{"key":"2026030705400572000_2026.03.02.709072v1.12","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.med.49.1.275"},{"key":"2026030705400572000_2026.03.02.709072v1.13","doi-asserted-by":"publisher","DOI":"10.1126\/science.1224577"},{"key":"2026030705400572000_2026.03.02.709072v1.14","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.67.3.1368-1378.1999"},{"key":"2026030705400572000_2026.03.02.709072v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2318"},{"key":"2026030705400572000_2026.03.02.709072v1.16","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1016\/j.bbagen.2011.06.003","article-title":"Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH","volume":"1820","year":"2012","journal-title":"Biochim Biophys Acta"},{"key":"2026030705400572000_2026.03.02.709072v1.17","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.1128\/iai.65.4.1455-1461.1997","article-title":"Ehrlichia chaffeensis inclusions are early endosomes which selectively accumulate transferrin receptor","volume":"65","year":"1997","journal-title":"Infect Immun"},{"key":"2026030705400572000_2026.03.02.709072v1.18","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.67.5.2258-2265.1999"},{"key":"2026030705400572000_2026.03.02.709072v1.19","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.62.11.4804-4810.1994"},{"key":"2026030705400572000_2026.03.02.709072v1.20","doi-asserted-by":"crossref","unstructured":"Yan Q , Zhang W , Lin M , Teymournejad O , Budachetri K , Lakritz J , Rikihisa Y . 2021. 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