Keys to prevent iron hijacking and infection by intracellular bacteria

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The gram-negative obligatory intracellular bacterium Ehrlichia chaffeensis (Ech) infects monocytes- macrophages, and causes the emerging tick-borne zoonosis human monocytic ehrlichiosis (HME). Our long- term goal is to identify targets for intervention that can prevent and treat HME. As an obligatory intracellular aerobe, Ech is dependent upon host iron for survival. We found that pretreating human macrophages with the iron chelator deferoxamine resulted in a complete block of Ech infection, suggesting Ech acquires iron from the host labile cellular iron (LCI) pool. Paradoxically, the host LCI pool catalyzes formation of reactive oxygen species (ROS), which is a key mechanism of host defense against intracellular pathogens. Our project's objective is to determine the mechanism by which Ech modulates the host LCI pool to acquire iron for its growth, while averting ROS-induced host cell response. By understanding the process by which Ech acquires iron, we may be able to prevent or limit infection. We previously found functional links between Type IV Secretion System (T4SS, VirB/D), iron, and superoxide dismutatses of Ech and host cells. Ferritin contains heavy chain (FTH) and light chain (FTL) subunits; in our preliminary study we found that the T4SS effector, Ehrlichia translocated factor (Etf)-3 interacts directly with FTL, and colocalizes with LC3 (ATG8), a maker of autophagosomes. Thus, o ur central hypothesis is that Ech induces ferritinophagy, a form of selective autophagy that degrades ferritin and increases the LCI pool by secreting Etf-3, and safely captures iron. We will test our central hypothesis with three Specific Aims: Aim 1. Analyze the interaction between Etf-3 and FTL: Etf-3 binding kinetics to human native ferritin; temporal pattern of Etf-3-FTL binding during the course of Ech infection; Etf-3 binding to other molecules in Ech-infected and uninfected cells, including nuclear receptor coactivator 4 (NCOA4); and cellular co-localization of Etf-3 and ferritin. Aim 2. Determine if Ech induces ferritinophagy that coincides with lowering ROS via T4SS, and if Etf-3-induced ferritinophagy is required for productive Ech infection; determine the roles of FTL, FTH, and NCOA4 in Ech infection; compare Etf-3-induced ferritinophagy to Etf-1-induced Rab5-regulated autophagy; determine if NCOA4 mediates Etf-3-induced ferritinophagy; and map the Etf-3 domains/segments that induce ferritinophagy. Aim 3. Determine if blocking Etf-3 expression and binding to FTL inhibits Ech-induced ferritinophagy and Ech infection. Elucidating how intracellular Ech acquires iron will 1) further our understanding of intracellular bacterial proliferation and survival, and 2) reveal the role of iron homeostasis that may be a critical target for development of new approaches to prevent or limit Ech infection. If our hypothesis is supported, the results will also reveal a unique molecular mechanism of ferritinophagy that may be inhibited, benefiting the broader fields of infectious diseases and iron homeostasis.