IroT/MavN is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by In‐vitro Real‐time Transport

Abstract

Iron plays a vital role in all living organisms due to its key role as an enzyme co‐factor, central to diverse metabolic processes. However, because of its high reactivity, speciation, and insolubility in oxidative environments, organisms have evolved sophisticated networks for intercellular iron acquisition without reaching toxic levels. In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, the causative agent of Legionnaires’ disease, a putative transmembrane protein, IroT/MavN, is inserted onto the surface of the host pathogen‐containing vacuole to facilitate intravacuolar iron acquisition from the host, bypassing the problem of Fe(III) insolubility and mobilization.In this work, we have developed a platform for purification and the functional reconstitution of IroT/MavN in artificial lipid bilayer vesicles (proteoliposomes) to identify and characterize the IroT/MavN mediated metal transport properties. By encapsulating the fluorescent reporter probe Fluozin‐3, by real‐time metal transport assays, we revealed that IroT/MavN is a high‐affinity and high‐capacity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains and loops important for substrate recognition and rapid‐kinetic translocation. In addition, by encapsulating the pH indicator pyranine, we demonstrated that Fe(II) translocation is coupled to H+ counter‐transport, suggesting that IroT/MavN is a Fe(II)‐H+ antiporter.This work establishes the substrate transport properties involved in a novel transporter family important for iron acquisition at the host‐pathogen intravacuolar interface and provides chemical tools for comparative investigation of the translocation properties in other iron transporter families.Support or Funding InformationThe work was supported by the Robert A. Welch Foundation (AT‐1935‐20170325 to G.M.) and by the National Institute of General Medical Sciences of the National Institutes of Health (R35GM128704 to G.M.).