9.A.42 The Ferrous Iron Transporter (IroT/MavN) Family
Legionella pneumophila is a pathogenic bacterium commonly found in water. It can be transmitted to humans via inhalation of contaminated aerosols. Iron is known as a key requirement for the growth of L. pneumophila in the environment and within its hosts.Transcriptomic analyses were performed in standard versus iron restricted conditions, and lpp_2867 proved to be highly induced under iron-restricted conditions (Portier et al. 2015). Mutants lacking this gene were not affected for siderophore synthesis or utilization but were defective for growth on iron-depleted solid media and for ferrous iron uptake. As an 8 TMS membrane protein, an involvement in ferrous iron transport was suggested, and thus, it was named IroT, for iron transporter. iroT mutants are strongly impaired with respect to intracellular growth within their environmental hosts, Acanthamoeba castellanii and human macrophages.
The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system (TC# 3.A.7.9.1) targets the bacterial-derived IroT, also called MavN (more regions allowing vacuolar colocalization N) protein to the surface of the Legionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition (Isaac et al. 2015). The ΔmavN mutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ΔmavN mutant (Isaac et al. 2015). This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron.
As noted above, IroT/MavN facilitates intravacuolar iron acquisition from the host cytoplasm, bypassing the problem of Fe3+ insolubility and mobilization. Abeyrathna et al. 2019 developed a platform for purification and reconstitution of IroT in artificial lipid bilayer vesicles (proteoliposomes). By encapsulating the fluorescent reporter probe Fluozin-3, they found that IroT is a high-affinity ferrous iron transporter with very low transport capabilities for other essential transition metals. Mutational analysis revealed important residues in the transmembrane helices, soluble domains, and loops important for Fe2+ recognition and translocation. An iron:proton antiport mechanism was demonstrated, showing that IroT is a secondary carrier, using the high H+ intravacuolar concentration to drive iron uptake.