Abstract
Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.
Highlights
Transition metals are essential micronutrients for most organisms, including many bacterial pathogens, and one of the host immune strategies is to starve phagocytosed microorganism from transition metal micronutrients [1,2]
Activity of the purified MntH2 in proteoliposomes was confirmed by a fluorescent assay with calcein-loaded proteoliposomes (LPR 70:1 to 110:1, w/w) using a similar assay as reported for Natural Resistance-Associated Macrophage Protein (Nramp)-type transporters from S. capitis and D. radiodurans [16,17]
Electrophysiology of solid-supported membranes monitors all electrogenic processes that are related to transport activity, which can include substrate binding, conformational change and transport [7]
Summary
Transition metals are essential micronutrients for most organisms, including many bacterial pathogens, and one of the host immune strategies is to starve phagocytosed microorganism from transition metal micronutrients [1,2]. A transition metal transporter expressed in the phagosomal membranes of macrophages, SLC11A1, is thought to reduce the metal bioavailability to pathogens as part of the immune defence system [3,4,5]. This transporter belongs to the Natural Resistance-Associated. Members of this family are proton-coupled transporters of divalent transition metals that do not transport the earth metals Ca(II) and Mg(II). Metal binding by these dyes is often pH dependent, while quenching and metal affinity varies between transition metals. This can limit the type of experiments that can be performed or the experimental conditions that can be investigated
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