The general transition metal (Tro) and Zn2+ (Znu) transporters in Treponema pallidum: Analysis of metal specificities and expression profiles

Abstract

Acquisition of transition metals is central to the struggle between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum encodes a cluster-9 (C9) ABC transporter (troABCD) whose solute-binding protein component (TroA) ligands Zn2+ and Mn 2+ with essentially equal affinities. Bioinformatic analysis revealed that T. pallidum encodes an additional C9 transporter (tp0034-36) orthologous to Zn2+-uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His-rich tract characteristic of C9 Zn 2+-binding proteins. Metal analysis and metal-reconstitution studies demonstrated that ZnuA is a Zn2+-binding protein; parallel studies confirmed that TroA binds Zn2+, Mn2+ and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn2+. Using isothermal titration calorimetry (ITC), we demonstrated that TroA binds Zn2+ and Mn2+ with affinities approximately 100-fold greater than those previously reported. ITC analysis revealed that ZnuA contains multiple Zn2+-binding sites, two of which are high-affinity and presumed to be located within the binding pocket and His-rich loop. Quantitative reverse transcription polymerase chain reaction of tro and znu transcripts combined with immunoblot analysis of TroA and ZnuA confirmed that both transporters are simultaneously expressed in T. pallidum and that TroA is expressed at much greater levels than ZnuA. Collectively, our findings indicate that T. pallidum procures transition metals via the concerted utilization of its general metal (Tro) and Zn2+ (Znu) transporters. Sequestration of periplasmic Zn2+ by ZnuA may free up TroA binding capacity for the importation of Fe and Mn2+. © 2007 The Authors.