Evidence for an ABC-Type Riboflavin Transporter System in Pathogenic Spirochetes

Abstract

Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B2). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein’s fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host.

Identification and Characterization of RibN, a Novel Family of Riboflavin Transporters from Rhizobium leguminosarum and Other Proteobacteria

“…Thus, it can be speculated that a number of bacterial riboflavin transporter families remain undiscovered by current screening methods. Recently, using structural and functional characterization of ligand-binding components, Deka et al (35) identified the substrate binding component of the ABC transporter system RfuABCD from spirochetes that has the ability to bind riboflavin, which is highly suggestive of a riboflavin transport function for this system.…”

The TP0796 Lipoprotein of Treponema pallidum Is a Bimetal-dependent FAD Pyrophosphatase with a Potential Role in Flavin Homeostasis

“…However, this mechanism is not likely to be applicable to T. pallidum. Unlike most bacteria, T. pallidum lacks the riboflavin biosynthesis pathway and relies on its uptake from the host via an ABC-type transport system (76). We therefore propose that periplasmic FMN is supplied by the action of TP0796 on exogenously acquired FAD (Fig.…”

Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi

“…Furthermore, purified, recombinant BB0319, TP0298, and TDE0951, the putative substrate binding proteins from the B. burgdorferi, T. pallidum, and T. denticola transport systems, respectively, have been demonstrated to bind riboflavin. These data suggest that genes bb0319 to bb0316 may encode a ligand binding-dependent ABC transport system for riboflavin uptake (29).…”

“…Gene bb0318 is located on the B. burgdorferi chromosome within a predicted operon comprised of genes bb0319 to bb0316 (1,29) and possibly bb0321 and bb0322 (30,31). Genes bb0319 to bb0316 are tightly organized in the genome, with no nucleotides separating the adjacent open reading frames (1).…”

“…The bb0318 gene is located within a putative operon including genes bb0319 to bb0316 (1,29) and possibly bb0321 and bb0322 (30,31). Genes bb0319 to bb0316, but not bb0321 and bb0322, are conserved in Treponema pallidum and Treponema denticola, and the homologs have been shown to be cotranscribed in T. pallidum (29). Furthermore, purified, recombinant BB0319, TP0298, and TDE0951, the putative substrate binding proteins from the B. burgdorferi, T. pallidum, and T. denticola transport systems, respectively, have been demonstrated to bind riboflavin.…”