The hFbpABC Transporter from Haemophilus influenzae Functions as a Binding-Protein-Dependent ABC Transporter with High Specificity and Affinity for Ferric Iron

AUTOR(ES)

Anderson, Damon S.

FONTE

American Society for Microbiology

RESUMO

Pathogenic Haemophilus influenzae, Neisseria spp. (Neisseria gonorrhoeae and N. meningitidis), Serratia marcescens, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe3+ transport assay, we established an apparent Km = 0.9 μM and Vmax = 1.8 pmol/107cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe3+ and selectivity for trivalent metals, including Ga3+ and Al3+, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli. Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity Kd = 5.2 × 10−4 M−1, ∼14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, ∼35% of wild-type transport, with Km = 1.2 μM and Vmax = 0.5 pmol/107cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe3+) binding and membrane transport.

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