Selective inhibition of NikA mediated Ni(II) import in E. coli by the Indium(III)-EDTA complex.

Abstract

Nickel is a required nutrient for bacteria to produce [NiFe]-hydrogenase and urease enzymes. [NiFe]-hydrogenase catalyzes the reversible conversion of hydrogen into protons and electrons and urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia - both key in bacterial pathogenesis. As such, nickel trafficking and homeostasis are interesting targets for potential antibacterial strategies. In E. coli, NikA binds a Ni(II)-(L-His)2 chelate in the periplasm and delivers this complex to the NikBCDE transporter. Blocking Ni(II) uptake by NikA would prevent the biosynthesis of active [NiFe]-hydrogenase. Fe(III)-EDTA is a potent ligand for NikA, however due to the potential for reduction of Fe(III) to Fe(II) it has limited utility. Using Fe(III)-EDTA as a starting point for inhibitor design, similar stable complexes of Bismuth(III), Lutetium(III) and Indium(III) were investigated. The In(III)-EDTA complex is a potent inhibitor of cellular [NiFe]-hydrogenase activity (IC50 of 600 μM ± 100 μM) while being non-toxic to bacterial growth. The mechanism of In(III)-EDTA hydrogenase inhibition was confirmed by the inhibition of Ni(II)-dependent processing of HycE (hydrogenase-3), which could be rescued with the addition of exogenous nickel. To elucidate the binding affinity of In(III)-EDTA to NikA, isothermal titration calorimetry(ITC) was carried out, revealing stoichiometric 1:1 binding with a Kd of 17.3 µM ± 3.0 µM. Indium concentrations determined by inductively coupled plasma mass spectrometry (ICP-MS) in E. coli cells in the presence or absence of NikA showed no discernable difference further supporting the competitive inhibition of nickel uptake by blocking NikA.