Fluorescence-based metalloproteomic strategy for metallodrug-protein interaction : mining protein targets of metallo-antimicrobial drugs

Abstract

Metallodrugs have been clinically utilized to treat various microbial infections for decades. Nevertheless, the mechanism of action of these drugs has not been elucidated clearly. Exploration of the actions of the therapeutics will definitely progress the development of pharmacology. In this thesis, fluorescent-based techniques were applied to examine the interaction of bismuth-based metallodrug, colloidal bismuth subcitrate (CBS) with proteins in Helicobacter pylori, owing to its convenience in providing real-time monitoring of protein localizations and dynamic activities in relatively non-destructive manner.

H. pylori is a pathogenic microbe that inhabits in human gastrointestinal tract. Genetically-encoded fluorescence resonance energy transfer (FRET) sensors, CYHpnl and CYHpnl_1-48, were fabricated to examine the interaction of CBS and a nickel storage protein, Hpnl, in H. pylori. CYHpnl and CYHpnl_1-48 exhibited stronger responses towards Ni(II), Zn(II) and Bi(III) ions in vitro and in Escherichia coli, with comparable binding affinities for Ni(II) and Zn(II) ions (Kd ~ 4.6 and 3.4 μM, respectively) but a much weaker affinity to Bi(III) ions (Kd ~ 115 μM). The FRET assay confirmed the role of Hpnl in sequestering Ni(II) ions in bacteria. Importantly, the FRET study disclosed the possible specific interaction of Hpnl with the antimicrobial drug, CBS.

To monitor the metallodrugs-protein interaction in cells, NTA-AC, a fluorescent ligand with metal-binding ability, was designed. Ni-bound NTA-AC (Ni-NTA-AC) was developed to track intracellular His-tagged proteins. By using the functional domain of Xeroderma pigmentosum group A (XPA122) and the Red Fluorescent Protein (RFP) genetically encoding with a His-tag, it was demonstrated that Ni-NTA-AC precisely illustrated the subcellular localization of the His-tagged proteins in the bacterial and mammalian cells. The excellent membrane permeability but low cytotoxicity of Ni-NTA-AC signified its potential application in cell biology and biomedical science, which was further proved in the latter part of this thesis.

The Bi-bound fluorescent ligand (Bi-NTA-AC) was employed to mine Bi(III)- binding proteins in H. pylori, which identified alkylhydroperoxide reductase subunit C (AhpC) being one of them. Inhibition of AhpC in decomposing the reactive oxidative species (ROS) and in assisting the refolding of denatured protein were found upon treatment with Bi(III) due to the interaction with the functional Cys49 and Cys169 on AhpC and protein aggregation. Ni-NTA-AC (vide supra) indicated that translocation of AhpC was hindered upon Bi(III)- binding, while increase in susceptibility to ROS killing was observed in ahpC gene knocked-out E. coli strain, suggesting that CBS could effectively eradicate microbes by disrupting important peroxiredoxins.

Mining with Bi-NTA-AC also identified the essential pH buffering urease system. Enzymatic assay strongly supported that Bi(III) perturbed the maturation of urease through urease accessory protein UreG rather than by interacting with urease itself. Cys48 and Cys66 were confirmed to coordinate Bi(III) ions, resulting in protein aggregation that affected the GTPase activity of UreG and the subsequent urease maturation process.

Fluorescence-based techniques have been manipulated to explore the actions of Bi-based metallodrugs. This thesis clearly corroborated the Bi-protein interactions, affecting the survival of H. pylori. Disturbance on metal homeostasis, imbalance of cellular redox system and interruption in maturation of crucial enzyme would be resulted, exterminating microbial infections.