Copper as an Antibacterial Agent and Disruptor of Protein Stability

Abstract

The emergence of resistance to existing antibiotic drugs necessitates the development of new strategies to treat bacterial infections. Copper (Cu) has been used since ancient times to inhibit bacterial growth and has recently experienced a resurgence in its clinical utility as an antimicrobial coating for surfaces in hospitals. Small molecule chelators that bind Cu have also been shown to have antibacterial activity and are believed to disrupt metal homeostasis within the microbes they kill. Molecules called ionophores shuttle Cu into the cell to poison it. However, the antibacterial modes of action behind Cu and small molecule ionophores are not well understood. In this work, we employ a variety of biological, spectrometric, and proteomic techniques to study how Cu and a small molecule ionophore called pyrithione (PT) kill bacteria. First, we present antibacterial susceptibility assays that demonstrate PT and a β-lactamase-activated prodrug of PT called PcephPT kill bacteria in a Cu-dependent manner. Cu hyperaccumulated in cells that were cotreated with low-micromolar Cu and either PT or PcephPT, demonstrating their activity as metal-shuttling ionophores. Next, proteome-wide protein expression level and stability measurements were used to probe treatment-induced cellular changes after E. coli were exposed to Cu in the absence and presence of PT or PcephPT. The stability-based study identified key protein targets such as the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase and isocitrate dehydrogenase, whose activities were confirmed to be inhibited by PT-induced copper toxicity in enzymatic assays. Finally, the impact of Cu on the proteome was further investigated in a metal-induced protein precipitation experiment. Unlike other divalent first row transition metals, low millimolar Cu induced complete protein precipitation from E. coli lysate. Protein solubility was restored by addition of Cu chelators, showing that Cu-induced protein precipitation is reversible. We then obtained Cu precipitation curves for over 800 proteins and saw that some were more sensitive while others were more tolerant to precipitation by Cu. Finally, we analyzed the data set to better understand what biophysical characteristics of the proteins may contribute to making them sensitive or tolerant to precipitation by Cu.