Protein Folding Stability Changes Across the Proteome Reveal Targets of Cu Toxicity in E. coli

Abstract

The ability of metal ionophores to induce cellular metal hyperaccumulation endows them with potent antimicrobial activity; however, the targets and mechanisms behind these outcomes are not well understood. This work describes the first utilization of proteome-wide measurements of protein folding stability in combination with protein expression level analysis to identify protein targets of copper, thereby providing new insight into ionophore-induced copper toxicity in E. coli. The protein folding stability analysis employed a one-pot protocol for pulse proteolysis (PP) in combination with a semi-tryptic peptide enrichment strategy for proteolysis procedures (STEPP) to generate stability profiles for proteins in cell lysates derived from E. coli exposed to copper with and without two ionophores, the antimicrobial agent pyrithione and its β-lactamase-activated prodrug, PcephPT. As part of this work, the above cell lysates were also subject to protein expression level analysis using conventional quantitative bottom-up proteomic methods. The protein folding stability and expression level profiles generated here enabled the effects of ionophore vs copper to be distinguished and revealed copper-driven stability changes in proteins involved in processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (∼3×) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies.

Membrane Transporters Involved in the Antimicrobial Activities of Pyrithione in Escherichia coli

“…Evidence for Cu’s toxic effects is abundant, and its direct impact on the proteins required for metabolism, respiration, and protein synthesis is steadily emerging, as revealed, for example, by a study in Staphylococcus aureus that found that Cu stress targets proteins involved in central carbon metabolism [ 61 ]. Additionally, a recent proteomics study from one of us used protein folding stability measurements to identify protein hits of pyrithione in combination with Cu [ 33 ]. In that study, we found a number of protein hits involved in glycolysis, the citric acid cycle, and protein biosynthesis that were distinctly driven by the Cu delivered to the cell by pyrithione [ 33 ].…”

“…Additionally, a recent proteomics study from one of us used protein folding stability measurements to identify protein hits of pyrithione in combination with Cu [ 33 ]. In that study, we found a number of protein hits involved in glycolysis, the citric acid cycle, and protein biosynthesis that were distinctly driven by the Cu delivered to the cell by pyrithione [ 33 ]. In the context of the current results identifying the ΔcopA knockout strain as highly sensitive to pyrithione, a picture emerges that pyrithione’s mechanism of action is directly linked to its facilitation of Cu gaining unregulated access to the cytoplasm of E. coli , consistent with its activity in yeast [ 62 ].…”

“…However, the overexpression of membrane transporters is known to be mostly deleterious [ 73 , 74 ]. Pyrithione itself has been shown to induce the overexpression of CopA and CueO [ 33 ]. It could be that excess CopA, as provided genetically in the Aska-CopA strain, might have impaired the homeostatic machinery to such a degree that the exposure to pyrithione only worsened downstream cytotoxicity.…”

“…It is also possible that Cu stress could cause the release of internal Zn stores that become problematic when ZntA is missing. Interestingly, ZntA was one of the strongest over-expression and protein-stability hits from our previous proteomics study [ 33 ]. The analysis of these data suggested that pyrithione, not Cu, drove the increase in protein stability.…”

“…One strategy to combat this mechanism of drug resistance could be to provide a substance that is not itself antibacterial in the absence of any β-lactamase activity but becomes so in its presence. Noting that copper compounds can be significantly antibacterial [ 27 , 28 , 29 , 30 , 31 , 32 , 33 ], an embodiment of this strategy was recently proposed that exploited the ability of β-lactamases to activate a prochelator compound (PcephPT, phenylacetamido-cephem-pyrithione) to selectively release pyrithione (2-mercaptopyridine N-oxide) and thereby facilitate its known copper-dependent cytotoxicity [ 34 , 35 ]. In that work, we found that the combination of Cu(II) and pyrithione was indeed highly bacteriotoxic.…”