Analysis of copper-induced protein precipitation across the E. coli proteome.

Metal cations have been exploited for their precipitation properties in a wide variety of studies, ranging from differentiating proteins from serum and blood to identifying the protein targets of drugs. Despite widespread recognition of this phenomenon, the mechanisms of metal-induced protein aggregation have not been fully elucidated. Recent studies have suggested that copper's (Cu) ability to induce protein aggregation may be a main contributor to Cu-induced cell death. Here, we provide the first proteome-wide analysis of the relative sensitivities of proteins across the Escherichia coli proteome to Cu-induced aggregation. We utilize a metal-induced protein precipitation (MiPP) methodology that relies on quantitative bottom-up proteomics to define the metal concentration-dependent precipitation properties of proteins on a proteomic scale. Our results establish that Cu far surpasses other metals in promoting protein aggregation and that the protein aggregation is reversible upon metal chelation. The bulk of the Cu bound in the protein aggregates is Cu1+, regardless of the Cu2+ source. Analysis of our MiPP data allows us to investigate underlying biophysical characteristics that determine a protein's sensitivity to Cu-induced aggregation, which is independent of the relative concentration of protein in the lysate. Overall, this analysis provides new insights into the mechanism behind Cu cytotoxicity, as well as metal cation-induced protein aggregation.

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

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 ß-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.

A Cephalosporin Prochelator Inhibits New Delhi Metallo-ß-lactamase 1 without Removing Zinc.

Antibacterial drug resistance is a rapidly growing clinical threat, partially due to expression of ß-lactamase enzymes, which confer resistance to bacteria by hydrolyzing and inactivating ß-lactam antibiotics. The increasing prevalence of metallo-ß-lactamases poses a unique challenge, as currently available ß-lactamase inhibitors target the active site of serine ß-lactamases but are ineffective against the zinc-containing active sites of metallo-ß-lactamases. There is an urgent need for metallo-ß-lactamase inhibitors and antibiotics that circumvent resistance mediated by metallo-ß-lactamases in order to extend the utility of existing ß-lactam antibiotics for treating infection. Here we investigated the antibacterial chelator-releasing prodrug PcephPT (2-((((6R,7R)-2-carboxy-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)thio) pyridine 1-oxide) as an inhibitor of New Delhi metallo-ß-lactamase 1 (NDM-1). PcephPT is an experimental compound that we have previously shown inhibits growth of ß-lactamase-expressing E. coli using a mechanism that is dependent on both copper availability and ß-lactamase expression. Here, we found that PcephPT, in addition to being a copper-dependent antibacterial compound, inhibits hydrolysis activity of purified NDM-1with an IC50 of 7.6 µM without removing zinc from the active site and restores activity of the carbapenem antibiotic meropenem against NDM-1-producing E. coli. This work demonstrates that targeting a metal-binding pharmacophore to ß-lactamase-producing bacteria is a promising strategy for inhibition of both bacterial growth and metallo-ß-lactamases.

Copper Influences the Antibacterial Outcomes of a ß-Lactamase-Activated Prochelator against Drug-Resistant Bacteria.

The unabated rise in bacterial resistance to conventional antibiotics, coupled with collateral damage to normal flora incurred by overuse of broad-spectrum antibiotics, necessitates the development of new antimicrobials targeted against pathogenic organisms. Here, we explore the antibacterial outcomes and mode of action of a prochelator that exploits the production of ß-lactamase enzymes by drug-resistant bacteria to convert a nontoxic compound into a metal-binding antimicrobial agent directly within the microenvironment of pathogenic organisms. Compound PcephPT (phenylacetamido-cephem-pyrithione) contains a cephalosporin core linked to 2-mercaptopyridine N-oxide (pyrithione) via one of its metal-chelating atoms, which minimizes its preactivation interaction with metal ions and its cytotoxicity. Spectroscopic and chromatographic assays indicate that PcephPT releases pyrithione in the presence of ß-lactamase-producing bacteria. The prochelator shows enhanced antibacterial activity against strains expressing ß-lactamases, with bactericidal efficacy improved by the presence of low-micromolar copper in the growth medium. Metal analysis shows that cell-associated copper accumulation by the prochelator is significantly lower than that induced by pyrithione itself, suggesting that the location of pyrithione release influences biological outcomes. Low-micromolar (4-8 µg/mL) minimum inhibitory concentration (MIC) values of PcephPT in ceftriaxone-resistant bacteria compared with median lethal dose (LD50) values greater than 250 µM in mammalian cells suggests favorable selectivity. Further investigation into the mechanisms of prochelators will provide insight for the design of new antibacterial agents that manipulate cellular metallobiology as a strategy against infection.