Toxicity and assimilation of cellulosic copper nanoparticles require α-arrestins in S. cerevisiae.

The increased use of antimicrobial compounds such as copper into nanoparticles changes how living cells interact with these novel materials. The increased use of antimicrobial nanomaterials combats infectious disease and food spoilage. Fungal infections are particularly difficult to treat because of the few druggable targets, and Saccharomyces cerevisiae provides an insightful model organism to test these new materials. However, because of the novel characteristics of these materials, it is unclear how these materials interact with living cells and if resistance to copper-based nanomaterials could occur. Copper nanoparticles built on carboxymethylcellulose microfibril strands with copper (CMC-Cu) are a promising nanomaterial when imported into yeast cells and induce cell death. The α-arrestins are cargo adaptors that select which molecules are imported into eukaryotic cells. We screened α-arrestins mutants and identified Aly2, Rim8, and Rog3 α-arrestins, which are necessary for the internalization of CMC-Cu nanoparticles. Internal reactive oxygen species in these mutants were lower and corresponded to the increased viability in the presence of CMC-Cu. Using lattice light-sheet microscopy on live cells, we determined that CMC-Cu were imported into yeast within 30 min of exposure. Initially, the cytoplasmic pH decreased but returned to basal level 90 min later. However, there was heterogeneity in response to CMC-Cu exposure, which could be due to the heterogeneity of the particles or differences in the metabolic states within the population. When yeast were exposed to sublethal concentrations of CMC-Cu no resistance occurred. Internalization of CMC-Cu increases the potency of these antimicrobial nanomaterials and is likely key to preventing fungi from evolving resistance.

Metallomic and lipidomic analysis of S. cerevisiae response to cellulosic copper nanoparticles uncovers drivers of toxicity.

Nanotechnology is a promising new technology, of which antimicrobial metal nanocomposites are predicted to become valuable in medical and food packaging applications. Copper is a redox-active antimicrobial metal that can become increasingly toxic depending on the target biomolecule's donor atom selectivity and the chemical species of copper present. Mass is the traditional measurement of the intrinsic elemental chemistry, but this practice fails to reflect the morphology and surface area reactivity of nanotechnology. The carboxymethyl cellulose copper nanoparticles (CMC-Cu) investigated in this study have unique and undefined toxicity to Saccharomyces cerevisiae that is different from CuSO4. Cellular surface damage was found in scanning electron micrographs upon CMC-Cu exposure. Further investigation into the lipids revealed altered phosphatidylcholine and phosphatidylethanolamine membrane composition, as well as depleted triacylglycerols, suggesting an impact on the Kennedy lipid pathway. High levels of reactive oxygen species were measured which likely played a role in the lipid peroxidation detected with CMC-Cu treatment. Metal homeostasis was affected by CMC-Cu treatment. The copper sensitive yeast strain, YJM789, significantly decreased cellular zinc concentrations while the copper concentrations increased, suggesting a possible ionic mimicry relationship. In contrast to other compounds that generate ROS, no evidence of genotoxicity was found. As commonplace objects become more integrated with nanotechnology, humanity must look forward past traditional measurements of toxicity.

Proteomic and genetic analysis of the response of S. cerevisiae to soluble copper leads to improvement of the antimicrobial function of cellulosic copper nanoparticles.

Copper (Cu) was used in antiquity to prevent waterborne and food diseases because, as a broad-spectrum antimicrobial agent, it generates reactive oxygen species, ROS. New technologies incorporating Cu into low-cost biodegradable nanomaterials built on cellulose, known as cellulosic cupric nanoparticles or c-CuNPs, present novel approaches to deliver Cu in a controlled manner to control microbial growth. We challenged strains of Saccharomyces cerevisiae with soluble Cu and c-CuNPs to evaluate the potential of c-CuNPs as antifungal agents. Cells exposed to c-CuNPs demonstrated greater sensitivity to Cu than cells exposed to soluble Cu, although Cu-resistant strains were more tolerant than Cu-sensitive strains of c-CuNP exposure. At the same level of growth inhibition, 157 µM c-CuNPs led to the same internal Cu levels as did 400 µM CuSO4, offering evidence for alternative mechanisms of toxicity, perhaps through ß-arrestin dependent endocytosis, which was supported by flow cytometry and fluorescence microscopy of c-CuNPs distributed both on the cell surface and within the cytoplasm. Genes responsible for genetic variation in response to copper were mapped to the ZRT2 and the CUP1 loci. Through proteomic analyses, we found that the expression of other zinc (Zn) transporters increased in Cu-tolerant yeast compared to Cu-sensitive strains. Further, the addition of Zn at low levels increased the potency of c-CuNPs to inhibit even the most Cu-tolerant yeast. Through unbiased systems biological approaches, we identified Zn as a critical component of the yeast response to Cu and the addition of Zn increased the potency of the c-CuNPs.