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

Abstract

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 to 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-CuNP led to the same internal Cu levels as did 400 CuSO4, offering evidence for alternative mechanisms of toxicity, perhaps through β-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 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-CuNP to inhibit even the most Cu-tolerant yeast. Through unbiased systems biological approaches, we identified Zn as a critical component of yeast response to Cu and the addition of Zn increased potency of the c-CuNPs.

Copper blocks V‐ATPase activity and SNARE complex formation to inhibit yeast vacuole fusion

“…To show copper toxicity in growing Saccharomyces cerevisiae , Sowada et al used millimolar concentrations of CuCl 2 . Finally, a recent proteomic and genetic analysis of the response to soluble copper by yeast used ≥200 μM CuSO 4 to screen their collection of strains . Granted, these are the examples of whole cells, which can process greater levels of copper in comparison to purified vacuoles.…”

“…Finally, a recent proteomic and genetic analysis of the response to soluble copper by yeast used ≥200 μM CuSO 4 to screen their collection of strains. 18 Granted, these are the examples of whole cells, which can process greater levels of copper in comparison to purified vacuoles. That said, we must consider that the vacuole serves as a storage compartment for multiple cations including calcium, zinc, iron and copper.…”

Pick Your Poison: Benzalkonium Chloride and Copper Enable Nanocellulose Derivatives to Form Antimicrobial Properties Against a Spectrum of Microorganisms

“…Both BZK and copper result in the eventual lysis of fungi, but through different mechanisms. Inside yeast, the copper released from CMC-Cu induces reactive oxygen species (ROS) causing free radical damage to biological molecules including protein, DNA, and lipids (Rong-Mullins, et al 2017). BZK is an amphiphilic molecule and interaction with cellular membranes induces membrane disorganization, leakage of intracellular material, and degradation of nucleic acids/proteins leading to cell death.…”

“…BZK is an amphiphilic molecule and interaction with cellular membranes induces membrane disorganization, leakage of intracellular material, and degradation of nucleic acids/proteins leading to cell death. Copper is an important fungicide and through repeated exposure many agricultural yeast strains are copper tolerant (Rong-Mullins, et al 2017). The most common mechanism of copper tolerance is a genomic amplification of the CUP1 locus (Karin et al 1984).…”

“…The difference in synergy between scaffold and biocidal agent suggests that pairing of the scaffold and agent is an important consideration for its intended use as a PVA plastic. Copper generates free radicals through Fenton reactions when it interacts with water and previous work demonstrated the antimicrobial activity of CMC-Cu hybrid suspensions against S. cerevisiae (Rong-Mullins et al 2017). BZK disrupts the cellular membrane's surface charge and bacterial studies have shown that Pseudomonas aeruginosa copes with benzalkonium chlorides (BAC) in three ways; decreasing porin expression related to BAC, increasing efflux pump activity, and stabilizing the cell surface charge (Krishnan et al 2018).…”