Studying Copper Nanoparticle-Induced Programmed Cell Death in Bacteria

Summary

Copper nanoparticles act as antimicrobial agents by generating reactive oxygen species. Here, procedures are presented demonstrating that copper nanoparticles are effective against three clinically relevant pathogens and that certain programmed cell death pathways are involved in this bactericidal process.

Abstract

Recently, concerns over multidrug-resistant pathogens and incurable infections have increased due to the overuse and misuse of antibiotics. Nanomaterials, such as metallic and metallic oxide nanoparticles, have gained popularity in the biomedical field as potential new strategies to combat multidrug-resistant pathogens. This study investigated the use of copper nanoparticles (CuNPs) as a bactericide against three common hospital-acquired opportunistic pathogens-Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), and Staphylococcus aureus (S. aureus)-which are increasingly developing drug resistance. Detailed protocols are presented for synthesizing CuNPs of two sizes (20 nm and 60 nm) and evaluating their bactericidal efficacy through colony assays. The mechanisms of antimicrobial action underlying CuNPs were explored by assessing changes in reactive oxygen species production. Additionally, four modulators that inhibit human protein functions were applied to study the potential involvement of programmed cell death (PCD) pathways in bacterial killing. Through this approach, the potential emergence of copper-resistant strains is suggested, building on research into copper homeostasis proteins, including copper-dependent transcriptional regulators. These findings provide a comprehensive methodology for studying the bactericidal effects of CuNPs and their potential role in addressing antibiotic resistance.

Introduction

Drug-resistant bacteria are a serious cause of concern in medicine. Their rapid emergence has reduced the efficacy of conventional antibiotics, resulting in more clinical complications. They pose a major threat to public health and create an urgent need for new antimicrobial agents. One avenue of research is nanomaterials. Nanomaterials possess unique physicochemical properties that allow them to interact with microbes in ways that compromise their viability. For instance, silver nanoparticles (AgNPs) induce oxidative stress in bacteria, resulting in protein dysfunction, membrane disruption, DNA damage, and ultimately cell death¹. Gold nanoparticles (AuNPs), on the other hand, are known for their antifungal properties and can enhance the bactericidal effect of antibiotics by serving as carriers².

Additionally, copper nanoparticles (CuNPs) have also attracted considerable attention due to their potent antimicrobial effect and low production cost. Studies suggest that CuNPs exhibit broad-spectrum bactericidal activity by disruption of enzymatic activity and the generation of reactive oxygen species (ROS)³. The positive charge of CuNPs facilitates their penetration into the bacteria, enhancing their cellular uptake⁴. This mechanism makes CuNPs a promising option for surface coating, such as on implants, to prevent infections³. One interesting finding, however, is that the bactericidal effect of CuNPs appears to be size-dependent. Some studies have found that smaller CuNPs exhibit higher antibacterial activity, probably due to their superior surface area-to-volume ratio⁵.

ROS generation causes widespread damage to cells and bacteria, including lipid peroxidation, protein dysfunction, DNA fragmentation, and inhibition of gluconeogenesis/glycogenolysis, and is involved in necrosis or programmed cell death (PCD)^6,7,8. Recent studies have revealed that PCD systems exist in bacteria, with action modes and effectors similar to those in eukaryotic systems⁹. Bacterial communities can induce PCD in response to stress, including oxidative stress, through a toxin-antitoxin (TA) system¹⁰. In simple terms, the toxin-antitoxin system consists of toxins that can disrupt essential cellular processes and antitoxins that can form stable complexes with the toxins to inhibit their toxicity under normal growth conditions. Most bacteria and archaea contain TA loci in their genomes, often present in multiple copies of extrachromosomal and chromosomal DNA. There are several types of TA systems, with type II TA (known as MazE/MazF module) being of particular interest. Under stress conditions, antitoxins are degraded, allowing toxins to inhibit their cellular targets. In E. coli and S. aureus, the toxin MazF is activated in response to stress conditions such as oxidative stress, high temperature, and amino acid starvation. Consequently, the expression of the antitoxin MazE is reduced, releasing the toxin MazF¹⁰. Studies have found that MazF enables the synthesis of proteins that allow a small sub-population to survive under adverse conditions, while most of the population undergoes mazEF-mediated cell death. This cell death can be either ROS-dependent, where ROS induces transcriptional or translational inhibition, or ROS-independent, where DNA damage triggers the death pathways¹¹.

This study explores the mechanisms by which CuNPs induce bacterial death. Rather than focusing solely on the TA system, four PCD modulators, previously used in our research^7,12, were employed to investigate potential PCD pathways in bacteria.

By examining the bactericidal effects of CuNPs of two different sizes (20 and 60 nm) at varying concentrations, and utilizing methods such as colony assays, ROS detection, and PCD modulators (SBI, Z-VAD, NSA, and Wortmannin), this research highlights that PCD is not exclusive to multicellular organisms but also occurs in bacterial communities under stress. By providing detailed protocols, this work aims to enable researchers to evaluate CuNP efficacy and bactericidal mechanisms in their own systems. Furthermore, these findings advance the understanding of bacterial PCD and support the development of CuNP-based therapies to combat antibiotic-resistant bacteria.

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Preparation of copper nanoparticle

1. Obtain commercial copper nanopowders (25 nm and 60-80 nm) from a commercial source.
2. Use 1.0 mM sodium dodecyl sulfate (SDS) as a dispersant for two sizes of 1 mg/mL nanoparticles.
3. Disperse the nanoparticles using an ultrasonic bath for at least 30 min at room temperature. The fully dispersed nanoparticles are then ready for use in subsequent experiments.

2. Preparation of bacteria

1. Obtain E. coli (Migula) Castellani and Chalmers strain 25922 and A. baumannii Bouvet and Grimont strain from the American Type Culture Collection. Obtain S. aureus from the Bioresource Collection and Research Center.
2. Culture the bacteria in Luria-Bertani (LB) broth under aerobic conditions at 37 °C.
3. Dilute the bacterial cultures in LB medium to an optical density at 600 nm (OD₆₀₀) of approximately 0.5.

3. Cell viability assessment

1. Colony assay
   1. Use stock CuNP solutions (1 mg/mL) to prepare various concentrations of two sizes of CuNPs, including 0 µg/mL, 1 µg/mL, 5 µg/mL, 10 µg/mL, 50 µg/mL, and 100 µg/mL.
   2. Split the bacterial cultures prepared in step 2.3 into microcentrifuge tubes and centrifuge at 3300 × g for 10 min at room temperature.
   3. Retain the bacterial pellets and add different concentrations of two sizes of CuNPs, respectively, with gentle pipetting.
   4. Treat bacterial pellets with PBS and 70% alcohol as the negative and positive controls, respectively.
   5. Incubate all treated bacteria with shaking at 200 rpm at 37 °C for 24 h.
   6. After incubation, wash all treated bacteria with PBS and spread them on LB agar plates. Place the plates in a 37 °C incubator for 24 h.
   7. Count the colony numbers in each treatment group the next day and perform statistical analysis. It is recommended to conduct this in triplicate for statistical accuracy.
2. Bactericidal mechanism study
   1. Prepare bacteria as described in step 3.1.2 and treat them with either 5 µM of SBI-0206965 (SBI) for 2 h, 0.5 µM of necrosulfonamide (NSA) for 1 h, 100 nM of wortmannin (Wort) for 30 min, or 100 nM of Z-VAD-FMK (Z-VAD) for 30 min.
   2. Co-treat the bacteria with different concentrations of two sizes of CuNP solutions, as described in step 3.1.1, in the presence or absence of 5 µM of SBI, 0.5 µM of NSA, 100 nM of Wort, and 100 nM of Z-VAD.
   3. Centrifuge the bacteria after modulator treatments (step 3.2.1) and remove the supernatants.
   4. Resuspend the bacterial pellets in solutions prepared in step 3.2.2 and incubate them with shaking at 200 rpm at 37 °C for 24 h.
   5. Treat the bacteria with 70% ethanol and PBS as the positive and negative controls, respectively. Use a solution without CuNPs as the CuNP blank control (0 µg/mL; mock) under the same inhibitor conditions for each group. Incubate all samples for an additional 24 h.
   6. After incubation, add cell viability reagent to the cultures at a 1:10 volume ratio. Incubate the cultures for another 2 h with shaking at 37 °C.
   7. Centrifuge the cultures (step 3.1.2) after the 2 h incubation. Transfer the fluorescent supernatants to 96-well plates. Measure fluorescence using an excitation wavelength of 560 nm and an emission wavelength of 590 nm with a microplate reader.
   8. Dilute the remaining supernatant to 10^-5 and 10^-4 and spread it onto LB agar plates for culturing.
   9. Count single colonies the following day.

4. Detection of reactive oxygen species

1. Prepare bacterial cultures as described in step 2.3 and split them into microcentrifuge tubes.
2. Treat the bacteria with various stress conditions as ROS-inducing positive control groups (data not shown in the results). The treatments are described in steps 4.2.1-4.2.4.
   1. Expose the bacteria to 405 nm UV light for 3 h. Incubate bacteria at 45 °C for 2 h.
   2. Next, incubate bacteria at 4 °C for 2 h.
   3. Treat bacteria with 3% H₂O₂ for 30 min.
   4. Maintain the bacteria at 37 °C in LB broth as the negative control.
3. Prepare various concentrations of CuNPs as described in step 3.1.1, and treat the bacteria with 20 nm or 60 nm CuNPs at concentrations of 1 µg/mL, 5 µg/mL, 10 µg/mL, and 100 µg/mL for 24 h.
4. Wash the incubated bacteria twice with PBS to remove any remaining nanoparticles.
5. Prepare 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) dye in PBS at a final concentration of 5 µM.
6. Resuspend the bacterial pellets in 5 µM of H₂DCFDA and measure the fluorescence intensity at 520/30 nm emission using a flow cytometer.
   NOTE: The intensity of FL1 green fluorescence correlates with the ROS level in the treated culture. It is recommended to perform this in triplicate for statistical accuracy.

Results

Antimicrobial activities of two-size CuNPs in three pathogens
Three opportunistic pathogens (E. coli, S. aureus, and A. baumannii) were used to test the bactericidal activities of CuNPs. The bacteria were treated with 0 µg/mL, 1 µg/mL, 5 µg/mL, 10 µg/mL, 50 µg/mL, and 100 µg/mL of 20 nm or 60 nm CuNPs, and the bactericidal activities were determined using the minimum bactericidal concentration (MBC) derived from colony counts. Our results showed positive bactericidal effect...

Discussion

This study investigated the antimicrobial effects and mechanisms of CuNPs at two sizes and various concentrations against E. coli, S. aureus, and A. baumannii. Using the established protocols, it was observed that CuNP-induced bactericidal effects involve oxidative stress and potential PCD activation. However, the interplay between metal homeostasis and bacterial stress responses remains largely unexplored. Previous studies have identified bacterial resistance strategies, such as copper efflux ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acinetobacter baumannii Bouvet and Grimont strain	American Type Culture Collection (ATCC), Manassas, VA, USA	17978	Bacteria for CuNP toxocity experiment
Bio-Rad iMark Microplate Reader	Bio-Rad Laboratories, Hercules, CA, USA	168-1130	Used to measure absorbance in bacterial viability assays.
cell-permeant 2’,7’-dichlorodihydrofluorescein diacetate  (H2DCFDA)	Sigma-Aldrich, Saint Louis, MO, USA	D6883	Used for detecting reactive oxygen species (ROS) in treated bacterial cells.
Copper nanoparticles (CuNPs) 25 nm	Sigma-Aldrich, St. Louis, MO, USA	774081	Used to prepare CuNP stock solution
Copper nanoparticles (CuNPs) 60-80 nm	Sigma-Aldrich, St. Louis, MO, USA	774103	Used to prepare CuNP stock solution
Escherichia coli (Migula) Castellani and Chalmers	American Type Culture Collection (ATCC), Manassas, VA, USA	25922	Bacteria for CuNP toxocity experiment
Gallios flow cytometer	Beckman Coulter, Brea, CA, USA		Used for flow cytometric analysis in multiple experiments, including reactive oxygen species detection.
LB agar	FocusBio, Miaoli, Taiwan	LBA500	Used for culturing bacteria
Luria-Bertani (LB) broth	Becton, Dickinson and Company, Sparks, MD, USA	244620	Used for culturing bacteria
Necrosulfonamide (NSA)	Sigma-Aldrich, St. Louis, MO, USA	480073	Used as a modulator for pretreatment in bacterial death pathway studies.
PrestoBlue Cell Viability Reagent	Invitrogen, Carlsbad, CA, USA	P50200	Used for assessing cell viability via fluorescence.
SBI-0206965 (SBI)	BioVision, Milpitas, CA, USA	9580	Used as a modulator for pretreatment in bacterial death pathway studies.
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich, St. Louis, MO, USA	L4509	Used as a dispersant for copper nanoparticles to reduce aggregation.
Staphylococcus aureus	American Type Culture Collection (ATCC), Manassas, VA, USA Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan	13567	Bacteria for CuNP toxocity experiment
Varioskan LUX multimode microplate reader	Thermo Fisher Scientific, Waltham, MA, USA	VLBLATGD2	Used for measuring fluorescence in cell viability assays
Wortmannin (Wort)	Abcam, MA, USA	ab120148	Used as a modulator for pretreatment in bacterial death pathway studies.
Z-VAD-FMK (Z-VAD)	Sigma-Aldrich, St. Louis, MO, USA	V116	Used as a modulator for pretreatment in bacterial death pathway studies.