Antimicrobial Activity of Graphene Oxide Contributes to Alteration of Key Stress-Related and Membrane Bound Proteins

Introduction: Antibacterial activity of graphene oxide (GO) has been extensively studied, wherein penetration of the bacterial cell membrane and oxidative stress are considered to play a major role in the bactericidal activity of GO. However, the specific mechanism responsible for the antibacterial activity of GO remains largely unknown. Hence, the goal of this study was to explore the mode of action of GO, via an in-depth proteomic analysis of the targeted bacteria. Methods: Staphylococcus aureus was grown in the presence of GO and samples were collected at different growth phases to examine the cell viability and to analyze the changes in protein expression. Antimicrobial efficiency of GO was tested by assessing bacterial viability, live/dead staining and scanning electron microscopy. The intracellular reactive oxygen species (ROS) induced by GO treatment were examined by fluorescence microscopy. Label-free quantitative proteomics analysis was performed to examine the differentially regulated proteins in S. aureus after GO treatment. Results: GO treatment was observed to reduce S. aureus viability, from 50 ± 17% after 4 h, to 93 ± 2% after 24 h. The live/dead staining confirmed this progressive antimicrobial effect of GO. SEM images revealed the wrapping of bacterial cells and their morphological disruption by means of pore formation due to GO insertion. GO treatment was observed to generate intracellular ROS, correlating to the loss of cell viability. The proteomics analysis revealed alteration in the expression of cell membrane, oxidative stress response, general stress response, and virulence-associated proteins in GO-treated bacterial cells. The time-dependent bactericidal activity of GO correlated with a higher number of differentially regulated proteins involved in the above.-mentioned processes. Conclusion: The obtained results suggest that the time-dependent bactericidal effect of GO is attributed to its wrapping/trapping ability, ROS production and due to physical disruption of the cell membrane. Keywords: cell wrapping, membrane disruption, oxidative stress, proteomics