Interaction with teichoic acids contributes to highly effective antibacterial activity of graphene oxide on Gram-positive bacteria

https://doi.org/10.1016/j.jhazmat.2021.125333Get rights and content

Highlights

  • GO exhibited higher antibacterial activity to Gram+ bacteria than Gram− bacteria.

  • GO had higher adsorption affinity to teichoic acid than peptidoglycan.

  • Expression of autolysin after GO exposure was suppressed by teichoic acids addition.

  • Adsorption of teichoic acid on GO stimulated cell death by increasing autolysis.

Abstract

Graphene oxide (GO) has high-efficient antibacterial activity to diverse pathogenic bacteria. However, the detailed antibacterial mechanism of GO is not fully clear. Herein the antibacterial properties of GO against model Gram-positive (Gram+) (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Gram−) bacteria (Pseudomonas aeruginosa and Escherichia coli) were compared by plate count method. Results showed that 4 mg/L of GO induced the mortality of Gram+ and Gram− bacteria by > 99% and < 25%, respectively. GO had greater adsorption affinity to teichoic acids, the unique components existing in the cell wall of Gram+ bacteria, mainly via π−π interaction. The adsorption efficiency of teichoic acids was 27 times higher than that of peptidoglycan when they were simultaneously exposed to 100 mg/L GO. The superior adsorption of teichoic acids onto GO increased one order of magnitude of atlA expression, the autolysin related gene. As a result, these accelerated bacterial death by hydrolyzing peptidoglycan in cell walls. Exogenous addition of 50 mg/L teichoic acids could impair 4–5 fold of antibacterial activity of GO against S. aureus. These new findings illuminate the antibacterial mechanism of GO against Gram+ bacteria, which paves the way for the further application of graphene-based materials in water disinfection and pathogen control.

Introduction

Graphene, owing to its unique sp2-bonded 2D atomic monolayer carbons arranged in hexagonal network structure and excellent electronic, mechanical, and thermal properties, facilitates in addressing universal concerns in water security, energy production and biomedicine (Dziewięcka et al., 2020, Lu et al., 2017, Zhao et al., 2014). Graphene oxide (GO), as the most concerned derivative of hydrophilic graphene, has great potentials in environmental applications such as photocatalysis, contaminant removal, membrane-based separation and electrochemical sensing (Huang et al., 2019a, Malina et al., 2020, Mu et al., 2019, Tang et al., 2020, Ye et al., 2020). Meanwhile, its applications in water disinfection and pathogen control are subjects of current interests (Huang et al., 2020, Panda et al., 2018, Zhao et al., 2018). Prior reports have demonstrated that the oxygenated and hydrophilic GO usually had a broad antibacterial spectrum against various kinds of bacteria (e.g. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa)), regardless of its diverse sources and characteristics (Anand et al., 2019, Chen et al., 2014, He et al., 2015, Keshvardoostchokami et al., 2020). Most of researches showed that the antibacterial activities of GO against Gram-positive (Gram+) bacteria (e.g. S. aureus and Streptococcus mutans) were higher than that against Gram-negative (Gram−) bacteria (e.g. P. aeruginosa and Escherichia coli (E. coli)) in in vitro culture experiments (Anand et al., 2019, He et al., 2015, Pulingam et al., 2019). Akhavan and Ghaderi (2010) also showed that GO nanosheets exhibited stronger antibacterial activity against S. aureus than E. coli, with an inactivation efficiency of 74% and 59%, respectively, after 1 h exposure at 37 °C.

A series of mechanisms have been proposed for the antibacterial activity of GO. (1) The physical interaction between GO and bacterial cells, including “wrapping” by GO, cutting into bacterial membrane and photothermal ablation by absorbing infrared light (Dallavalle et al., 2018, He et al., 2015, Perreault et al., 2015); (2) The inductions of reactive oxygen species (ROS) or carbon radical (·C), which further caused lipid peroxidation (Li et al., 2016, Panda et al., 2018, Yao et al., 2020); (3) Interactions between GO and biological macromolecules such as proteins, lipids or DNA (Liu et al., 2011, Qi et al., 2019). The antibacterial ability of GO against different bacteria was divergent, following the order S. aureus Enterococcus faecalis E. coli P. aeruginosa (Pulingam et al., 2019). However, the known mechanisms could not explain why the antibacterial ability of GO against Gram+ bacteria is superior to Gram− ones.

It is well-known that Gram+ and Gram− bacteria are distinct in the cell envelope architectures (Akhavan and Ghaderi, 2010, Pulingam et al., 2019). The cell wall of Gram+ bacteria is mainly composed of peptidoglycan, teichoic acids and some proteins. By contrast, the cell wall of Gram− bacteria contains peptidoglycan, lipopolysaccharide and some protein but not teichoic acid (Caudill et al., 2020). The formation of pores in the bacterial cell wall, initiating osmotic imbalance and cell death, was reported to govern the antibacterial activity of graphene (Pham et al., 2015). A previous study illuminated that the interactions between different bacterial components and antibacterial substance contributed to divergent antibacterial activities, which means that the interaction of antibacterial peptides with teichoic acid rather than peptidoglycan could reduce the antibacterial activity (Malanovic and Lohner, 2016). Pulingam et al. (2019) elucidated that the different morphology changes of bacterial cells after exposure of GO contributed to divergent antibacterial activity of GO, which cell entrapment and membrane disruption were observed for Gram+ and Gram− bacteria upon GO treatments, respectively. A proposed explanation for the greater antibacterial activity of GO towards Gram+ than Gram− bacteria was the differences in components of their cell walls, that is the Gram+ bacteria lacking the outer membrane were demonstrated to be more vulnerable to the cell membrane damage than Gram− bacteria with an outer membrane (Akhavan and Ghaderi, 2010, Vi et al., 2020). However, the systematic molecular-level understanding of GO interactions with different components of cell walls is still limited, which would mislead its ideal applications in environment and biomedicine. In particular, teichoic acids play critical roles in the regulation of cell morphology and division, protecting Gram+ bacteria from autolysis and sustaining cation homeostasis for the cell (Brown et al., 2013, Caudill et al., 2020, Schlag et al., 2010). The quantitative comparisons of interaction affinities between GO and diverse cell walls together with membrane components, especially the unique components of teichoic acid existing in Gram+ bacteria, at molecular level may provide an insight in mechanism elucidation.

In this study, the antibacterial activities of GO against two types of Gram+ bacteria, S. aureus, Staphylococcus epidermidis (S. epidermidis), and two types of Gram− bacteria, P. aeruginosa and E. coli were investigated. Interaction mechanisms between GO and bacterial cell walls were further characterized using scanning electron microscopy (SEM), transmission electron micrographs (TEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) spectra analyses. To better elucidate the underlying antibacterial mechanisms, for the first time, the adsorption affinities of teichoic acid and peptidoglycan to GO were compared in vitro, and the relative expression of autolysin of S. aureus (AtlA) after exposure to GO was explored. The superior antibacterial ability of GO against Gram+ versus Gram− bacteria is hypothesized to be attributed to its higher adsorption affinity for teichoic acid, a distinctive component of Gram+ bacterial cell wall which regulates the activity of autolysin. Our findings would explain the enhanced antibacterial activity of GO against Gram+ bacteria and provide guidance for practical water disinfection and pathogen control applications of graphene-based materials.

Section snippets

Graphene oxide and bacterial strains

GO (~99.8%) was purchased from XFNANO Materials Tech Inc. (Nanjing, China, product no. XF224). TEM (Hitachi H-7650, Japan), AFM (Alpha300R-2018, Germany), FTIR (Nicolet AVA TAR370, USA) and Raman spectroscopy (LabRAM HR Evolution, France) were employed to characterize the morphology and property of GO.

S. aureus (ATCC29213) and S. epidermidis (KB-3) were selected as model Gram+ bacteria; P. aeruginosa (PAO1) and E. coli (DH5α) were chosen as representative Gram− bacteria. S. aureus (ATCC29213),

Characterization of graphene oxide

The TEM, AFM, FTIR and Raman characterizations of GO are exhibited in Fig. S1. The TEM image of GO presented a typical two-dimensional flake with obvious wrinkles (Fig. S1a). AFM image (Fig. S1b) showed that the lateral dimensions of GO ranged from nanometers to micrometers and the thickness of GO was ~ 2.0 nm, indicating multi-layer distribution of GO nanosheets (Yao et al., 2020). Detailed functional groups of GO were explored via the FTIR spectrum. As shown in Fig. S1c, the intense peak at

Conclusion

GO exhibited a higher antibacterial activity on Gram+ than Gram− bacteria, due to its higher adsorption efficiency with teichoic acid, a unique component of Gram+ bacteria cell walls, than peptidoglycan, in which π−π interaction is the main driving force. GO adsorbed teichoic acid preferentially, which promoted the wrap of GO on Gram+ bacterial cell surfaces. Whereas, the adhesion did not occur with Gram− bacteria. The role of teichoic acid in antibacterial mechanisms to Gram+ bacteria was

CRediT authorship contribution statement

Meizhen Wang: Funding acquisition, Resources, Visualization, Writing - review & editing. Zhangqiang Li: Methodology, Formal analysis, Investigation, Software. Yunyun Zhang: Investigation, Formal analysis, Writing - original draft. Yue Li: Investigation. Na Li: Software. Dan Huang: Visualization, Supervision, Writing - review & editing. Baile Xu: Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was funded by the National Science Foundation of China (Grant no. 22076167, 21906145 and 21876155) and National Key R&D Program of China (Grant no. 2018YFE0110500). B. Xu acknowledges the scholarship from China Scholarship Council and Deutscher Akademischer Austauschdienst.

References (65)

  • T. Malina et al.

    The environmental fate of graphene oxide in aquatic environment—complete mitigation of its acute toxicity to planktonic and benthic crustaceans by algae

    J. Hazard. Mater.

    (2020)
  • L. Mu et al.

    Graphene oxide quantum dots stimulate indigenous bacteria to remove oil contamination

    J. Hazard. Mater.

    (2019)
  • T. Pulingam et al.

    Graphene oxide exhibits differential mechanistic action towards Gram-positive and Gram-negative bacteria

    Colloids Surf. B Biointerfaces

    (2019)
  • K. Sahu et al.

    Atomic force microscopic study on morphological alterations induced by photodynamic action of Toluidine Blue O in Staphylococcus aureus and Escherichia coli

    J. Photochem. Photobiol. B Biol.

    (2009)
  • H. Tang et al.

    pH-Dependent adsorption of aromatic compounds on graphene oxide: an experimental, molecular dynamics simulation and density functional theory investigation

    J. Hazard. Mater.

    (2020)
  • Y. Vicente-Martínez et al.

    Graphene oxide and graphene oxide functionalized with silver nanoparticles as adsorbents of phosphates in waters. A comparative study

    Sci. Total Environ.

    (2020)
  • J. Wang et al.

    Adsorption and coadsorption of organic pollutants and a heavy metal by graphene oxide and reduced graphene materials

    Chem. Eng. J.

    (2015)
  • B. Ye et al.

    Graphene oxide enhanced ozonation of 5-chloro-2-methyl-4-isothiazolin-3-one: kinetics, degradation pathway, and toxicity

    J. Hazard. Mater.

    (2020)
  • L. Zarate-Reyes et al.

    Naturally occurring layered-mineral magnesium as a bactericidal against Escherichia coli

    Appl. Clay Sci.

    (2017)
  • L. Zarate-Reyes et al.

    Antibacterial clay against Gram-negative antibiotic resistant bacteria

    J. Hazard. Mater.

    (2018)
  • O. Akhavan et al.

    Toxicity of graphene and graphene oxide nanowalls against bacteria

    ACS Nano

    (2010)
  • A. Anand et al.

    Graphene oxide and carbon dots as broad-spectrum antimicrobial agents-a minireview

    Nanoscale Horiz.

    (2019)
  • R. Biswas et al.

    Proton-binding capacity of Staphylococcus aureus wall teichoic acid and its role in controlling autolysin activity

    PLoS One

    (2012)
  • S. Brown et al.

    Wall teichoic acids of Gram-positive bacteria

    Annu. Rev. Microbiol.

    (2013)
  • K. Bush

    Antimicrobial agents targeting bacterial cell walls and cell membranes

    OIE Rev. Sci. Tech.

    (2012)
  • J. Campbell et al.

    Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus

    ACS Chem. Biol.

    (2011)
  • J. Chen et al.

    A new function of graphene oxide emerges: inactivating phytopathogenic bacterium Xanthomonas oryzae pv. Oryzae

    J. Nanopart. Res.

    (2013)
  • J. Chen et al.

    Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation

    Nanoscale

    (2014)
  • Y. Chong et al.

    Light-enhanced antibacterial activity of graphene oxide, mainly via accelerated electron transfer

    Environ. Sci. Technol.

    (2017)
  • M. Dallavalle et al.

    Functionalization pattern of graphene oxide sheets controls entry or produces lipid turmoil in phospholipid membranes

    ACS Appl. Mater. Interfaces

    (2018)
  • M. Dziewięcka et al.

    Graphene oxide as a new anthropogenic stress factor - multigenerational study at the molecular, cellular, individual and population level of Acheta domesticus

    J. Hazard. Mater.

    (2020)
  • I. Fedtke et al.

    A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity

    Mol. Microbiol.

    (2007)
  • Cited by (33)

    • Chirality-influenced antibacterial behavior of gold nanoclusters

      2024, Colloids and Surfaces A: Physicochemical and Engineering Aspects
    • Time-dependent plastic behavior of bacteria leading to rupture

      2023, Journal of the Mechanical Behavior of Biomedical Materials
    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text