| Popis: |
Nanosilver (NAg) is currently one of the most commercialized antimicrobial nanoparticles although its mode of actions on bacteria are not fully understood. The unclear origins and mechanisms of NAg toxicity is one of the underlying reasons for the much need and yet, still challenging risks versus benefit assessment of NAg use. Numerous studies have in general recognized cellular oxidative stress as one of the main NAg toxicity paradigms on bacteria. However, only little is known in regard to the bacterial toxicological responses to the NAg-derived components, i.e. the leached soluble Ag and the solid Ag particulates that remain after leaching, as well as on the potential cellular targets of NAg attacks. The current thesis aimed to clarify the toxicity origins of NAg on bacteria, studying the unique toxicity characteristics of the soluble and solid components derived from the nanoparticle. The thesis then aimed to identify the types of intracellular reactive oxygen species (ROS) that were induced by the NAg exposure and finally, investigating at molecular level, the NAg-induced structural changes on bacterial cell envelope components. Our studies found that solid Ag particulates are capable of rapid stimulation of lethal levels of cellular oxidative stress, while the leached Ag+ ions being locked in organic complexes only exerts an apparent ROS-independent sub-lethal toxicity on the model bacteria. Using electron paramagnetic resonance (EPR) analysis, we identified an elevated presence of superoxide radical (O2•) inside the NAg-exposed bacteria. Overwhelming the bacteria innate anti-oxidant defence systems, this is thought to subsequently lead to the generation of the highly destructive hydroxyl radical (OH•), also detected inside the bacteria. A spectroscopy analysis (attenuated total reflectance-fourier transform infrared) then revealed attacks on the bacteria envelope component peptidoglycan. The attack specifically targets the peptide chains and the amino sugar groups most likely causing degradation of peptidoglycan, a rigid polymer envelope structure that are present in both Gram-positive (outermost layer) and Gram-negative bacteria. The study also identified attacks on the phosphate groups of phosphatidylethanolamine, one of the major phospholipid building blocks of cell membrane in both Gram-positive (inner layer) and Gram-negative (outer and inner layers) bacteria. It is apparent that this attack on the hydrophilic phosphate ‘head’ component of the phospholipid causes highly disordered states in the hydrophobic fatty acid ‘tail’ component of the phospholipid and therefore for the cell membrane to lose its amphiphilic characters, possibly leading to membrane leakage. We also detected structural changes in lipopolysaccharide (present in the outer layer of Gram-negative bacteria), as well as in lipoteichoic acid (present in the outer and inner layer of Gram-positive bacteria). However these hydrogen bond- and ligand exchange-associated structural changes do not seem to relate to NAg toxicity. In summary, the work provides greater insights into the multiple origins and targets of NAg toxicity on bacteria. The generated knowledge of the microbiological activity of the NAg-derived components as well as their cellular targets is crucial for the better assessment of NAg impact in the environmental and clinical setting, in particular in the wake of the phenomena of bacterial adaptation to the toxicity. |
