Bactericidal mechanisms of nanoparticles and microbial defence strategies

Abstract

Manufactured nanoparticles can be toxic to living organisms. This work aims to study the interaction of nanoparticles with bacteria as a model organism. The first objective was, to determine the mechanistic pathways of nanotoxicity with an emphasis on ions and oxidative stress as two key contributors and the second objective, was to investigate what mechanisms bacteria have developed as a strategy to protect themselves against nanotoxicity. The thesis further explores the role of environmental variables such as water chemistry, organic matter and other microorganisms, all of which can potentially change speciation of nanoparticles through their transformation into less toxic species. KEIO deletion mutants lacking genes encoding proteins which mediate resistance to oxidative stress and ionic toxicity were screened and found to be sensitive to both ionic silver and silver nanoparticles. A bioreporter to detect silver ions was constructed. This was found not to be induced by silver nanoparticles, yet showed reduced viability; this observation also indicates that besides ionic silver there are other toxicity pathways. E. coli strains capable of mediating resistance to oxidative stress by overexpression of certain proteins and bio reporters that could detect oxidative stress were constructed. The biosensor cells provide some but not too significant signals. Overexpression of proteins like superoxide dismutase and catalase reduces cell growth, hence, cell viability assays do not provide a realistic measure of protective impact, and thus this strategy is not suited to detect the nature of nanotoxicity. The protective role of extracellular polymeric substances (EPS) was studied by developing an engineered strain of E. coli that overproduces the EPS colanic acid, and use of mutant strains of Sinorhizobium meliloti, a free-living N2 fixing bacterium. Nanoparticle exposure studies reveal that overproduction of EPS mitigates silver nanotoxicity. EPS encapsulates the cells and leads to aggregation of nanoparticles, as shown by microscopy and dynamic light scattering. Furthermore, addition of xanthan, an EPS analogue also produces a similar effect. Lastly, x-ray absorption spectroscopy (XAS) of microcosms amended with silver and zinc oxide nanoparticles show rapid transformation of nanoparticles into corresponding oxides and sulphides. The microcosms show a significant presence of dissimilatory sulphate reducing bacteria (DSRB), and display only marginal change in bacterial community composition on exposure to nanoparticles. These findings suggest that nanomaterials will undergo changes in speciation dependent on the sediment chemistry and the metabolic activities of bacteria in the environment. This process will influence the impact of nanoparticles and the outcomes could be quite different from controlled in vitro exposure studies.