Abstract:
Colloidal suspensions of gold particles of nanometre (nm) sizes are termed gold nanoparticles (AuNPs). Although stable, AuNPs have been reported to be toxic to E. coli cells by collapsing the bacterial cell membranes and promoting protein misfolding. An understanding of biodistribution in drug delivery and the effects AuNPs have on the function and structure of proteins such as heat shock proteins is important. Heat shock proteins facilitate protein folding and are particularly important during cellular stress. At high concentrations, AuNPs are thought to promote protein aggregation. Heat shock proteins are thought to alleviate cell stress induced by AuNPs. This study explored the role of heat shock proteins in conferring cytoprotection to E. coli against the effects of AuNPs. Citrate-AuNPs were synthesized and their integrity was validated at 520 nm by ultraviolet-visible-near infrared spectroscopy (UV-Vis-NIR). Crystallinity was confirmed by X-Ray diffraction (XRD), while dynamic light scattering (DLS) estimated the size distribution at 13 nm. Furthermore, transmission electron microscopy (TEM) and scanning electron microscope (SEM) revealed the spherical shape and crystal lattice surface morphology of citrate-AuNPs respectively. A complementation assay was conducted using cells deficient of DnaK function (E. coli ΔdnaK52). E. coli ΔdnaK52 was transformed with a recombinant dnaK before examining both DnaK deficient and transformed cells using TEM. E. coli O157:H7 was exposed to citrate-AuNPs (0 – 50 μg/ml) and allowed to grow at 37 oC before protein expression was analysed using electrophoresis followed by LC-MS analysis. This led to the identification of highly expressed proteins such as DnaK, GAPDH, ClpX, DnaJ, and GroEL. Subsequent co-affinity assay revealed possible interaction protein partners of DnaK. These were identified as ClpB, HtpG, GroEL, DnaJ, and SurA proteins. Furthermore, circular dichroism and fluorescence spectroscopy established that recombinant DnaK is stable at citrate-AuNPs concentrations less than 10 μg/ml and the protein was unstable at concentrations beyond 10 μg/ml citrate-AuNPs. In addition, the ATPase activity of recombinant DnaK increased in the presence of citrate-AuNPs at 2.5 μg/ml. The ability of DnaK to suppress aggregation of MDH in vitro was abrogated by the presence of >10 μg/ml citrate-AuNPs. The findings suggest that at low concentrations (<10 μg/ml) citrate-AuNPs seem to stabilize proteins and similarly at elevated concentrations (>10 μg/ml), citrate-AuNPs destabilizes protein conformation and function. Altogether the findings suggest that DnaK in cooperation with its network partners is implicated in E. coli cytoprotection against citrate-AuNPs toxicity.
