Antibacterial Activity of Glutathione-Coated Silver Nanoparticles against Gram Positive and Gram Negative Bacteria

In the present paper, we study the mechanism of antibacterial activity of glutathione (GSH) coated silver nanoparticles (Ag NPs) on model Gram negative and Gram positive bacterial strains. Interference in bacterial cell replication is observed for both cellular strains when exposed to GSH stabilized colloidal silver in solution, and microbicidal activity was studied when GSH coated Ag NPs are (i) dispersed in colloidal suspensions or (ii) grafted on thiol-functionalized glass surfaces. The obtained results confirm that the effect of dispersed GSH capped Ag NPs (GSH Ag NPs) on Escherichia coli is more intense because it can be associated with the penetration of the colloid into the cytoplasm, with the subsequent local interaction of silver with cell components causing damages to the cells. Conversely, for Staphylococcus aureus, since the thick peptidoglycan layer of the cell wall prevents the penetration of the NPs inside the cytoplasm, the antimicrobial effect is limited and seems related to the interaction with the bacterial surfaces. Experiments on GSH Ag NPs grafted on glass allowed us to elucidate more precisely the antibacterial mechanism, showing that the action is reduced because of GSH coating and the limitation of the translational freedom of NPs

Kinetic analyses of hRecProl-WT and its pathological variants.

<p>Michaelis-Menten plots obtained using Gly-Pro as substrate. The graphs of hRecProl-E412 and hRecProl-G448 were magnified in the insets for clarity.</p

Prolidase metal content.

<p>Mn(II) and Zn(II) content was analyzed by ICP-MS after 48 h dialysis against metal free buffer based on their presence in the enzyme active site as previously demonstrated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058792#pone.0058792-Besio2" target="_blank">[4]</a>.</p

Prolidase thermal denaturation and dimerization follow up.

<p>(A) Protein melting temperatures in the presence of the cofactor as revealed by Thermofluor Technology. The solvatochromic dye SYPRO orange was used as an indicator of protein unfolding (fluorescence excitation λ = 492 nm; fluorescence emission λ = 568 nm). (B) The ratio between prolidase activity and protein concentration was plotted against protein concentration.</p

Mutant prolidase forms <i>in silico</i> modeling.

<p>(A) Human prolidase dimer: the two prolidase interfacing monomers are reported as a green cartoon (molecule A) and as a grey surface (molecule B), with Mn(II) shown as dark grey spheres. The magenta color was chosen to identify mutated residues. On molecule A, Mn(II) coordinating residues (D276, D287, E412 and E452) and the phosphate group (orange) are highlighted as sticks. Y231 residues from molecules A and B are located on the C-terminal portion of two α-helices arranged in a symmetric and anti-parallel manner, allowing their interaction with E223 of the interfacing protomer. (B) Destabilization of prolidase core structural elements upon G448R mutation. R448 substituenting residue is reported in spheres to highlight its steric hindrance. The protein portion that buries the mutated residue is shown as a grey surface. A red dashed line indicates the distance between the average position of Mn(II) ions and the C_α of the substituted residue. (C) E412K substitution does not impair ions binding. Solvent molecules occupy a small cavity near the active site, and are represented with red crosses. This small cavity can host the side chain of the mutated K412 (reported in sticks with green carbon atoms and the nitrogen atom in blue) which substitutes E412 (magenta carbon and red oxygen atoms). The putative location of the K412 side chain here proposed may be set by the electrostatic repulsion exerted by residue R450 (shown in sticks with grey carbon atoms).</p

Human prolidase activity and protein expression.

<p>(A) Mutations and clinical phenotype of the PD patients used for the study. (B) Prolidase activity levels, and (C) prolidase expression levels in control (WT) and PD patients fibroblasts lysates (231delY, E412K, G448R). Densitometric analysis of Western blots (C, top) shown in the lower part of the panel. (D) SDS-PAGE (top) and Western blotting (bottom) of purified recombinant WT and mutant forms. (E) Prolidase activity of the recombinant proteins.</p

Secondary structure composition as estimated by the CD spectra of the recombinant prolidase variants.

<p>Secondary structure composition as estimated by the CD spectra of the recombinant prolidase variants.</p

Kinetic properties of hRecProl-WT and its pathological variants.

<p>Kinetic properties of hRecProl-WT and its pathological variants.</p