DnaK protein alleviates toxicity induced by citrate-coated gold nanoparticles in Escherichia coli

Abstract

A number of previously reported studies suggest that synthetic gold nanoparticles (AuNPs) are capable of stabilising proteins against heat stress in vitro. However, it remains to be understood if AuNPs confer stability to proteins against cellular stress in vivo. Heat shock proteins (Hsps) are conserved molecules whose main role is to facilitate folding of other proteins (chaperone function). Hsp70 (called DnaK in prokaryotes) is one of the most prominent molecular chaperones. Since gold nanoparticles exhibit chaperone-like function in vitro, we investigated the effect of citrate-coated gold nanoparticles on the growth of E. coli BB1553 cells that possess a deleted dnaK gene. We further investigated the effects of the AuNPs on the solubility of the E. coli BB1553 proteome. E. coli BB1553 cells exposed to AuNPs exhibited cellular defects such as filamentation and plasma membranes pulled off the cell wall. The toxic effects of the AuNPs were alleviated by transforming the E. coli BB1553 cells with a construct expressing DnaK. We also noted that cells in which DnaK was restored exhibited distinct zones to which the nanoparticles were restricted. Our study suggests a role for DnaK in alleviating nanoparticle induced stress in E. coli.

Fig 1. TEM and HRTEM images of synthesised citrate-coated gold nanoparticle 0.3 mM gold salt was reduced with 136 mM tri-sodium citrate.

(A) Absorption spectra of citrate capped AuNPs; the insert shows the red wine suspensions obtained. (B) TEM images of the citrate AuNPs (top panel), and HRTEM images of AuNPs (lower panel). The presence of visibly defined lattice fringes confirmed the crystal morphology of the nanoparticles produced. (C) Bar graph representing the frequency of the citrate capped AuNPs by size.

Fig 2. Citrate-AuNPs induce cytotoxicity in E. coli DnaK deficient cells.

TEM images showing E. coli DnaK^- cells exposed to 40 μgmL^-1 AuNPs. The images show various degrees of cell damage: (A) cell that has internalised AuNPs; (B) AuNPs complexed with cytosolic material; (C) cell matrix disintegrating in the presence of AuNPs; (D) extensive cytosolic disintegration; (E) dead cell showing membrane pooled off the cell.

Fig 3. Comparative analysis of the effects of citrate AuNPs on E. coli DnaK^- and E. coli DnaK⁺ cells.

TEM images showing E. coli DnaK^- and E. coli DnaK⁺ cells exposed to citrate-AuNPs. A comparative population overview of E. coli DnaK^- cells cultured in the presence of AuNPs is shown (panel A1) versus E. coli DnaK⁺ cells cultured under similar conditions (panel A2). Panels B1-B2 and C1-C2 illustrate comparative morphological features of E. coli DnaK^- cells versus E. coli DnaK⁺ cells, respectively. AuNP aggregates are seen as black spots inside the cells. Note the evident delineation zones in panels C2 and D2, associated with E. coli DnaK⁺ cells that are missing in E. coli DnaK^- cells (panels C1and D1).

Fig 4. Expression and solubility profiles of E. coli BB1553 cells exposed to AuNPs.

SDS-PAGE analysis representing protein expression and solubility profiles of E. coli BB1553 cultured in the absence and presence of AuNPs (25–75 μgmL^-1). Various fractions of E. coli cells were obtained: (A) E. coli DnaK^- cells cultured at 30°C in the absence and presence of variable AuNPs; (B) E. coli DnaK⁺ cells cultured at 30°C in the absence and presence of variable levels of AuNPs; (C) E. coli DnaK⁺ cells cultured at 40°C in the absence and presence of variable levels of AuNPs. Lanes representing total cell lysate (TC); cell pellet (P) and soluble (S) fractions, respectively, are shown. Western blot analyses were conducted to confirm production of heterologously expressed DnaK (D); and endogenous GroEL (E). Pellet and soluble fractions of E. coli BB1553 that had been exposed to AuNPs at the given concentrations were sampled for the Western analyses.

Fig 5. Citrate-coated gold nanoparticles suppress malate dehydrogenase aggregation in a concentration dependent manner.

(A) 1 μM MDH was suspended in assay buffer in the absence or presence of various levels of AuNPs (2.5–7.5 μgmL^-1). (B) The assay was repeated in the presence of 10 fold higher levels of AuNPs (25–75 μgmL^-1). The suspensions were subjected to heat stress at 48°C for 20 minutes. The soluble fraction (S) was separated from pellet fraction (P) by centrifugation. Samples were analysed by SDS-PAGE analysis.

Fig 6. Suppression of MDH aggregation by citrate-coated gold nanoparticles combined with Hsp70.

1 μM MDH was suspended in assay buffer in the absence or mixed with 1.3 μM Hsp70 or AuNPs (10 μgmL^-1); and a combination of Hsp70 and AuNPs, respectively. The suspensions were subjected to heat stress at 48°C for 20 minutes. The soluble fraction (S) was separated from pellet fraction (P) by centrifugation. Samples were analysed by SDS-PAGE analysis.

Fig 7. Spectrophotometric analysis for the heat-induced aggregation of MDH in the presence of AuNPs and Hsp70.

1 μM MDH was suspended in assay buffer in the absence or mixed with high concentrations of AuNPs (0–100 μgmL^-1) (a); the assay was repeated in the presence of 1.3 μM Hsp70 (b). The suspensions were subjected to heat stress at 48°C for 20 minutes. Absorbance values were measured at 340 nm in triplicates using a 96-well micro titre plate. Data are presented as mean and standard deviations.

Fig 8. Proposed model illustrating the effects of citrate-coated gold nanoparticles in vitro and in E. coli cells.

The model describes the proposed effects of single species versus agglomerated species of citrate-gold nanoparticles on the integrity of proteins in vitro and in E. coli cells that are deficient of DnaK and in which DnaK function is restored.