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

Makumire S, Revaprasadu N, Shonhai A - PLoS ONE (2015)

Bottom Line: We further investigated the effects of the AuNPs on the solubility of the E. coli BB1553 proteome.The toxic effects of the AuNPs were alleviated by transforming the E. coli BB1553 cells with a construct expressing DnaK.Our study suggests a role for DnaK in alleviating nanoparticle induced stress in E. coli.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Mathematics & Natural Sciences, University of Venda, Thohoyandou, South Africa; Department of Biochemistry & Microbiology, University of Zululand, KwaDlangezwa, South Africa.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0121243.g004: 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.

Mentions: E. coli DnaK+ and E. coli DnaK- cells were exposed to citrate AuNPs at variable levels (25–75 μgmL-1) and incubated at 30°C. Based on the SDS-PAGE data, E. coli DnaK- cells exposed to AuNPs had a more enriched pellet fraction compared to that isolated from cells that had not been exposed to AuNPs (Fig. 4A). This suggests that the presence of AuNPs marginally promoted the aggregation of proteins in E. coli DnaK- cells. GroEL, a chaperone that cooperates with DnaK in E. coli was expressed in E. coli DnaK- cells cultured at 30°C in the absence and presence of AuNPs. However, its production was more enhanced in E. coli DnaK- cells in the presence of AuNPs. The over-production of GroEL in E. coli DnaK- cells exposed to increasing levels of AuNPs further testifies that the presence of AuNPs further subjected the cells to additional stress. DnaK was evidently over-produced in E. coli DnaK+ cells (Fig. 4B). As expected, GroEL was not over-expressed in E. coli DnaK+ cells. This is in agreement with DnaK playing the most prominent role in protein folding and thus GroEL was over-expressed in the absence of DnaK as a compensatory measure [34]. The solubility profile of the proteomic constituents of E. coli DnaK+ cells cultured in the presence and absence of AuNPs that were incubated at 30°C was similar (Fig. 4B). This suggests that the presence of DnaK reduced the detrimental effects of the AuNPs. A batch of E. coli DnaK+ cells cultured in the presence of AuNPs was subjected to heat stress at 40°C. We had expected that the possible detrimental effects of the AuNPs on the proteome of E. coli DnaK+ cells would be more apparent at higher temperatures. However, based on the SDS-PAGE data, there was no difference in the protein solubility profile in E. coli DnaK+ cells cultured at 40°C in the presence and absence of AuNPs (Fig. 4C). Altogether, the findings suggest that AuNPs at comparable levels (25–75 μgmL-1) promoted protein aggregation in E. coli cells that lacked DnaK and that restoration of DnaK in these cells conferred cytoprotection. The expression of endogenous GroEL by E. coli DnaK- cells that were exposed to various levels of AuNPs was validated by Western blotting (Fig. 4D). Similarly, the expression of DnaK by E. coli DnaK+ cells exposed to AuNPs was confirmed by Western blot analysis (Fig. 4E). The Western blot data suggests that GroEL occurred in the soluble fraction of the cells, whilst DnaK was spilt between the soluble and insoluble fraction of the cells (Fig. 4D-E).


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

Makumire S, Revaprasadu N, Shonhai A - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383610&req=5

pone.0121243.g004: 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.
Mentions: E. coli DnaK+ and E. coli DnaK- cells were exposed to citrate AuNPs at variable levels (25–75 μgmL-1) and incubated at 30°C. Based on the SDS-PAGE data, E. coli DnaK- cells exposed to AuNPs had a more enriched pellet fraction compared to that isolated from cells that had not been exposed to AuNPs (Fig. 4A). This suggests that the presence of AuNPs marginally promoted the aggregation of proteins in E. coli DnaK- cells. GroEL, a chaperone that cooperates with DnaK in E. coli was expressed in E. coli DnaK- cells cultured at 30°C in the absence and presence of AuNPs. However, its production was more enhanced in E. coli DnaK- cells in the presence of AuNPs. The over-production of GroEL in E. coli DnaK- cells exposed to increasing levels of AuNPs further testifies that the presence of AuNPs further subjected the cells to additional stress. DnaK was evidently over-produced in E. coli DnaK+ cells (Fig. 4B). As expected, GroEL was not over-expressed in E. coli DnaK+ cells. This is in agreement with DnaK playing the most prominent role in protein folding and thus GroEL was over-expressed in the absence of DnaK as a compensatory measure [34]. The solubility profile of the proteomic constituents of E. coli DnaK+ cells cultured in the presence and absence of AuNPs that were incubated at 30°C was similar (Fig. 4B). This suggests that the presence of DnaK reduced the detrimental effects of the AuNPs. A batch of E. coli DnaK+ cells cultured in the presence of AuNPs was subjected to heat stress at 40°C. We had expected that the possible detrimental effects of the AuNPs on the proteome of E. coli DnaK+ cells would be more apparent at higher temperatures. However, based on the SDS-PAGE data, there was no difference in the protein solubility profile in E. coli DnaK+ cells cultured at 40°C in the presence and absence of AuNPs (Fig. 4C). Altogether, the findings suggest that AuNPs at comparable levels (25–75 μgmL-1) promoted protein aggregation in E. coli cells that lacked DnaK and that restoration of DnaK in these cells conferred cytoprotection. The expression of endogenous GroEL by E. coli DnaK- cells that were exposed to various levels of AuNPs was validated by Western blotting (Fig. 4D). Similarly, the expression of DnaK by E. coli DnaK+ cells exposed to AuNPs was confirmed by Western blot analysis (Fig. 4E). The Western blot data suggests that GroEL occurred in the soluble fraction of the cells, whilst DnaK was spilt between the soluble and insoluble fraction of the cells (Fig. 4D-E).

Bottom Line: We further investigated the effects of the AuNPs on the solubility of the E. coli BB1553 proteome.The toxic effects of the AuNPs were alleviated by transforming the E. coli BB1553 cells with a construct expressing DnaK.Our study suggests a role for DnaK in alleviating nanoparticle induced stress in E. coli.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Mathematics & Natural Sciences, University of Venda, Thohoyandou, South Africa; Department of Biochemistry & Microbiology, University of Zululand, KwaDlangezwa, South Africa.

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.

No MeSH data available.


Related in: MedlinePlus