Changes in protein patterns of Staphylococcus aureus and Escherichia coli by silver nanoparticles capped with poly (4-styrenesulfonic acid-co-maleic acid) polymer

The extracted proteins of bacteria gathered from every incubation time were digested prior to analysis by LC/MS–MS. The proteins of NS-treated E. coli cells, untreated E. coli cells, NS-treated S. aureus cells, and untreated S. aureus cells were identified based on the NCBI database. There were a total number of 860 bacterial proteins matched with proteins deposited in the database. However, 26 proteins belonged to E. coli (treated or untreated) and 3 proteins belonged to S. aureus (treated or untreated) were further identified for protein functions as shown in Table 1.

From proteomes analysis, AgNPs-PSSMA were able to downregulate long-chain fatty acid transport protein (fadL) of S. aureus and quinolinate synthase A (nadA) and protein YhgF (yhgF) of E. coli. For S. aureus, only 3-deoxy-d-manno-octulosonic-acid transferase protein (waaA) was upregulated after exposed to NS (Table 1). In contrast, eight proteins of E. coli were expressed after exposed to NS. The E. coli proteins mentioned were functioned with metabolism (srlB, fliE, murD), DNA replication (dinG), DNA–RNA transcription (yrdD), RNA–protein translation (rplD), molecular transport (sapF), and signal transduction (tdcF).

The upregulated genes and downregulated genes of the bacteria were further studied for protein–protein interaction data via String database. The bioinformatics result was shown as a network of predicted functional associations for a group of proteins. The network nodes represent the proteins while the edges (lines) are the predicted associations of protein (Figure 2). The connection among proteins in S. aureus represented genes mainly involved in metabolic process, while those found in E. coli were genes mostly implicated mainly in the transporter system, metabolic activity, transcription, and translation. Interestingly, for E. coli, there were four unmatched proteins, such as family transcriptional regulator (iclR), HAD-superfamily hydrolase (yqaB), threonine dehydrogenase, and related Zn-dependent dehydrogenase (srlB) and probable ATP-dependent helicase DinG (dinG). As srlB and dinG genes were expressed in NS-treated E. coli, they were represented as biomarkers for antibacterial effect of AgNPs-PSSMA.

Figure 2

In this study, we investigate the role of AgNPs-PSSMA on the changes in protein patterns of Gram-positive S. aureus and Gram-negative E. coli, which eventually lead to bacterial cell death. A decrease in the cell survival of the bacteria treated with NS was observed while the untreated control cells grew exponentially after an hour of incubation (Figure 1A,B). From the previous reports on the antimicrobial activity of NS [28, 29], it is anticipated that AgNPs-PSSMA when penetrating into the cells make cell death by causing structural changes and damage, which could disturb vital cell functions. From our study, the changes in cellular protein after exposed to AgNPs-PSSMA were obviously seen from gel electrophoresis results, especially the E.coli cells (Figure 1C,D). Protein pattern modification leads to bacterial death by interference with metabolic function and synthesis of cell wall, nucleic acid, and protein [6].

We used the proteomic approach as a tool to explore the bacterial proteins of the NS-treated cells. For S. aureus, protein fadL was downregulated by NS. Protein fadL is associated with protein receptor and translocation of long-chain fatty acids across the outer membrane, the first part to confront with NS [30]. AgNPs-PSSMA might affect the protein receptors leading to lipid transport deficiency and ultimately cell death [2, 31, 32]. In contrast, 3-deoxy-d-manno-octulosonic acid (Kdo) transferase or waaA, a protein locating at an inner cell membrane and involved in lipopolysaccharide (LPS) bio-synthesis [33, 34], was upregulated in NS-treated S. aureus. Cellular oxidative response has been reported to promote the function of LPS biosynthesis [35], and NS is evident to cause oxidative stress. It was then not unexpected that AgNPs-PSSMA induce waaA gene.

For NS-treated E. coli, quinolinate synthase A (nadA) and protein YhgF (yhgF) were downregulated. Protein nadA is in a cytoplasm involved in the cell metabolism. It catalyzes the condensation of iminoaspartate with dihydroxyacetone phosphate to form quinolinate (pyridine nucleotide biosynthesis) [36, 37, 38]. As the appearance of hydrogen peroxide (H₂O₂) has known to depress quinolinate synthetase, which is used for quinolinate production [39], a release of H₂O₂ from NS [40] could possibly lead to inhibition of nadA protein. Protein yhgF is the respiratory protein involved in cyclic di-GMP signaling [41]. Accordingly, NS could interfere with the respiratory process of the bacteria, finally leading to cell death.

In contrary to the downregulation, NS triggered the expression of approximately eight proteins in E. coli cells (Table 1). It seems that when E. coli cells are perturbed, a higher number of genes dealt with many cell functions are expressed [42]. In this study, the upregulated proteins found were involved in the metabolic function (srlB, fliE, and murD proteins), signal transduction (tdcF protein), and signal transcription (yrdD protein).

Interestingly, we have found the upregulation of proteins dinG, rplD, and sapF, which are suggested to play a role in antimicrobial resistance. The ATP-dependent helicase dinG (dinG) is related to DNA repair and replication [43]. The process of DNA repair after exposure to NS might be consequences of cell resistance to antibacterials. Protein rplD is the 50S ribosomal protein functions as ribosome-mediated translation of mRNA into a polypeptide [44]. Protein sapF is an ATP-binding protein in plasma membrane involved in a peptide transport system [45]. The upregulation of proteins rplD and sapF of E. coli perhaps indicates a change in protein synthesis after confront with antibacterial agent.

The previous proteomic study in E. coli cells was usually focused on the membrane protein. The expression of outer membrane precursors (OmpA, OmpC, OmpF, OppA) of E. coli cells was reported to be enhanced by NS [10]. The rfa gene necessary for outer member lipopolysaccharide was expressed in E. coli mutant strain [12]. The bacterial response to NS could be different and perhaps dependent on a type of stabilizer. For example, genes functioning in quinone binding and ubiquinone biosynthesis were found in E. coli mutant strain treated with AgNPs stabilized by polyethylene imine polymer but not found in those treated with polyvinylpyrrolidone polymer-capped AgNPs [12]. Herein, we reported the additional information about the effect of AgNPs stabilized by PSSMA polymer on gene expression of S. aureus and E. coli cells. Finally, the association of bacterial proteins was determined via String database. We have found that srlB and dinG proteins expressed in E. coli after confronted with NS were not found for any protein association (Figure 2). The finding makes proteins srlB and dinG be considered as novel biomarkers for antibacterial effect of such NS.

The stable AgNPs-PSSMA could be used as antibacterial agents to Gram-positive and Gram-negative bacteria. NS caused both downregulation and upregulation of proteins in bacterial cells with the latter was more obviously found in E. coli cells.

In this study, the NS without capping agent added was not performed due to the reasons dealing with the stability of AgNPs. The data obtained from the present study were the antibacterial effect AgNPs capped with PSSMA. The AgNPs could be further applied clinically as materials for antibacterial purposes. As the bacterial proteins change after AgNPs exposure seemed to be depend on the type of stabilizers [12], the further study should be on the comparison of bacterial protein change of AgNPs capped with different types of polymer. The information could be used to select the appropriate stabilizing polymer for the targeted bacterial proteins.