Nanocompartments combine with silver ions in vitro
A general feature of nanocompartments is their high stability, which enables them to endure in culture supernatants[22, 23]. An encapsulin nanocompartment resembles a virus capsid in terms of its mechanical properties. In order to determine whether the nanocompartments can combine with silver ions, we incubated the purified nanocompartments with silver ions in vitro. It was found that the nanocompartments did combine with silver ions, appearing darker than nanocompartments without silver ions when observed by transmission electron microscopy (TEM) (Fig. 1A and B). Energy dispersive X-ray (EDX) analysis also detected silver ions in the nanocompartments (Fig. 1C, D, and E), confirming that a single nanocompartment can stably combine with silver ions in vitro.
Agglomeration/dispersion of nanocompartment combined with silver ions
We have shown that nanocompartments can combine with silver ions, but the process of combination cannot be dynamically observed. So we used SPRi, which is an unlabeled in situ detection technique[24-26], to quantify the agglomeration of nanocompartments and silver ions. SPRi was conducted using a high aperture optical objective[27, 28]. Supporting Information Fig. S2 shows that when nanocompartments and silver ions combined, they attached to the coated sensor chip, but the nanocompartments without silver ions did not attach to the surface of the chip (Supporting Information Fig. S3). Fig. 2A and B show the transmission and plasmonic images of the nanocompartment particles with silver ions, it should be noted that the plasma image is V-shape. Different light intensity positions in the image show different colors. The color bar normalized the light intensity, with the display range from 0 to 1.
It has been reported that the stability of a nanocompartment is dependent on the pH of its environment . The nanocompartment particles seemed to be stable at pH 5-9, which is in agreement with the previous reports (Supporting Information Fig. S4) [29, 30]. In our system, the nanocompartment particles were able to attach to the surface, but were also allowed to agglomerate/disperse. So, the buffer pH was reduced from 7 to 4 and the particles were incubated for 750 min. Fig. 2C shows the agglomeration/dispersion information of the nanocompartment particles combined with silver ions. At pH 7, the SPRi signal increased over time, which was likely caused by the combination of the nanocompartments with silver ions. The nanocompartments landed on the surface of the sensor chip, resulting in the increased SPRi signal. When the pH of the solution was changed to 4, the SPRi signal decreased over time, suggesting that the nanocompartment particles were dispersing and the silver ions were being released back into the solution (see the Supporting Information Animation, Additional file 2). In order to provide further evidence supporting our hypothesis, the surface of the sensor chip was analyzed by scanning electron microscopy (SEM-EDX). The measured points included nanocompartment particles as well as other sections of the sensor chip (Fig. 2D and E). Empty encapsulin nanocompartments could not be observed by SEM. It was found that there were silver ions where there were nanocompartment particles, but sections without nanocompartments did not have silver ions.
Nanocompartment centered resistance to silver ion stress in model organism
Our experiments have shown that the nanocompartments were able to combine with silver ions, but it is not known whether they can protect normal bacteria from the effects of silver ions. So, we used E. coli as a model organism to explore the functions of nanocompartments in resisting silver ions stress. The process of silver ions binding to E. coli BL21 that were immobilized on the surface of the sensor chip was imaged using the surface plasmon resonance microscope. Supporting Information Fig. S5 shows that the E. coli BL21 cells were tethered to the sensor chip. Fig. 3A and B show the transmission and plasmonic images of the bacterial cells. The V-shape formed by a single bacteria matches the position of the bacteria in the optical image.
Then the effects of silver ions on E. coli BL21 were studied by adding AgNO3 (final concentration 20 µΜ). To test whether the nanocompartments were effective in bacteria, we established two different types of bacteria, using genetic and molecular techniques. One of them was genetically modified bacteria (GMB, with nanocompartments), and the other was wild-type bacteria (WB, without nanocompartments). Then we passed a solution of AgNO3 through the bacteria at a rate of 3 μl/s for about 1,000 seconds. With the addition of silver ions, the image intensity of the bacteria increased significantly. The intensity of the image signal was proportional to the mass density of the sensor surface. So, the data showed that silver ions were absorbed by the bacteria cells. Importantly, there was a significant difference between the image intensities of GMB and WB (Fig. 3C) which indicated that the nanocompartments placed in normal bacteria were able to combine with silver ions.
It has been reported that silver ions can damage cell membranes and DNA by producing reactive oxygen species (ROS). We speculated that nanocompartments would reduce the sensitivity of bacteria to silver ions by reducing ROS production. To verify this, we investigated the effects of nanocompartments on the survival of E. coli under ROS stress caused by silver ion exposure. Shown in Fig. 3D, as the concentration of silver ions increased, the bacterial survival rate decreased. When exposed to 30 μM AgNO3, the survival rate of GMB was 86%, while WB’s survival rate was 59%, which suggested that GMB had a higher resistance to silver ions than WB.
The possible mechanisms by which bacteria resist silver ion stress using nanocompartments
To further explore the protective effects of nanocompartments in bacteria, the effects of nanocompartments on the growth curve of E. coli experiencing silver ion-caused ROS stress was investigated. As shown in Fig. 4A, the beginning of Phase I the growth curves of E. coli from all groups were similar. However, in Phase II, following dosing with silver ions (WB + 0µM AgNO3 (WB), GMB + 0µM AgNO3 (GMB), WB + 120µM AgNO3 (WB+Ag+), and GMB + 120µM AgNO3 (GMB+Ag+)), the growth rates in WB+Ag+ and GMB+Ag+ were lower than that in WB and GMB (Fig. 4A). The growth curve of GMB is no different from that of WB in the presence of no silver ions. Notably, when both were exposed to silver ions, the growth rate of the genetically modified bacteria was faster than the wild-type bacteria, which is consistent with our previous result (the survival rate of GMB is higher than WB). As predicted, the encapsulin protein (about 42-kDa) was found to be highly expressed in a band from the genetically modified bacteria when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Supporting Information Fig. S6). These results support the hypothesis that nanocompartments help bacteria resist silver ion stress.
High-throughput proteomics was used to get a comprehensive picture of the proteomic changes in the WB, WB+Ag+, and GMB+Ag+ groups. As shown in Fig. 4B, a total of 2679, 2680, and 2653 proteins were expressed in WB, WB+Ag+, and GMB+Ag+, respectively. Among them, 2440 proteins were shared by all three groups, suggesting that the majority of the proteins didn't change among the three groups.
In order to clarify the function of differentially expressed proteins in WB, WB+Ag+, and GMB+Ag+, Gene Ontology (GO) annotation analysis was used to analyze the quantified proteins. GO annotation analysis included biological processes, cellular components, and molecular functions. As shown in Fig. 4C and D, in the biological process category, many of the differentially expressed proteins were involved in cellular processes, metabolic processes, and single-organism processes. It indicated that silver ions can inhibit many catabolic processes and some biosynthetic processes in bacteria. These results suggested that silver ions have intrinsic functions as toxic metals.
In terms of cellular components, a large number of the up-regulated proteins were from cell parts. Intriguingly, up-regulated proteins, not down-regulated proteins, were more commonly associated with cell membrane components. In E. coli, it has been found that silver ions affect membrane proteins, leading to cell lysis. In terms of molecular functions, the up-regulated proteins and down-regulated proteins were mainly concentrated in catalytic activity and binding, suggesting that differentially expressed proteins were involved in protein processing.
We then performed a GO enrichment analysis of up-regulated proteins between specific groups, i.e. ‘WB+Ag+ vs WB’ and ‘GMB+Ag+ vs WB’. As shown in Fig. 5A and B, we saw that the GO terms “structural constituent of ribosome” and “integral component of plasma membrane” were significantly enriched among the up-regulated proteins in ‘WB+Ag+ vs WB’. These results suggested that wild-type bacteria responded to the silver stress by increasing the expression of ribosome-associated proteins and membrane proteins, without success. We then found that the GO terms “single-organism transport” and “integral component of plasma membrane” were significantly enriched among the up-regulated proteins in ‘GMB+Ag+ vs WB’. Interestingly, the GO term “single-organism transport” was significantly enriched in ‘GMB+Ag+ vs WB’, but not in ‘WB+Ag+ vs WB’. These results indicated that genetically modified bacteria likely have other ways of regulating silver ion stress, ways which have been proven successful. Therefore, we hypothesized that nanocompartments induced the up-regulation of transporter proteins in bacteria, thus helping to transport silver ions out of cells, thereby reducing silver ion stress.
We then subjected the up-regulated proteins identified in ‘WB+Ag+ vs WB’ and ‘GMB+Ag+ vs WB’ to KEGG pathway enrichment analyses. Among the enriched KEGG pathways from ‘WB+Ag+ vs WB’ , we identified 5 significantly enriched metabolic pathways (P < 0.05) including the “ribosome”, “protein export”, “bacterial secretion system”, “phosphotransferase system”, and “oxidative phosphorylation” (Fig. 6A). Of the 12 significantly enriched metabolic pathways from ‘GMB+Ag+ vs WB’, 5 were shared with ‘WB+Ag+ vs WB’, and 7 were unique (Fig. 6B). Therefore, those 7 metabolic pathways may be crucial in helping bacteria resist silver ions. Those 7 metabolic pathways were “fatty acid degradation”, “valine, leucine and isoleucine biosynthesis”, “butanoate metabolism”, “starch and sucrose metabolism”, “pantothenate and CoA biosynthesis”, “caprolactam degradation”, and “limonene and pinene degradation”. These results indicated that detoxification was associated with protein processing and secondary metabolites.