Exposure to High Dosage of Gold Nanoparticles Accelerates Growth Rate by Modulating Ribosomal Protein Expression.

doi:10.21203/rs.3.rs-2446306/v2

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Exposure to High Dosage of Gold Nanoparticles Accelerates Growth Rate by Modulating Ribosomal Protein Expression.

https://doi.org/10.21203/rs.3.rs-2446306/v2

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published 06 Aug, 2023

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Gold nanoparticles (AuNPs) have been utilized in various biomedical applications, including diagnostics and drug delivery. However, cellular and metabolic responses of cells to these particles remain poorly characterized. In this study, we used bacteria (Escherichia coli and Bacillus subtilis) and a fungus (Saccharomyces cerevisiae) as model organisms to investigate the cellular and metabolic effects of exposure to different concentrations of citrate-capped spherical AuNPs with diameters of 5 and 10 nm. Our data showed the stability of synthesized AuNPs in different growth media and revealed comparable uptake by microorganisms. An exposure to a high concentration of AuNPs (10¹² particles) resulted in reduced cell-division time and a two-fold increase in cell density in both bacteria and unicellular eukaryote. The exposed cells exhibited a decrease in average cell size and an increase in the cell-division marker protein (FtsZ), further supporting an accelerate growth rate. Notably, exposure to such high dosage of AuNPs did not induce DNA damage, envelope stress, or a general stress response in bacteria. Differential whole proteome analysis demonstrated modulation of ribosomal protein expression upon exposure to AuNPs in both E. coli and S. cerevisiae. Interestingly, the accelerated growth observed in the presence of AuNPs was sensitive to sub-MIC dosages of a drug that specifically targets ribosome assembly and recycling. Based upon these findings, we argue that exposure to high concentrations of AuNPs induces stress on the translation machinery. This leads to an increase in protein synthesis rate by modulating ribosome assembly, which results in the rapid proliferation of cells.

Gold Nanoparticles

Growth Rate

Stress Response

Ribosomal Proteins

Aminoglycosides

Translational rate

Gold nanoparticles (AuNPs) remain the preferred choice of metal for various biomedical applications primarily because of their chemical inertness, tunable size, and variable surface functionalization^1,2. The most commonly used method for synthesizing spherical AuNPs in the range of 3-100 nm involves the use of sodium citrate as both a reducing and stabilizing agent^3,4. Spherical AuNPs with diameters ranging from 3 to 13 nm are ideal for drug delivery and diagnostic applications^5–9 because of their rapid uptake ^10,11 and ability to be excreted out of the body¹². However, studies investigating the biocompatibility (cytotoxicity) of AuNPs in various model systems, including bacteria^13,14, yeast¹⁵, in vitro cell lines¹⁶, fruit flies¹⁷, and murine models¹⁸, have produced conflicting biocompatibility data. This conflict in data can be explained by variations in the size, shape, surface charge, and stabilizing agents of AuNPs¹⁹, which regulate the distinct biochemical and biophysical properties of the particles (SI-Table-1). Most biocompatibility studies have focused on determining the effect of AuNPs on cell viability, but very few have investigated the cellular and underlying metabolic response^20–22 to understand the true interaction between particles and biological systems.

The cell cycle of microorganisms includes birth, growth, division, and death, which are controlled by the availability of nutrients and signals from the extracellular environment²³. Under favourable conditions, microorganisms grow in three distinct phases: (a) a lag phase, acclimatization of cells to a new environment without increasing the cell number; (b) log phase, rapid cellular growth and cell division as the cell’s metabolic machinery is tuned to the available nutrients and environmental cues; and (c) stationary phase, a state of nutrient deprivation and stressed environmental cues, where the birth rate equals the death rate ²³. Upon exposure to toxins, antibiotics, and heavy metals, cells can experience damage to DNA, RNA, proteins, and cell membranes, which arrests cell cycle progression. Survival (with or without genetic alteration in the progeny) or death of microorganisms is determined by whether the stress and metabolic response enables cells to adapt to the new environment by modulating the quantity and diversity of the synthesized proteins²⁴. Proteins are the functional currency of cellular and metabolic machinery across life, and the modulation and regulation of their expression play an important role not only in maintaining the proliferation rate, but also in adaptation across various niches²⁵. Instead of transcriptional regulation, controlling the rate of protein synthesis with an available pool of mRNA is an effective strategy to rapidly adapt to changes in the extracellular environment²⁶.

The process of protein synthesis carried out by the ribosomes, consists of four major steps: initiation, elongation, termination, and ribosomal recycling. These steps are facilitated by the collective actions of ribosomal proteins and RNA molecules within the ribosome. The initiation step, which is highly regulated, can occur through either canonical or non-canonical mode ^27,28. Under favourable conditions, initiation primarily occurs via the canonical mode, which involves the stepwise assembly of smaller ribosomal subunits on mRNA and the subsequent recruitment of initiation factors in a licensing process²⁸. However, under unfavourable conditions, cells can utilize the non-canonical mode of protein synthesis to modulate the quantity and spectrum of proteins. Non-canonical mode includes mechanisms such as internal ribosome entry sites (IRESs), cap-independent translation enhancers (CITEs), ribosome shunting, and m6A-dependent translation^29,30. For example, in E. coli, the overexpression of the MazF toxin, an endoribonuclease, can induce the assembly of stress ribosomes and promote the translation of leaderless mRNA³¹. Similarly, in Saccharomyces cerevisiae, the accumulation of misfolded or unfolded peptides in the endoplasmic reticulum can activate the unfolded protein response (UPR) and promote translation via non-canonical mode^32,33. Notably, cancer cells that exhibit rapid proliferation under hypoxic conditions exploit both canonical and non-canonical modes of translation initiation to increase protein production³⁰. These examples highlight the flexibility of cells to exploit the translation machinery to adapt to various stress conditions and optimize protein synthesis.

In this study, we examined the cellular and metabolic responses of four bacterial model organisms (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus) and a unicellular fungus (Saccharomyces cerevisiae) upon exposure to two citrate-capped gold nanoparticles (AuNPs) having a diameter of less than 10 nm. Our findings revealed that exposure to high doses of AuNPs led to a substantial increase in growth rate, with a ~2-fold increase in cell density. Interestingly, despite this accelerated growth, we observed no activation of the known stress response pathways, including envelope stress, general stress, or DNA damage. Differential whole proteome analysis revealed that exposure to high doses of AuNPs led to the underexpression of a majority of ribosomal proteins in involved in protein synthesis via canonical mode. Furthermore, treatment with sub-MIC ribosome-targeting drugs, such as aminoglycosides for bacteria and rapamycin for yeast, effectively inhibited accelerated cellular growth rate. These findings strongly suggest that exposure to high concentrations of AuNPs induces stress on the translational machinery to accelerate protein synthesis to promote rapid proliferation and meet the cellular metabolic demand.

Stability of citrate stabilized spherical AuNPs and their uptake by microorganisms

Two monodisperse, spherical, citrate-capped gold nanoparticles (AuNPs) with diameters of 4.2 ± 1 nm and 9.2 ± 1 nm (hereafter referred to as 5 nm and 10 nm AuNPs, respectively) were synthesized using the Turkevich method, with some modifications. The synthesized AuNPs appeared wine red in color, with absorbance maxima peaks at 514 nm and 525 nm for 5 nm and 10 nm AuNPs, respectively (Figure 1G.). High-resolution transmission electron microscopy (HR-TEM) confirmed the spherical shape and core size of each AuNP (Figure 1A & C). Dynamic light scattering (DLS) analysis of over 1000 particles showed a monomodal curve with polydispersity indices (PDI) of 0.4 and 0.7 and mean hydrodynamic radii of 7 nm and 12 nm for 5 nm and 10 nm AuNPs, respectively (S.I. Figure 1A.). The final concentration and zeta potential of 5 nm and 10 nm AuNPs were 4.1x10¹³and 1x10¹³particles/mL (38 nM and 10 nM) and -31mV and -21mV, respectively (S.I. Figure 1B). It is noteworthy that attempts to concentrate AuNPs via centrifugation or lyophilization resulted in the aggregation of particles. In contrast, freshly synthesized AuNPs that were washed and resuspended in Milli-Q water remained stable for more than a month.

Prior to investigating the effect of AuNPs on microorganism viability and the subsequent cellular and metabolic responses, the stability of AuNPs in different growth media commonly used for microorganism culture was examined. Both 5 and 10 nm AuNPs did not aggregate or agglomerate at the highest concentration of 10¹² NPs/mL, except in Spiz media (minimal media for Bacillus species) (S.I. Figure 2). The observed aggregation of AuNPs can be attributed to the presence of a high concentration of sodium citrate in the spiz medium. Therefore, for growth studies on B. subtilis, M9 medium supplemented with 0.2% glucose and 1.0% casamino acids was used as a substitute. To investigate the uptake of AuNPs of different sizes across various microorganisms (varying in membrane composition and charge), we incubated the highest concentration (10¹² particles) of both 5 and 10 nm AuNPs with increasing numbers of gram-negative (E. coli), gram-positive (B. subtilis), and unicellular fungi (S. cerevisiae) (S.I. Figure 3A). A gradual decrease in AuNP absorbance in the supernatant was observed with an increasing number of cells over time, with the maximum uptake observed after four hours (Figure 1E & F, S.I. Figure 3B-D). Given that the loss of absorbance in the supernatant did not distinguish whether the AuNPs were internalized, diffused, or remained attached to the outer cellular membrane, we functionalized surfaces of both 5 and 10 nm AuNPs with a fluorescent dye (Rhodmine-6G) and determined its uptake by microorganisms, using fluorescence microscopy. Single-cell analysis revealed that both 5 and 10 nm AuNPs were internalized into the cytosol (Figure 1I & J). As reported previously for 2 nm AuNP³⁴, maximum internalization of our synthesized particles (<10 nm) was achieved within 4 hours and further incubation did not change the amount of intracellular AuNPs. (Figure 1 & S.I. Figure 3). These findings suggest that despite the differences in the membrane composition and surface charge (-110³⁶, -21.9³⁷, and -38.4³⁸ mV in B. subtilis, E. coli, and S. cerevisiae, respectively) of the microorganisms, citrate-capped spherical AuNPs with diameters of <10 nm can be internalized.

Exposure to AuNPs accelerates growth rate

To determine whether exposure to varying concentrations of AuNPs (< 10 nm) affects the growth kinetics and viability of microorganisms, the growth rate of different microorganisms was monitored in the presence of varying concentrations of AuNPs (0 - 10¹² particles/mL) (S.I. Figure 4). Across microorganisms, the growth rate and cell viability (Colony Forming Unit, CFU) analysis showed no significant change after exposure to a concentration of 10¹⁰ particles (Figure 2B & C), regardless of the size of the nanoparticles (5 or 10 nm). However, exposure to a high concentration of AuNPs (10¹¹ & 10¹²particles) resulted in accelerated growth across microorganisms, as evidenced by a reduction in cell division time (t_d) (S.I. Figure 5). Additionally, there was an increased number of viable cells (C.F.U) in the stationary phase of E. coli, B. subtilis, and S. cerevisiae cultures, indicating a higher cell density or biomass, compared to unexposed cells (p < 0.0001) (Figure 2 D - F, S.I. Figure 5). Both brightfield and scanning electron microscopy (SEM) analyses confirmed that accelerated growth rates were achieved without compromising the symmetrical of cell-division or the outer membrane integrity. In addition, no visible or characteristic alterations in the cellular phenotype, surface morphology, or visible aggregation of particles on the cell membrane were observed (Figure 2D - F).

Furthermore, accelerated growth rate and high cell-density as observed, irrespective of the cell-cycle phase (lag phase or log phase), suggesting that exposure to AuNPs elicits consistent cellular and metabolic responses, regardless of the metabolic or physiological state of the cells. A similar increase in growth rate and high cell density was observed in other gram-negative (P. aeruginosa) and gram-positive (S. aureus) bacteria upon exposure to AuNPs (p < 0.01 and < 0.001 respectively) (S.I. Figure 5).

To further confirm the accelerated growth, we monitored the following: (a) average cell length, (b) expression of FtsZ, a cell-division marker protein and (c) live vs dead cells after exposure to AuNPs and compared them with unexposed cells. Cells were grown to log phase (O.D₆₀₀~0.2) and exposed to 10¹² particles. After 4 hours of exposure, cell length was measured using flow cytometry (population-based analysis) and microscopy (Brightfield microscopy). Analysis of 50,000 cells after 4 hour of exposure showed a downward shift in the FSC-A and SSC-A plots compared with unexposed cells, implying an increase in the number of small-size cells (Figure 3B). Similarly, an increase in the smaller cell population was observed after 4 hours of exposure to 10¹² particles in S. cerevisiae (Figure 3D). Single-cell analysis of ~1,000 E. coli cells also revealed a significant reduction in the average cell-size from 2.39 µm to 1.88 µm and 1.85 µm after 4 hours of exposure to 5 nm and 10 nm AuNPs, respectively (Figure 3C). These findings suggest that the accelerated growth rate is accompanied by an increase in the number of new-born cells (post cell-division). To further confirm an increase in cell division rate, we monitored the per-cell intensity of the GFP-tagged FtsZ protein (FtsZ-GFP). Single cell analysis revealed an increase in the expression of FtsZ-GFP proteins by approximately 1.17-fold and 2.14-fold in cells exposed to 5 nm and 10 nm AuNPs, respectively (Figure 3E; S.I. Figure 6). Further, a live and dead assay, including C.F.U. and propidium iodide (PI) staining analysis was performed to rule out accumulation of dead cells after AuNP exposure. In PI staining assay, uptake of propidium iodide dye by cells (due to loss of membrane integrity) is used as an indicator for dead cell accumulation and for this paraformaldehyde treated cells (loss of membrane integrity) were used as control. Both, C.F.U. and PI staining analysis, confirmed the absence of a significant accumulation of dead cells after AuNP exposure (Figure 3F – H; S.I. Figure 7), suggesting an increase in the number of newly born cells, which ultimately contributes to a higher cell density observed in the stationary phase.

Known stress responses are not induced or required for accelerated growth rate

To determine whether exposure to high concentrations of AuNPs induces DNA damage or envelope stress in the cells. We utilized E. coli strains harbouring recA-gfp and ϕʎ (rpoHP3::lacZ)³⁵, indicator of DNA damage and envelope stress response (RpoE expression), respectively. These strains were grown to logarithmic phase and then exposed to 10¹² AuNPs (Figure 4A). The induction of RecA-GFP expression was measured using flow cytometry and fluorescence microscopy, and RpoE expression was determined by measuring the change in ß-galactosidase activity. Our findings revealed that exposure to AuNPs did not induce DNA damage or result in the expression of RpoE (envelope stress response) in E. coli (Figure 4B & C; S.I. Figure 10).

To determine whether the known stress response plays a role in facilitating accelerated growth, the growth rate of E. coli strains lacking following stress response, such as envelope stress (ΔpspC, ΔrseA, ΔbaeS, ΔrcsF and ΔcpxA)³⁶; DNA damage and repair responses (ΔrecA and ΔrecN)³⁷; and general stress (ΔrpoS)³⁸ were determined. Similarly, for B. subtilis strains lacking DNA damage and repair response (ΔrecA)³⁷ and general stress (ΔsigM)³⁹ were used (S.I. Fig 8). Our data demonstrated that accelerated growth was not contingent upon any known stress response (S.I. Figure 8; Table-1). Furthermore, we screened whether accelerated growth required free-radical quenching proteins, namely SodA and SodB in E. coli ⁴⁰, to protect it against oxidative stress and Sod1 in S. cerevisiae. However, mutant strains of both E. coli (ΔsodA & ΔsodB strains) and S. cerevisiae (Δsod1) also showed accelerated growth and higher cell density upon exposure to AuNPs (S.I. Fig 9, S. cerevisiae data not shown). These findings confirm that exposure to AuNPs does not induce DNA damage or membrane stress and also the observed accelerated growth rate is not dependent upon the known stress responses.

Exposure to AuNPs results in the altered-expression of the ribosomal proteins

To investigate the metabolic responses of E. coli and S. cerevisiae, we employed a hypothesis-free Liquid Chromatography – Mass Spectroscopy (LC-MS) based whole-proteome analysis. E. coli and S. cerevisiae cells were grown until the log phase (O.D_600nm~0.2) and exposed to two different concentrations (10¹¹ and 10¹² particles/mL) of both 5 and 10 nm AuNPs (Figure 5A & H). After 4 hours the cells were harvested, lysed in lysis buffer, digested with trypsin to generate peptides, and subjected to LC-MS for peptide identification. Three independent cultures (biological replicates) were used for each AuNP concentration, and the unexposed cell lysate protein profile was used as a baseline to determine the differential expression of protein levels across concentrations (S.I. Figure 11 & 12).

In E. coli, exposure to 5 and 10 nm AuNPs significantly altered (p<0.05) the expression of 41 and 18 proteins, respectively, compared to unexposed cells (Figure 5B and E). A protein-protein interaction network (Figure 5C and F) revealed exclusive enrichment of proteins involved in ribosome assembly/biogenesis (Figure 5D and G), irrespective of the size and dosage.

Exposure to smaller (5 nm) AuNPs altered the expression of proteins involved in various metabolic processes, including glycolysis, protein folding, and amino acid synthesis. Interestingly, irrespective of size, exposure to a low dosage (10¹¹ particles) resulted in the under-expression of most ribosomal and metabolic proteins, with the exception of a few (Figure 5D). On the other hand, exposure to a higher dosage, irrespective of size, resulted in partial restoration of the expression of the majority of proteins, which was comparable to that in unexposed cells (Figure 5 D & G ,S.I. Figure 11C & F) . Irrespective of dosage exposure to 5 nm AuNP resulted in the overexpression of RplU (50s ribosomal subunit protein), RplC (50s ribosomal subunit protein), BipA (ribosome binding GTPase), Bcp (peroxiredoxin), and GnsA (suppressor of SecG involved in lipid synthesis⁴¹) proteins in E. coli.

In the case of 10 nm, overexpression of PNPase protein (an endoribonuclease involved in mRNA⁴² stability) was observed compared to that in unexposed cells (Figure 5G). Interestingly, overexpression of the PNPase protein in cells with MazF toxins, has shown to generate stressed ribosomes, which promotes protein synthesis via the non-canonical mode³¹. Also, it is important to highlight that exposure to higher dosages of AuNPs resulted in the overexpression of stress associated proteins such as RpsO, BipA, OmpT, RpsC, RpsA, RpsN, and YqhD^43–48. Based upon this data, we propose that exposure to AuNPs leads to an under-expression of most ribosomal proteins and over-expression of stress associated proteins in a size and concentration dependent manner.

In S. cerevisiae, when compared across dosage exposure to 5 and 10 nm AuNPs significantly altered (p<0.05) the expression of 27 proteins in each, compared to unexposed cells (Figure 5I and L). Regardless of the size of the AuNPs, a protein-protein interaction network revealed exclusive enrichment of proteins involved in ribosome assembly/biogenesis (Figure 5J & M). Unlike E. coli the metabolic response of yeast is more consistent, while the expression of ribosomal and metabolic proteins was altered in a dose-dependent manner, with few exceptions (Figure 5K & N). Irrespective of size and dosage, under-expression of a majority of ribosomal proteins was quite evident, with the exception of RPS25A (40s ribosomal subunit) and RPL19 (60s ribosomal subunit) for 5 nm and RPS3, RPS30B (40s); RPL17B, RPL24A, RPL24ap (60s) in the case of 10 nm. Interestingly, overexpression of ribosomal protein homologs, such as RPS3, RPL19, RPL24, and RPL17, has been reported in different types of cancers^49,50. Also, overexpression of RPS3 and RPS30 proteins can alter mRNA selection for translation initiation (non-canonical mode) and thus modulate protein synthesis^51,52. Further, the exclusive overexpression of proteins such as ILV5 (involved in branch amino acid synthesis), PDC1 (pyruvate decarboxylase), ENO2 (phosphopyruvate hydratase) and HSP12 (chaperone, involved in peptide folding), indicated an increase in cellular metabolic activity that can be correlated with an accelerated growth rate of the cells. Furthermore, the expression of RPL19, RPS25A, RPL24A, PDC1, and HSP12 has been implicated in the unfolded protein stress response (UPR) and cap-independent mRNA translation initiation via IRESs (non-canonical mode) ^53–58.

Based upon this data, we propose that in both bacteria and fungi, exposure to high-dose of AuNPs exerts stress on translation machinery as majority of ribosomal proteins are under-expressed in cells with increased cell-division rate. Interestingly, no known stress response is induced or required to do so. Further, an increased expression of proteins involved in carbohydrate metabolism, amino-acid synthesis, protein folding, supported an increase in cellular metabolic activity upon AuNP exposure. Moreover, an increased expression of some stress proteins involved in stress ribosome assembly, UPR, cold stress and in non-canonical mode of translation, it supports our argument that cells experience stress on its translation machinery thus exploits both, the canonical and non-canonical, mode of protein synthesis and increase its rate of protein synthesis. This might be an adaptive response to optimize protein synthesis under stressful conditions, which leads to rapid cell proliferation.

AuNP-induced accelerated growth rate is sensitive to sub-MIC of ribosome-targeting drugs Given an accelerated cellular growth demands an increased proteins synthesis rate, under-expression of the majority of ribosomal proteins was counter intuitive. But, it has been well documented that both mRNA and ribosomes are always in surplus⁵⁹, and that only a selected pool of ribosomes and mRNA is involved in translation⁶⁰. Interestingly, the observed accelerated growth was exclusive to nutrient-poor media, not to the nutrient-rich media such as LB broth. The further acceleration in growth rate wasn’t possible in LB broth as cells have already achieved the highest possible metabolic rate with cell division time of ~27 minutes. Based on these observations, we argue that exposure to high concentrations of AuNPs exploits the existing pool of ribosomes and RNA to accelerate protein synthesis rate through both canonical and non-canonical modes. This argument is supported by the following observations: (a) overexpression of PNPase in E. coli (Fig 5D), known to be involved in stress ribosome assembly and in promotion of translation via the non-canonical mode; (b) overexpression of RPS3 and RPS30 in S. cerevisiae (Figure 5N), which modulates the selection of mRNA for translation initiation, and (c) under-expression of ribosomal proteins involved in the canonical mode of translation initiation (Fig 5D, G, K & N). To test our hypothesis, we treated bacterial cells with sub-MIC dosages of drugs that targets ribosome assembly and recycling, such as aminoglycosides (Kanamycin, Tetracycline & Chloramphenicol) for bacteria, and rapamycin for yeast, which inhibits the mTOR pathways involved in ribosome assembly⁶¹. Ampicillin and Tebuconazole, membrane-targeting drugs commonly used in bacteria and yeast respectively, were used as controls to confirm the specific role of the available functional ribosomal pool.

Growth rate and cell-viability of E. coli and S. cerevisiae exposed to 10¹² AuNPs and varying sub-MIC dosages of aminoglycosides at log phase was monitored (Figure 6A & S.I. Figure 13). The data revealed that treatment with sub-MIC dosages of ribosome-targeting antibiotics/drugs, such as Kanamycin (1.25 & 7.5 µg/mL for 5 nm and 10 nm, respectively), Tetracycline (0.25 & 0.5 µg/mL for 5 nm and 10 nm, respectively), Chloramphenicol (3.75 µg/mL for 5 nm and 10 nm both) in E. coli, and Kanamycin (15 µg/mL for 5 nm and 10 nm both) and Tetracycline (3 µg/mL for 5 nm and 10 nm both) in B. subtilis, inhibited the observed accelerated growth rate as well as higher cell-density (S.I. Figure 13). Similarly, treatment with a sub-MIC dosage of Rapamycin (25 µg/mL for 5 nm and 10 nm both ) inhibited the accelerated growth rate of S. cerevisiae cells upon exposure to a high dose of AuNPs. However, treatment with sub-MIC doses of non-ribosome-targeting antibiotics/drugs did not have an impact on the accelerated growth rate and higher cell density in both bacteria and S. cerevisiae (Figure 6 E & F and S.I. Figure 13). The specific suppression of accelerated growth only in the presence of sub-MIC dosage of aminoglycosides provides further support for our hypothesis that the increased growth rate is achieved through modulation of the protein synthesis rate, particularly by increasing the pool of functional ribosomes. Similar adaptive strategy has been reported in cancer cells, which downregulates canonical modes of translation initiation and upregulates translation initiation via non-canonical mode to increase the amount and spectrum of expressed proteins. By doing so, under nutrients limitation and hypoxic environment, cancer cells are able to meet the heightened demands of rapid proliferation (Sriram A, EMBO Reports 2018).

In summary, this study provides valuable insights into the accelerated cellular growth observed upon exposure to AuNPs, which is likely to be achieved through the modulation of the functional pool of ribosomes within the cell. Still, there are some unanswered questions regarding the mechanisms underlying the effects of AuNPs on cellular growth. Specifically, it remains unclear (a) how cells sense the presence of AuNPs, (b) whether AuNPs interact directly or indirectly with the ribosomes, and (c) why the threshold concentration of AuNPs (10¹² particles), which corresponds to approximately 10⁷ particles per cell, is critical for the observed effects.

These findings have several important implications. First in therapeutics (a) there is a need to optimize the dosage of AuNPs for drug delivery (chemotherapeutic drugs or Antibiotics), to prevent proliferation of cancer cells or pathogenic bacteria, and (b) AuNPs might act as a proliferating agent for in vitro cell-culture or stem-cells to promote rapid growth. Second in biotechnology industry it might act as a supplement to increase biomass production in fermentation as well as in recombinant DNA technology.

Strains and Chemicals

All strains used in the study are listed in Table-2. Gold (III) chloride hydrate (HAuCl₄) and Tannic acid were purchased from Sigma-Aldrich. Citric acid trisodium salt dihydrate was obtained from Amresco, whereas potassium carbonate (K₂CO₃) was purchased from Thermo Fisher Scientific India Pvt. Ltd. Luria Bertani (LB) agar powder and Yeast Peptide Dextrose (YPD) were obtained from HiMedia (India). M9 minimal salts (5x) and Cas Amino acids (CAA) were purchased from Sigma-Aldrich. The growth media used were M9 salts with 0.2% glucose (M9 glu) for E. coli and P. aeruginosa; M9glu supplemented with 1.0% CAA for B. subtilis; LB for S. aureus; and YPD for S. cerevisiae.

Synthesis and Characterization

We synthesized 5 nm and 10 nm gold nanoparticles (AuNPs) using a modified citrate reduction (Turkevich) method. For the synthesize 5 nm AuNPs, 0.15 mM of HAuCl₄.3H₂O was slowly added to a preheated (75°C) reducing solution containing 2.2 mM sodium citrate tribasic dihydrate and 1.56 mM tannic acid while constantly stirring at 650 rpm. To synthesize 10 nm AuNPs, we added 0.31 mM HAuCl₄.3H₂O drop-wise to a preheated (75°C) reducing solution containing 2.2 mM sodium citrate tribasic dihydrate and 0.62 mM tannic acid while stirring constantly at 650 rpm. The reaction was allowed to continue for 15 minutes after the appearance of the wine-red color and then stopped by adding potassium carbonate (K₂CO₃, 1 mM). The synthesized AuNPs were washed by centrifugation and resuspended in equal amounts of Milli-Q water. The AuNPs were characterized using various techniques, including ultraviolet-visible spectrophotometry (MultiSkan GO), transmission electron microscopy (Talos S), dynamic light scattering (Malvern Zetasizer), and Fourier-transform infrared spectroscopy (FTIR) (Bruker, Tensor 37).

For the synthesis of fluorescence-capped gold nanoparticles, Rhodamine 6 G dye was used. The synthesized gold nanoparticles (both 5 and 10 nm) were stirred at room temperature for 30 minutes followed by the addition of 0.01 mM Rho 6G dye. The solution was further stirred for 30 minutes. The synthesized AuNPs were washed by centrifugation and resuspended in equal amounts of Milli-Q water. The AuNPs were characterized using ultraviolet-visible spectrophotometry and fluorescence spectroscopy.

AuNPs stability and uptake

Various concentrations of AuNPs, ranging from 0 to 10¹²per mL, were added to different media including water, LB, M9 salts with 0.2% glucose (M9glu), YPD and Spiz broth, and their stability was assessed by comparing the absorbance spectra (300-700 nm). To determine the uptake of AuNPs by microorganisms, 10¹²AuNPs per mL were incubated with varying concentrations (ranging from 10⁶ to 10¹¹ CFUs per m) of Escherichia coli (MJ35), Bacillus subtilis (MJ476), and S. cerevisiae (MJ512) in water for 4 hours. After 4 hours, the cells were harvested, and the absorbance of the supernatant was measured at 514 and 518 nm for 5 and 10 nm diameters, respectively. The decrease in absorbance in the supernatant indicated the uptake of AuNps by microorganisms.

Growth rate and cell-viability

Escherichia coli (MJ35), Bacillus subtilis (MJ476), and S. cerevisiae (MJ512), were cultured in M9 Glucose, M9 Glucose with 0.1% casamino acids (37°C), and YPD broth (30°C), respectively, with constant shaking for 18 hours. Cells were sub-cultured (1:1000 dilution) in fresh media, and AuNps were added (0 and 10¹² AuNPs/mL) in U-bottom 96-well plates (Genaxy GEN-MTP-96U-S). Cell growth (OD₆₃₀ _nm) was monitored every hour in a BioTek 800 TS microplate reader at 37°C under constant shaking for 18 hours. After 18 hours, the cells were diluted and viable cell numbers were counted by spotting or spreading on M9glu agar plates. The absorbance of AuNPs alone (for each concentration) in the given media was subtracted before plotting the growth curve.

SEM

Escherichia coli (MJ35), Bacillus subtilis (MJ476), and S. cerevisiae (MJ512) were grown until mid-log and exposed to 10¹² AuNPs per mL. After 4 hours of exposure to AuNPs, the cells were washed three times with 1x phosphate buffer solution (PBS) at pH 7.0, and then fixed by incubation in 2.5% paraformaldehyde at 4°C for 16-18 hours. The fixed cells were washed, dried, and mounted on a stub. After sputter-coating with gold, the samples were observed under an EVO 18 Scanning Electron Microscope (SEM).

C.F.U (live cells counting) and Propidium Iodide staining (Dead cell imaging assay)

Escherichia coli (MJ35) cells were grown in M9 glucose until the mid-logarithmic phase (OD_600nm ~0.2) and then exposed to high concentrations of AuNPs (10¹²particles/mL) for 4 hours. The cells were harvested by centrifugation. For live cells determination, cells were serially diluted in 1x PBS and C.F.U per mL was determined. For dead cell staining, cells were resuspended in 1x phosphate buffer solution (PBS) at pH 7.0 and split into two groups. The control cells were fixed by incubation in 2.5% paraformaldehyde at 4°C. Both, fixed and non-fixed cells were incubated with 0.01 mg/mL for 4 hours. After incubation cells were centrifuged at 8,000 rpm for 5 minutes at room temperature. The pellet was washed with 1x PBS and the uptake of PI dyes was determined using spectrophotometer (Jasco V-660). All samples were excited at a wavelength of 493 nm and emission spectra were measured in the spectral range of 550-650 nm.

Flow cytometry

Escherichia coli (MJ35, MJ976 and MJ269) and S. cerevisiae (MJ512) were grown in M9 glucose and YPD until the mid-logarithmic phase (OD_600nm ~0.2) and then exposed to high concentrations of AuNPs (10¹²particles/mL) for 4 hours. The cells were then washed three times with 1x phosphate buffer solution (PBS) at pH 7.0, and fixed by incubating them in 2.5% paraformaldehyde at 4°C for 16-18 hours. The cells were analyzed using CytoFLEX (Beckman Coulter, USA). Flow cytometry data were analyzed using CytExpert (Beckman Coulter, USA).

Fluorescence Microscopy

For strains E. coli-MG1655 FtsZ-GFP (MJ976) and E. coli-MG1655 RecA-GFP (MJ269), cells were grown in M9gluc medium until the mid-logarithmic phase of the cell cycle and then exposed to the highest concentration of AuNPs (10¹² particles/mL). After 4 hours of exposure, cells were fixed with 2.5% paraformaldehyde, washed, and resuspended in 1x PBS (pH 7.0). The number of cells with FtsZ/RecA-GFP foci was counted using a Zeiss Axio-observer 7.0 microscope with a monochrome CCD camera.

RpoE activity

MJ476 (E. coli-MG1655; ϕʎ (rpoHP3::lacZ)) was grown in M9 glucose until the mid-logarithmic phase (O.D_600nm ~0.2) and was exposed to three different concentrations of AuNPs (0, 10¹¹ and 10¹²). RpoE activity of the strains after four hours of exposure was determined using a previously published protocol⁴⁰. ΔRseA strain was used as a positive control because it constitutively expresses the RpoE gene.

LC-MS

Escherichia coli (MJ35) and S. cerevisiae (MJ512) were grown in M9 glucose and YPD until mid-logarithmic phase (OD_600nm ~0.2) and then exposed to three different concentrations of AuNPs (0, 10¹¹and 10¹²) for 4 hours. Cells were harvested and lysed with 5% SDS + 50mM TE buffer (pH 8.0). Next, 50 µg of cell lysate protein was reduced with 5 mM TCEP, alkylated with 50 mM iodoacetamide, and digested with trypsin (1:50 trypsin/lysate ratio) for 16 hours at 37°C. The digested peptides were purified using a C18 silica cartridge and dried using a SpeedVac. The pellet was resuspended in buffer A (2% acetonitrile, 0.1% formic acid), and 1 µg was loaded onto a 50 cm C18 column with 3.0 µm Easy-spray particles (Thermo Fisher Scientific). Peptides were eluted with a 0–40% gradient of buffer B (80% acetonitrile, 0.1% formic acid) at a flow rate of 300 nl/min and then injected for mass spectrometry (MS) analysis using an EASY-nLC 1200 system (Thermo Fisher Scientific) coupled with a Thermo Fisher QExactive Plus instrument equipped with a nanoelectrospray ion source.

LC gradients were run for 100 minutes, and MS1 spectra were acquired using Orbitrap at 70,000 resolution. Dynamic exclusion was employed for 10 seconds, excluding all charge states for a given precursor. The MS2 spectra were acquired at resolution of 17,500. RAW files containing peptide information were generated and analyzed using Proteome Discoverer against the UniProt proteome database. For the Sequest and Amanda searches, the precursor and fragment mass tolerances were set at 10 ppm and 0.02 Da, respectively. The protease used to generate peptides was trypsin/P (cleavage at the C-terminus of “K/R” unless followed by “P”), with a maximum missed cleavages value of two. Carbamidomethyl on cysteine was set as a fixed modification, and the oxidation of methionine was considered a variable modification for database searches. Both the peptide spectrum match and the protein false discovery rate were set to 0.01 FDR. Raw abundance values were used for statistical analyses. The values were filtered based on valid values (quantified in at least two out of three replicates for each condition). Filtered values were log₂ standardized, followed by imputation of missing values based on the normal distribution. The values were normalized, Student’s t-test was applied, and the significance was calculated using p-values. All the proteins for which the significance score (p-value) was less than 0.05 were used as significant proteins and further used for data visualization. A protein-protein interaction map for significantly altered proteins was generated using the STRING database (https://string-db.org/).

Sub-MIC drug sensitivity of AuNP exposed cells

For screening sensitivity to sub-MIC dosage of drugs, cells were sub-cultured (1:1000 dilution) in fresh media in presence of AuNPs (0 and 10¹²/mL) and sub-MIC dosage of Kanamycin, Tetracycline, Chloramphenicol for Escherichia coli (MJ35) and Bacillus subtilis (MJ476) as well as sub-MIC dosages of Rapamycin (500 nM) & Tebuconocazole (1.6 µg/mL) for S. cerevisiae (MJ512) was optimized. Cell growth (OD_{630 nm}) was monitored every hour in a BioTek 800 TS microplate reader at 37°C under constant shaking for 18 hours. After 18 hours, the cells were diluted, and viable cell numbers were counted by spotting or spreading on M9glu agar plates.

ACKNOWLEDGEMENTS

The authors would like to acknowledge Jamia Millia Islamia for providing the necessary infrastructure to carry out this research. We also acknowledge Central Instrumentation Facility-Jamia Millia Islamia (CIF-JMI) for providing FTIR and Raman spectroscopy facilities, and Centre for Nanoscience and Nanotechnology, JMI for X-ray diffraction analysis. We also acknowledge JMI-UG/PG instruments grant for providing Microplate reader and other instrument facility at MCARS. We are grateful to Sophisticated Analytical Instrumentation Facility-All India Institute of Medical Science (SAIF-AIIMS, New Delhi) for providing SEM, TEM, and DLS facilities. We would like to thank Asim ul Islam (CIRBSc, JMI) for providing Fluorescence spectrophotometer instrument facility, Tanveer Ahmad (MCARS, JMI) for providing Rapamycin, Deepak Dutta (IIMTech Chandigarh) for providing Keio collection strains, Sabri Shankar (IISER-Trivandrum) for providing Bacillus strains, Rachna Chaba (IISER Mohali) for providing strains to measure RpoE activity, Krishnaveni Mishra (University of Hyderabad) for providing the yeast SOD1 knock out strain, and Ramajuam Srinivasan (NISER Bhubaneswar) for providing E. coli TZG (ftsZ-gfp::Cm) strain.

We would like to express our gratitude to Thelma B.K. (UDSC, New Delhi) for her unwavering support during the course of this research. Shabina Quadir acknowledges the Indian Council for Medical Research (ISMR) for providing her with a Senior Research Fellowship [NO.ISRM/11(87)/2019]. Deepak Kumar Singh acknowledges the University Grants Commission (UGC) for providing him with a Senior Research Fellowship (June18-319422). Amir Faraz is grateful to the Department of Biotechnology (DBT) for providing him with a Senior Research Fellowship (DBT/2019/JMI/1260). Nuha Abeer Khan would like to thank the Department of Biotechnology (DBT/2021-22/JMI/1585), DST-INSPIRE (IF191000), and PMRF (3302527) for providing her fellowship.

FUNDING

Dr. Mohan C. Joshi acknowledges the funding support provided by the Wellcome Trust/DBT India Alliance under grant number IA/I/15/2/502086, SERB-ECR (FILENO.ECR/2017/000976), and UGC-FRP (F.4-5/236 FRP (2015) BSR).

ASSOCIATED CONTENT

This work has been submitted to preprint server “Shabina Quadir, Nuha Khan, Deepak Singh, Amir Faraz, Gagan Jhingan, Mohan Joshi. Exposure to high dosage of gold nanoparticles accelerates growth rate in bacteria and yeast by modulating ribosomal protein expression. 2023, Research Square, DOI: https://doi.org/10.21203/rs.3.rs-2446306/v1”.

Table 1: Cell-division time

Strains		0 AuNP	5 nm AuNP (10¹² particles/ml)	10 nm AuNP (10¹² particles/ml)
		0 AuNP	5 nm AuNP (10¹² particles/ml)	10 nm AuNP (10¹² particles/ml)
		Td (min.)	Td (min.)	Td (min.)
Wild-type E.coli (MG1655)		89	79	69
DNA damage mutants	ΔrecA	173	109	120
DNA damage mutants	ΔrecN	103	66	64
Oxidative stress	ΔsodA	151	59	68
Oxidative stress	ΔsodB	99	74	88
Envelop stress mutants	ΔpspC	88	72	67
	ΔrseA	95	54	53
	ΔrcsF	95	59	55
	ΔbaeS	88	61	51
	ΔcpxA	111	68	47
Starvation stress mutants	ΔrpoS	128	103	92

Table 2: List of strains

S. No.	Strain No.	Genotype	Source
1	MJ 35	MG1655	DB1607
2	MJ 269	T28recA-gfp-Kan/recA wt	Construct from Christain Leseterlin
3	MJ380	ΔrecN::kan [P1 MJ78 (ΔrecN) in to MJ35 (MG1655]
4	MJ435	Staphylococcus aureus	MTCC1430
5	MJ437	Pseudomonas aeruginosa
6	MJ 467	E. coli (MG1655) ϕʎ (rpoHP3::lacZ))	Dr. Rachna Chaba, IISER Mohali
7	MJ472	ΔrecA::Kan [P1 MJ154 (ΔrecA) in to MJ35]	Made for this study
8	MJ474	ΔrpoS::kan [P1 MJ461 (ΔrpoS) in to MJ35]	Made for this study
9	MJ 476	PG1 (Bacillus subtilis)	MTCC121
10	MJ478	PG768 (B. subtilis ΔrecA)
11	MJ498	MG1655 ΔpspC::kan	This study
12	MJ500	MG1655ΔrseA::kan	This study
13	MJ502	MG1655ΔrcsF::kan	This study
14	MJ504	MG1655 ΔbaeS::kan	This study
15	MJ506	MG1655 ΔcpxA::kan	This study
16	MJ512	Yeast BY4741	Dr. Krishnaveni Mishra Univ. of Hyderabad
17	MJ518	MG1655 (ϕʎ (rpoHP3::lacZ))	This study
18	MJ807	ΔsodA, E. coli, Keio collection	Dr. D. Dutta IMTech.
19	MJ809	ΔsodB, E. coli, Keio collection	Dr. D. Dutta IMTech.
20	MJ904	ΔSOD1 (S. cerevisiae)	Dr. Krishnaveni Mishra Univ. of Hyderabad
21	MJ976	E. coli TZG (ftsZ-gfp::Cm)	Prof. Ramajuam Srinivasan NISER Bhubaneswar
22	MJ998	ΔsigB::kan, B. subtilis	168 ΔsigB::kan (Dr. Sabari IISER Thiru)
23	MJ1002	ΔsigM::kan, B. subtilis	168 ΔsigM::kan (Dr. Sabari IISER Thiru)

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Exposure to High Dosage of Gold Nanoparticles Accelerates Growth Rate by Modulating Ribosomal Protein Expression.

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