Metal Solubility and Production of Reactive Oxygen Species

The Oregon Blue clay completely kills a broad range of human bacterial pathogens, including antibiotic resistant strains (see Methods). To understand the antibacterial mechanism exhibited by the Blue clay, metal solubility and production of ROS from clay suspensions in water were measured and compared to metal toxicity from metal solutions alone. Antibacterial susceptibility testing was performed using the model Gram-negative bacterial species E. coli (ATCC 25922). The Blue clay mineral assemblage releases mM concentrations of metals (Fe2+, Fe3+, Al3+ and Ca2+) when hydrated with deionized water (Fig. 1A, Table S1), through pyrite oxidation, illite-smectite cation exchange and dissolution of plagioclase feldspar10. Other metal species (e.g., As, Ag, Hg, Pb, Cu, Zn, Ni) are present in nM to μM concentrations, below levels that inhibit bacterial growth14,18 (Table S1).

Figure 1 Clay suspensions provide extended metal release whereas aqueous leachates alone precipitate metals. (A) Major element concentrations leached from the Blue clays. (B) pH, MIC and MBC values for leachates (gray bars) and clay suspensions (blue bar) measured in MSA and LB media. The leachate pH is similar at MBC in both media, while clay suspensions kill bacteria at higher pH. A comparison of soluble metal concentrations (C,D) show clay suspensions kill bacteria by maintaining metal solubility over 24 h, while (E,F) leachates precipitate metals, only inhibiting growth. Full size image

Clay suspensions in deionized water and their aqueous leachates (equilibrated 24 h, the time it takes to kill bacteria) were prepared for antibacterial susceptibly testing. Recognizing that soluble elements speciate differently in various growth media, we compared the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of clay suspensions and leachates in two different growth media; minimal salts and amino acids (MSA) media and Lennox broth (LB) (Fig. 1B, Table 1). Increases in pH occurred when clay leachates were mixed with growth media due to metal speciation, causing Fe3+ and Al3+ precipitation, which reduces their bioavailability and toxicity (Fig. 1C–F). This is an important point because many toxicity studies have not considered precipitation and bioavailability of metals to bacteria14,18. Speciation calculations (Fig. S2) based on the major inorganic ions in each growth media were performed, organics from the growth media were not included due to the variability of oligopeptides and free amino acids available for reaction19. The potential formation of organic ligands with amino acids in the growth media could reduce the concentration of metals available to react with the bacteria. Organic ligands are not thought to contribute to the antibacterial action because the killing occurs at a similar rate in experiments performed with and without growth media8. Speciation modeling of clay suspensions at bactericidal pH and Eh conditions show that Fe2+ primarily exists in solution as Fe2+ and FeSO 4 in MSA media but in LB precipitates as Fe 3 O 4 . The speciation of Fe3+ in both media is dominated by insoluble Fe(OH) 2.7 Cl 0.3 . Aluminum is present as aqueous AlSO 4 + in MSA media but precipitates as Al(OH) 3 in LB. Calcium is present as aqueous Ca2+ and CaSO 4 in both media. However, these calculations assume that the system is in thermodynamic equilibrium. The antibacterial activity of the Blue clays occurs as the minerals approach a new oxidized equilibrium, with no antibacterial activity occurring in fully oxidized samples10. Therefore, thermodynamic speciation calculations will not provide a realistic portrayal of the speciation during the antibacterial mechanism. In order to gauge the concentration of soluble metals available to interact with the bacteria, we monitored the solubility of Fe2+, Fe3+ and Al3+ throughout the antibacterial process. These data are presented in Fig. 1C–F and Tables 1, 2.

Table 1 MIC, MBC and pH values for Blue clay leachates and aqueous metal mixtures (Fe2+, Fe3+, Al3+) reacted with E. coli growing in MSA and LB media for 24 hr at 37 °C. Full size table

Table 2 Individual metal MIC, MBC and pH values for E. coli measured in MSA and LB media. Full size table

Unbuffered clay leachates precipitate metals and thus require higher concentrations for toxicity (Fig. 1). However, the Blue clay suspensions maintain low pH, while sustaining Fe2+ and Al3+ release through mineral oxidation and dissolution, requiring a lower dose for toxic effect. Acid and base titrations of the growth media reveal that the LB has a greater buffering capacity compared to the MSA media (Fig. S3). We observed the highest levels of metal precipitation in the LB media due in part to the increased buffering capacity, which maintains a higher pH and precipitates Fe and Al solids (Fig. 1 and S2). The leachate MBC for E. coli occurs at pH 3.5, while clay suspension MBC reaches pH 4.2–4.6 (Fig. 1B); indicating that the pH is not the sole factor for killing bacteria. Synthetic metal chloride mixtures of Fe2+, Fe3+ and Al3+, simulating the clay leachate concentrations, produced similar MIC and MBC (Table 1), supporting Fe and Al as the primary bactericidal elements. Importantly, the metal combination is more toxic than single metal solutions (Tables 1, 2).

The hydrated Blue clay lowers solution pH < 5, at the MBC, where pyrite oxidation and soluble Fe2+ react with molecular oxygen to produce hydrogen peroxide, (H 2 O 2 )20 (Fig. 2). Without the clay assemblage, metal oxides precipitate and H 2 O 2 production stops. The oxidation state of the Blue clay aqueous suspension10 reaches Eh ≥ 500 mV. Under these conditions the oxidation of pyrite is known to generate H 2 O 2 (Fig. 2), which then reacts with Fe2+ to form hydroxyl radicals (·OH) through the Fenton reaction series16,20. Clay suspensions produced H 2 O 2 over 24 h, while aqueous leachates without minerals ceased H 2 O 2 production after 2 h in both MSA and LB media (Fig. 2A,B), showing that the clay is important as a prolonged source of reactants. Therefore, metal solutions alone are limited as a bactericide due to their inability to sustain H 2 O 2 production. Pyrite oxidation by molecular O 2 can directly generate H 2 O 2 on the crystal surfaces20. However, at low pH (<4.5) Fe3+ can also oxidize pyrite, releasing soluble Fe2+ that can produce H 2 O 2 3,20. The expandable smectite component of the Blue clays may serve as a reservoir for Fe2+, as divalent cations are preferred over trivalent cations in the smectite interlayer9, providing extended Fe2+ release when cation exchange occurs10 during rehydration for medicinal application. These combined oxidation reactions can explain the elevated H 2 O 2 levels observed in the Blue clay suspensions, leading to sustained ROS production and killing pathogens via geochemical processes (Fig. 2).

Figure 2 Generation of H 2 O 2 is maintained by clay suspensions in (A) MSA and (B) LB-media, while leachates cease production after 2 h. These data show that clay suspensions sustain metal release and H 2 O 2 production that leads to cell death. Full size image

Metal Hydrolysis

Pyrite is present in the Blue clays (3–5%), which can produce sulfuric acid (H 2 SO 4 ) during oxidation10,20. The hydrolysis of soluble metal cations can also generate acid as metals precipitate21. The pH and solubility of individual metal cations (Fe2+, Fe3+ and Al3+) were measured at the MIC and MBC in order to evaluate the role of metal hydrolysis reactions relating to metal solubility and toxicity (Table 2). Results show that bacterial metal toxicity is also linked to solution pH and metal solubility, with acidic metal cations (Al3+ and Fe3+) generating the most acid (Fig. 3). At MIC and MBC, the pH and solubility of individual metals correlate with their pKa (hydrolysis constant). Fe3+ and Al3+ hydrolysis produce acid that compounds their toxicity (eqn. 1), while precipitation occurs at pH values > pKa21. The amount of acid generated through metal hydrolysis is greatest for Fe3+ > Al3+ > Fe2+ > Ca2+. Thus, Fe3+ alone produced the lowest pH causing cell death at lower concentrations than Fe2+ and Al3+. Therefore, acid production from Fe3+ and Al3+ hydrolysis reaction are an overlooked aspect of metal toxicity and may enhance the toxicity of other metals with low pKa values (<5). We observed that leachates and synthetic metal mixtures produced similar pH, indicating that metal hydrolysis plays a greater role in lowering pH than pyrite oxidation in the Oregon Blue clays (Table 1).

Figure 3 Metal hydrolysis reactions generate acid; therefore, pH, metal solubility and toxicity correlate with pKa. These plots show average MIC and MBC for individual metals in MSA and LB media correlating with (A) pH and (B) soluble metal content. Full size image

Bioimaging

Bacteria adsorb a range of metals on their cell envelopes due to a high anionic charge density from phosphate and carboxyl groups22,23. We used elemental bioimaging to examine the adsorption, location and redox state of metals reacting with E. coli (Figs 4 and 5). Clay suspensions (100 mg/ml) reacted with E. coli were analyzed in hydrated state using synchrotron scanning transmission X-ray microscopy (STXM, Advanced Light Source 11.0.2) for C, K+, Ca2+, Fe2+ and Fe3+ (Fig. 4A–D). Near edge x-ray absorption fine structure (NEXAFS) spectra of C and K show K-rich particles adhered to cells (Fig. 4A). Illite-smectite (I-S) dominates the deposit mineralogy10 and is the likely source of K. Linear-regression fitting of Fe spectra with reference compounds24 (Fig. S1) shows that Fe is present in I-S particles predominantly as Fe2+ (81%). Most likely this Fe2+ is located in the I-S octahedral sites or interlayers (Fig. 4B). Soluble Fe2+ (89–99%) is adsorbed preferentially to cell envelopes (Fig. 4B,C), while Ca2+ remains in solution (Fig. 4D). In E. coli, Ca2+ and Mg2+ adsorbed to phosphate-rich lipopolysaccharides provide outer membrane stability; however, under acidic conditions H+ can displace these cations25.

Figure 4 STXM elemental maps of E. coli reacted with a clay suspension showing corresponding NEXAFS spectra beside each map (A-D). Elemental maps show (A) protein-C from cells (blue) and K (yellow) from illite-smectite (I-S), (B) Fe distribution, showing Fe2+ adsorption on E. coli (green) and Fe in I-S particles (purple), (C) E. coli cells after 12 h incubation showing preferred Fe2+ (green) adsorption, leaving Fe3+ (red) in extracellular solution. Percentages of Fe2+ and Fe3+ were calculated using linear-regression fitting (see supplementary information). (D) Ca2+ (light blue) remains in solution not on cells. (Variable scales: E. coli width = 0.5 μm). Full size image

Figure 5 NanoSIMS image of E.coli showing, (A) 12C map of cells, (B) 54Al 2 outside (yellow) and 56Fe (red) inside cells. (C) A cross section of ion counts across the white arrow in (B). (D) STEM image of E. coli showing intracellular nanoparticles (white spots) developed after 24 h incubation. (E) EELS spectra of nanoparticles showing O (K-edge) and Fe3+ (L 2/3 -edge) identifying white spots as Fe3+-oxide precipitates. (Scale: E. coli width = 0.5 μm). Full size image

Nano-scale secondary ion mass spectrometry (NanoSIMS) maps of C, Al and Fe were generated to determine their binding sites in the cell (Fig. 5A,B). Cells were measured after reaction with a 100 mg/ml Blue clay suspension for 12 hrs. A clump of E. coli cells were sputtered with the primary ion beam, exposing the cell interior and Fe and Al maps were generated (see Methods). NanoSIMS maps of Al and Fe (Fig. 5B,C) show that Al binds to bacterial membranes, while Fe enters the cell. E. coli that were not reacted with Blue clay showed no intracellular Fe (below detection limit), consistent with previous studies indicating 354 and 2662 ppm intracellular Fe concentrations in control and clay reacted cells, respectively8. The mechanism for Al toxicity is unknown, but it has been shown to compete with Ca and Mg for phosphate binding and may damage membranes17. Previous measurements of extracellular elements in E. coli reacted with the Oregon Blue clay leachates, indicated that Fe and Al replace membrane bound Ca and Mg8. STEM bioimaging of E. coli treated with Blue clay leachates show intracellular nanoparticles (electron dense white spots in Fig. 5D) after 24 hr incubation, coincident with cell death. Using scanning transmission electron microscopy-electron energy loss spectra (STEM-EELS) these spots were determined to be Fe3+-oxides (Fig. 5D,E).

The STXM and NanoSIMS bioimaging results reveal that bacterial membranes remain enriched in Fe2+ and Al3+ throughout the antibacterial process (Figs 4 and 5). The Blue clay suspensions maintain H 2 O 2 generation over 24 hrs (Fig. 2), which leads to production of toxic hydroxyl radicals (·OH) upon reaction with membrane adsorbed Fe2+ and encapsulates the cell in an oxidizing environment. The STEM-EELS images (Fig. 5D) showing Fe3+-oxide accumulations inside the cells likely formed from the oxidation of excess intracellular Fe2+ which may enter the cell through low affinity uptake systems14. The production of intracellular Fe3+-oxides requires oxidation, accompanied by formation of ·OH. Therefore, below we argue that the Fe3+ oxides reflect the transition to an oxidizing environment inside the cell, where proteins and DNA are damaged.

Protein Oxidation

Hydroxyl radicals are generated as Fe2+ is oxidized by H 2 O 2 through the Fenton reaction16,26. The proximity of ·OH generation to biomolecular targets is crucial to its toxicity because this radical exists only briefly (10–9 s half-lives) and diffuses only nanometers before reacting26. Thus, Fe2+-enriched bacterial membranes (Fig. 4C), reacting with H 2 O 2 formed outside the cells (Fig. 2), will generate ·OH in direct proximity to membrane proteins and lipids. Therefore, intracellular Fe2+ is required to generate ·OH that reacts with intracellular biomolecules, precipitating Fe3+-oxides where oxidative stress occurs (Fig. 5D).

We evaluated protein oxidation by measuring carbonyl content27 in separated membrane and soluble protein fractions of E. coli reacted with clay leachates and metal solutions (Fig. 6A). Membrane fractions of E. coli showed that the greatest protein oxidation (30–60 nmol-carbonyl/mg-protein) occurred upon exposure to leachates and metal solutions, while the soluble protein fractions (dominated by intracellular proteins) had lower carbonyl content (4–7 nmol/mg). This indicates that bacteria exposed to antibacterial clays first experience ROS stress targeting membranes, followed by intracellular protein oxidation in response to the influx of Fe2+.

Figure 6 Clay leachates produce high levels of envelope protein oxidation, activating σE and SOS stress responses. Plots show (A) protein carbonyl content (nmol-carbonyl/mg-protein) measured in envelope vs. soluble protein fractions of E. coli reacted with leachates and metal mixtures in MSA media, at MIC and MBC. (B) Envelope stress measured by σE-response from LacZ fusions in leachates compared to (C) single metal solutions. (D) DNA stress (SOS-response) measured on E. coli reacted with leachates and (E) single metal solutions. LacZ results are normalized to levels measured in control E. coli (Relative β-Gal Units). Full size image

Genetic Responses

Our results show that the synergistic activities of Fe2+, Fe3+ and Al3+ produce greater toxicity to bacteria at lower concentrations than individual metals. LacZ reporter gene fusions were employed to quantify bacterial stress responses to clay leachates and metals28,29,30. Genetic responses to bacterial envelope and DNA stress were evaluated in MSA-media using rpoHP3::lacZ (σE-response) and sulA::lacZ (SOS-response) gene fusion constructs (Fig. 6B–E). Outer membrane protein (OMP) misfolding from ROS can activate the σE-response, transcribing genes whose products regulate OMP proteolysis and folding28,29. Leachates caused the highest activation of σE, increasing rpoHP3::lacZ expression 4-fold upon reaction with a 1 mg/ml leachate solution (Fig. 6B,C). Weaker envelope stress occurred for individual Fe2+ and Al3+ solutions, requiring 77% more metal to reach stress levels similar to the clay leachate. This further supports the synergistic role of Al3+ and Fe2+ for membrane damage. Misfolding of OMPs by Al3+ may aid in protein oxidation by exposing amino acids to ·OH attack, supporting Al3+ as a pro-oxidant31. Pathogenic Gram-negative bacteria activate the σE-response to protect against ROS generated by host macrophages27. Our results demonstrate that metal binding to bacterial envelopes is capable of misfolding proteins, followed by ROS oxidation. Consequently, antibacterial clays may act in a manner similar to macrophages by engulfing and killing bacteria with metal-based ROS attack.

Intracellular oxidative stress can cause single strand breaks in DNA, triggering the SOS-response in E. coli, suspending cell growth until DNA is repaired30. Leachates and Fe2+ solutions produced similar DNA stress (Fig. 6D,E), while Fe3+ and Al3+ alone were not genotoxic. Thus, intracellular stress results from excess Fe2+ uptake. E. coli cells take in Fe2+ through high and low affinity uptake systems (Feo, ZupT), while trivalent cations (Fe3+) must enter through high affinity siderophore systems (TonB-ABC transporters)15. Al3+ substitution in Fe-S proteins, resulting in free intracellular Fe and ROS stress, has been proposed as a mechanism of Al3+ toxicity in Gram-negative bacteria32. However, our bioimaging (Fig. 5B,C) and genetic data show that Al3+ does not enter the cell or trigger the SOS-response. The toxicity of Al to cell membranes has been noted by several studies of acid rain17,33. In particular Williams17 (1999) found that Al3+ can only be transported into cells by chelators, as there are no pumps or channels for free Al3+ uptake or rejection. Because Al3+ cannot be reduced it accumulates at cell membranes where it bonds strongly to small oxygen donor ligands, such as phosphates17,33. Other researchers34,35 have observed that Al3+ alone does not produce lipid peroxidation, but enhances the Fe2+ peroxidation of phospholipids. Our research shows the combination of Fe2+ and Al3+ produces a greater genetic response to protein misfolding and oxidation in the outer membrane of E. coli, while genotoxicity only arises from the uptake and intracellular oxidation of Fe2+ (Fig. 6).