A Novel Silver-Ruthenium-Based Antimicrobial Kills Gram-Negative Bacteria Through Oxidative Stress-Induced Macromolecular Damage

Abstract

Amplified by the decline in antibiotic discovery, the rise of antibiotic resistance has become a significant global challenge in infectious disease control. Extraintestinal Escherichia coli (ExPEC), known to be the most common instigators of urinary tract infections (UTIs), represent such global threat. Novel strategies for more efficient treatments are therefore desperately needed. These include silver nanoparticles, which have been used as antimicrobial surface-coatings on catheters to eliminate biofilm-forming uropathogens and reduce the risk of nosocomial infections. AGXX^® is a promising silver coating that presumably kills bacteria through the generation of reactive oxygen species (ROS) but is more potent than silver. However, neither is AGXX^®'s mode of action fully understood, nor have its effects on Gram-negative bacteria or bacterial response and defense mechanisms towards AGXX^® been studied in detail. Here, we report that the bactericidal effects of AGXX^® are primarily based on ROS formation, as supplementation of the media with a ROS scavenger completely abolished AGXX^®-induced killing. We further show that AGXX^® impairs the integrity of the bacterial cell envelope and causes substantial protein aggregation and DNA damage already at sublethal concentrations. ExPEC strains appear to be more resistant to the proteotoxic effects of AGXX^® compared to non-pathogenic E. coli, indicating improved defense capabilities of the uropathogen. Global transcriptomic studies of AGXX^®-stressed ExPEC revealed a strong oxidative stress response, perturbations in metal homeostasis, as well as the activation of heat shock and DNA damage responses. Finally, we present evidence that ExPEC counter AGXX^® damage through the production of the chaperone polyphosphate.

Fig 1:. AGXX^®-stressed bacteria accumulate large amounts of ROS, which contribute to the bactericidal effects of this antimicrobial.

UPEC cells grown to mid-log phase were left untreated or treated with the indicated AGXX^®394C concentrations for 60 min before (A) intracellular ROS were quantified by H₂DCFDA, and (B; C) survival was determined by serially diluting cells in PBS and spotting 5μl onto LB agar for overnight incubation. (D) Intracellular superoxide levels were detected by DHE and (E) H₂O₂ quantified by Amplex ^™ Red. 70 mM thiourea was used to quench ROS; (n= 4-6, ±S.D., one-way ANOVA, Dunnet and Sidak’s multiple comparison test; ns = P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001).

Fig 2:. AGXX^® stress compromises bacterial membrane integrity.

(A) CFT073 cells in the mid-log phase were treated with the indicated AGXX^®394C concentrations for 60 min, washed in PBS, and stained with 0.5 μM PI. PI fluorescence (λ_Ex/Em: 535/617nm) was measured by spectrophotometry and normalized to untreated cells (n=3, ±S.D.). (B) Samples were washed in PBS after AGXX^®394C treatment, incubated with PI/Syto9 in the dark for 15 min at room temperature, and mounted on a glass slide with a 1% agarose pad for 63x imaging using inverted confocal microscopy. One representative image of 4 independent experiments is shown. (one-way ANOVA, Sidak’s multiple comparison test; ns = P > 0.05, * P < 0.05, ** P < 0.01).

Fig 3:. AGXX^® causes extensive protein aggregation and increases the cellular demand for molecular chaperones.

(A) Cellular IbpA-sfGFP fluorescence was monitored via flow cytometry after exposure of E. coli to sublethal AGXX^®394C treatment for 120 min; (n=5, ±S.D.). One representative image of five independent experiments is shown. (B) Exponentially growing cells were either left untreated or treated with sublethal AGXX^®394C concentrations for 90 min. Samples were harvested, washed with PBS, and incubated with DAPI (nucleic acid stain) and FM4-64 (membrane stain) in the dark for 15 min. Cells were mounted on a glass slide with a 1% agarose pad for imaging at 63x via inverted confocal microscopy. Arrows illustrate foci formed when IbpA binds to protein aggregates in vivo. One representative image of 4 independent experiments; [scale bar: 7.5 μm]. (C) Confocal images were quantified by counting IbpA-sfGFP foci (n=4) (one-way ANOVA, Sidak’s multiple comparison test; two-way ANOVA, Tukey’s multiple comparison test (compare total foci in untreated to AGXX treatment); ns = P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Fig 4:. AGXX^® causes DNA double-strand breaks.

(A) sulA mRNA levels of AGXX^®394C-treated UPEC CFT073 cells were determined by qRT-PCR. Transcript levels were normalized to the housekeeping gene rrsD and calculated as fold-changes based on the expression levels in the untreated control (n = 3, ±S.D.). (B) Cells expressing Gam-sfGFP were exposed to AGXX^®394C for 3 hours, washed, incubated with DAPI in the dark for 15 min, and visualized by confocal microscopy. Ciprofloxacin was used as a positive control. Arrows indicate Gam-sfGFP foci on the DAPI-stained DNA. One representative image of three independent experiments is shown [Scale bar: 5 μm]. (C) Gam-sfGFP foci were quantified by counting the number of foci per cell (n=3); [two-way ANOVA, Tukey’s multiple comparison test (compare total foci in untreated to AGXX^® treatment); ns = P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001].

Fig. 5:. UPEC strain CFT073 shows increased tolerance to AGXX^® compared to K-12 E. coli strain MG1655, which is independent of RcrB.

Overnight cultures of E. coli strains MG1655 and CFT073 were diluted into MOPSg to an OD₆₀₀~0.1 and grown to mid-log phase (OD₆₀₀~0.5). Cells were either left untreated or exposed to increasing concentrations of AGXX^®. Growth (A) and survival (B) were recorded. (A) AGXX^® concentrations that resulted in a growth arrest of MG1655 had no effect on CFT073. (B) After 60 min of AGXX exposure, cells were serially diluted and spot-titered onto LB agar plates for overnight incubation at 37 °C. One representative image of at least three independent experiments with similar outcomes. (C) rcrB mRNA levels of HOCl and AGXX^®-treated UPEC CFT073 cells were determined by qRT-PCR after 15 mins. Transcript levels were normalized to the housekeeping gene rrsD and calculated as fold changes normalized to that of untreated control (n = 3, ±S.D.). (D) Exponentially grown CFT073 WT and ΔrcrB cultures were either left untreated or treated with AGXX^®. After 60min, cells were serially diluted and spot-titered onto LB agar plates for overnight incubation at 37 °C. (n = 3, ±S.D.).

Fig 6:. AGXX^®-induced protein aggregation is less pronounced in members of the UPEC pathotype.

Mid-log phase cultures of K-12 strain MG1655 and UPEC CFT073 were exposed to the indicated AGXX^®823 concentrations for 30 min. (A) Total RNA was extracted, genomic DNA removed, and mRNA reverse-transcribed into cDNA. qRT-PCR analysis was performed for differential expression analyses of genes ibpA, dnaK, and ibpB, which were normalized to the housekeeping gene rrsD and the untreated cells; (n=5-7, ±S.D.) (B) The extent of protein aggregation was determined after harvesting and cell lysis. Protein aggregates and soluble proteins were separated, extracted, separated by SDS-PAGE, and visualized by Coomassie staining. One representative image of 5 biological replicates is shown.

Fig 7:. AGXX^® exposure of UPEC elicits significant changes in global gene expression.

(A) Exponentially growing CFT073 cells were incubated with a sublethal concentration of AGXX^®394C for 30 min. Transcription was stopped by the addition of ice-cold methanol. Reads were aligned to the CFT073 reference genome (accession number: AE014075). Data are visualized as a ratio/intensity scatter plot (M/A-plot) of differentially expressed genes in AGXX^®-treated CFT073 cells. Statistically significantly upregulated genes are depicted above the blue dashed line, whereas statistically significantly downregulated genes are presented as black dots below the black dashed line (M ≥1.5 or ≤ −1.5, P≤ 0.05). Light gray dots represent genes with no significant fold change in transcript level upon AGXX^® treatment (P> 0.5). Many of the upregulated genes can be categorized into metal ion homeostasis (blue IDs), protein homeostasis (green IDs), DNA damage (orange ID), and oxidative stress response (purple IDs), respectively. Transcriptome analysis was performed from three independent biological replicates. (B) Number of significantly differentially expressed genes of AGXX-stressed CFT073 grouped based on GO terms for biological processes. Genes with more than one biological process were assigned to their respective GO term from KEGG pathway database.

Fig 8:. Polyphosphate protects UPEC from AGXX^® stress.

The role of polyP for UPEC growth and survival during AGXX^®394C stress was determined in cells of the (A) exponential phase, (B) stationary phase, and (C) stationary phase cells diluted back into fresh MOPsg to OD₆₀₀=0.35. After 180, 240, and 150 min, samples were serially diluted in PBS, spot-titered on LB agar, and incubated for 20 hrs for CFU counts (n= 3-6, ±S.D.). (D) WT, ΔpolyP, and ΔpolyP supplemented with 4 mM PolyP cultures were cultivated in MOPSg media in the presence of the indicated AGXX^® concentrations. Growth was monitored at 600 nm for 16h and calculated as the area under the growth curve; (n=4, ±SD; student t-test; ns = P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.)