A Small-Molecule Modulator of Metal Homeostasis in Gram-Positive Pathogens

Abstract

Metals are essential nutrients that all living organisms acquire from their environment. While metals are necessary for life, excess metal uptake can be toxic; therefore, intracellular metal levels are tightly regulated in bacterial cells. Staphylococcus aureus, a Gram-positive bacterium, relies on metal uptake and metabolism to colonize vertebrates. Thus, we hypothesized that an expanded understanding of metal homeostasis in S. aureus will lead to the discovery of pathways that can be targeted with future antimicrobials. We sought to identify small molecules that inhibit S. aureus growth in a metal-dependent manner as a strategy to uncover pathways that maintain metal homeostasis. Here, we demonstrate that VU0026921 kills S. aureus through disruption of metal homeostasis. VU0026921 activity was characterized through cell culture assays, transcriptional sequencing, compound structure-activity relationship, reactive oxygen species (ROS) generation assays, metal binding assays, and metal level analyses. VU0026921 disrupts metal homeostasis in S. aureus, increasing intracellular accumulation of metals and leading to toxicity through mismetalation of enzymes, generation of reactive oxygen species, or disruption of other cellular processes. Antioxidants partially protect S. aureus from VU0026921 killing, emphasizing the role of reactive oxygen species in the mechanism of killing, but VU0026921 also kills S. aureus anaerobically, indicating that the observed toxicity is not solely oxygen dependent. VU0026921 disrupts metal homeostasis in multiple Gram-positive bacteria, leading to increased reactive oxygen species and cell death, demonstrating the broad applicability of these findings. Further, this study validates VU0026921 as a probe to further decipher mechanisms required to maintain metal homeostasis in Gram-positive bacteria.IMPORTANCEStaphylococcus aureus is a leading agent of antibiotic-resistant bacterial infections in the world. S. aureus tightly controls metal homeostasis during infection, and disruption of metal uptake systems impairs staphylococcal virulence. We identified small molecules that interfere with metal handling in S. aureus to develop chemical probes to investigate metallobiology in this organism. Compound VU0026921 was identified as a small molecule that kills S. aureus both aerobically and anaerobically. The activity of VU0026921 is modulated by metal supplementation, is enhanced by genetic inactivation of Mn homeostasis genes, and correlates with increased cellular reactive oxygen species. Treatment with VU0026921 causes accumulation of multiple metals within S. aureus cells and concomitant upregulation of genes involved in metal detoxification. This work defines a small-molecule probe for further defining the role of metal toxicity in S. aureus and validates future antibiotic development targeting metal toxicity pathways.

Keywords: MRSA; Staphylococcus aureus; antibiotics; cobalt; copper; manganese; metalloregulation.

FIG 1

Identification of a small molecule that is antimicrobial to S. aureus. (A) Workflow for identifying compounds of interest. (B) Percent growth inhibition by treatment with the indicated concentrations of compound ‘921 relative to vehicle control for S. aureus Newman or ΔmntH/C strain. Data are mean ± standard deviation from duplicate measurements. Statistical significance was determined by t test where * = P < 0.05 and ** = P < 0.01. (C) Relative growth at 12 h with 5 μM ‘921 ± MnCl₂ is graphed as % of growth without compound or MnCl₂. Data are mean ± standard deviation from triplicate measurements acquired on two separate days and combined (n = 6). Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test comparing each concentration of Mn to no-Mn control where *** = P < 0.001. (D) CFU recovered at 2-h intervals from growth curve of S. aureus Newman treated with vehicle, 5 μM ‘921, or 5 μM ‘921 + 1 mM MnCl₂ over time. Data are mean ± standard deviation from quadruplicate measurements. (E) CFU recovered following 24-h treatment of mid-exponential-phase S. aureus Newman cultures with the indicated combinations of vehicle, 1 mM MnCl₂, 100 μM ‘921, and 1 mM EDTA. Data are mean ± standard deviation from triplicate measurements acquired on two separate days combined (n = 6). Statistical significance was determined by one-way ANOVA with Sidak’s multiple-comparison test comparing each concentration of Mn to no-Mn control where * = P < 0.05 and *** = P < 0.001. Experiments in panels F and G were performed in an anaerobic chamber. (F) Growth of S. aureus Newman in the presence of vehicle, 1 mM MnCl₂, 5 μM ‘921, or 5 μM ‘921 + 1 mM MnCl₂. Data are mean ± standard deviation for triplicate measurements. (G) CFU recovered following treatment of mid-exponential-phase cultures of S. aureus with vehicle, 1 mM or 100 μM MnCl₂, 100 μM ‘921, or combinations of MnCl₂ + ‘921 for 24 h. Data are mean ± standard deviation for triplicate measurements.

FIG 2

VU0026921 induces a metal toxicity transcriptional response. RNA sequencing was performed on mid-exponential S. aureus Newman cultures exposed to vehicle, 1 mM MnCl₂, 100 μM ‘921, or 100 μM ‘921 + 1 mM MnCl₂ for 30 min. (A) Multidimensional scaling visualization of RNA sequencing biological replicates. The x and y axes are dimensionless units. The closeness of symbols to each other on the 2-dimensional plot represents the relatedness of the transcriptional profiles of the represented data sets. (B) Categories of genes whose transcription was significantly different between 100 μM ‘921 treatment alone and cotreatment with 1 mM MnCl₂. (C) Fold change (log₂) of transcript abundance for all S. aureus genes following treatment with VU0026921 compared to vehicle-treated controls. (D) Fold change (log₂) of transcript abundance for all S. aureus genes following treatment with VU0026921 and 1 mM MnCl₂ compared to ‘921 alone. For panels C and D, genes involved in metal homeostasis and reactive oxygen species (ROS) detoxification are highlighted. Dashed lines indicate genes with greater than 2-fold change and a false-discovery rate of less than 0.01.

FIG 3

VU0026921 causes metal accumulation and oxidative stress in S. aureus. Metal isotopes ⁵⁵Mn (A), ⁵⁹Co (B), ⁶³Cu (C), ⁵⁶Fe (D), and ⁶⁶Zn (E) were measured by ICP-MS in S. aureus Newman treated for 30 min with vehicle, 1 mM MnCl₂, 100 μM ‘921, or 100 μM ‘921 + 1 mM MnCl₂. Data are mean ± standard deviation normalized to ³⁴S to account for differences in growth of four biological replicates. Statistical significance was determined by one-way ANOVA where ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001. (F) ROS levels in S. aureus cultures treated with vehicle, 50 μM ‘921, 1 mM MnCl₂, or 50 μM ‘921 + 1 mM MnCl₂ for 6 h. Data are mean ± standard deviation for six biological replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test where ** = P < 0.01 and **** = P < 0.0001. (G) CFU recovered following 24-h treatment of mid-exponential-phase cultures of S. aureus Newman, ΔmntH/C, or ΔsodA/sodM with vehicle, 1 mM MnCl₂, 100 μM ‘921, or 1 mM Mn + 100 μM ‘921. Data are mean ± standard deviation combined from three independent triplicate experiments, performed on separate days (n = 9). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple-comparison test where ### = P < 0.001 compared to WT vehicle-treated bacteria and ** = P < 0.01 and *** = P < 0.001 for the comparisons indicated by the bars. (H) S. aureus Newman was treated with vehicle, 50 μM ‘921, 80 μM MitoTEMPO, or 50 μM ‘921 + 80 μM MitoTEMPO, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation for six biological replicates.

FIG 4

Mn and Co protect S. aureus against VU0026921 toxicity, while Cu exacerbates it. (A to D and F) CFU recovered following 4-h treatment of S. aureus Newman with vehicle, 100 μM ‘921, 100 μM FeSO₄ (A), ZnCl₂ (B), MnCl₂ (C), CoCl₂ (D), and CuSO₄ (F), or 10 μM ‘921 + 100 μM metal. Data presented are mean ± standard deviation from triplicate measurements. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test where ns = P > 0.05, *** = P < 0.001, and **** = P < 0.0001. (E and G) ROS levels in S. aureus cultures treated with vehicle, 50 μM ‘921, 100 μM CoCl₂ (E) or CuSO₄ (G), and 50 μM ‘921 + 100 μM metal for 6 h. Data are mean ± standard deviation for six biological replicates. Statistical significance was determined for each condition compared to ‘921 treatment by one-way ANOVA with Tukey’s multiple-comparison test where *** = P < 0.001 and **** = P < 0.0001. (H) USA300, an MRSA strain, and USA300 ΔcopAZ ΔcopBL (Δcop) were treated with vehicle, 5 μM ‘921, 10 μM CuSO₄, or 5 μM ‘921 + 10 μM CuSO₄, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation for six biological replicates.

FIG 5

Binding of divalent transition metals stabilized VU0026921. (A to H) Absorbance was measured from 280 nm to 600 nm in 10-nm increments of 0 or 100 μM ‘921 combined with vehicle (A) or 500 μM CoCl₂ (B), CuSO₄ (C), FeSO₄ (D), MnCl₂ (E), ZnCl₂ (F), CaCl₂ (G), or MgCl₂ (H). Absorbance measurements were taken at 0, 5, 15, 30, 60, or 120 min after the addition of metals. The final absorbance spectra are ‘921 + metal with spectra for metal alone subtracted. Spectra displayed are representative of a single experiment that was performed three times. The spectrum of ‘921 at 0 min was included in each panel as a reference of the compound absorbance alone.

FIG 6

Chemical features of VU0026921 required for toxicity. (A) Chemical structures of VU0026921 and analogs. (B) Relative potency of 100 μM (each) analog—VU0026921 (‘921), VU0849731 (‘731), VU0849732 (‘732), VU0849730 (‘730), or VU0849729 (‘729)—measured as growth of S. aureus as a percentage of untreated cells at 4 h for each compound tested at the indicated concentrations. Data are mean ± standard deviation from triplicate measurements. (C) S. aureus Newman was treated with vehicle, 100 μM ‘921, or 100 μM ‘921 analog, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation from triplicate measurements. (D) ROS levels in S. aureus cultures treated with vehicle, 50 μM ‘921, or 50 μM ‘921-2 for 6 h. Data are mean ± standard deviation for six biological replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test where ns = P > 0.05 and **** = P < 0.0001.

FIG 7

VU0026921 S. aureus killing is not through fatty acid biosynthesis inhibition. (A and B) MIC of irgasan (A), a FabI inhibitor that halts fatty acid biosynthesis and inhibits S. aureus strain RN4220, and VU0026921 (B). Irgasan has an increased MIC in S. aureus RN4220 mutants with fatty acid biosynthesis disabled when cotreated with oleic acid, while ‘921 (B) does not, indicating that ‘921 does not inhibit fatty acid biosynthesis. Note: no growth is observed for accD mutants in the absence of oleic acid. (C and D) Platensimycin (C), a FabF inhibitor, has an increased MIC in E. faecalis when cotreated with oleic acid, while ‘921 (D) does not, suggesting that ‘921 does not inhibit fatty acid biosynthesis in E. faecalis. All data are mean ± standard deviation for triplicate measurements. Note: 10 μg/ml was the highest concentration of platensimycin tested. The actual MIC of platensimycin against E. faecalis in the presence of oleic acid may be higher.

FIG 8

VU0026921 is growth inhibitory toward Gram-positive bacteria. (A to F) S. aureus MW2 (A), UAMS1 (B), HG003 (C), 8325-4 (D), RN6390 (E), and SH1000 (F) were treated with vehicle or 5 μM or 20 μM ‘921, and growth was monitored by optical density at 600 nm for 24 h. Data are mean ± standard deviation from triplicate measurements. (G and H) The Gram-positive organisms Bacillus anthracis (G) and Micrococcus luteus (H) were treated with vehicle or 5 μM ‘921, and growth was monitored by optical density at 600 nm for 16 h. Data are mean ± standard deviation from quadruplicate measurements.