Antimicrobial activity of the iron-sulfur nitroso compound Roussin's black salt [Fe4S3(NO)7] on the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

The iron-sulfur nitroso compound [Fe(4)S(3)(NO)(7)](-) is a broad-spectrum antimicrobial agent that has been used for more than 100 years to combat pathogenic anaerobes. Known as Roussin's black salt (RBS), it contains seven moles of nitric oxide, the release of which was always assumed to mediate its cytotoxicity. Using the hyperthermophilic archaeon Pyrococcus furiosus, it is demonstrated through growth studies, membrane analyses, and scanning electron microscopy that nitric oxide does not play a role in RBS toxicity; rather, the mechanism involves membrane disruption leading to cell lysis. Moreover, insoluble elemental sulfur (S(0)), which is reduced by P. furiosus to hydrogen sulfide, prevents cell lysis by RBS. It is proposed that S(0) also directly interacts with the membranes of P. furiosus during its transfer into the cell, ultimately for reduction by a cytosolic NADPH sulfur reductase. RBS is proposed to be a new class of inorganic antimicrobial agent that also has potential use as an inert cell-lysing agent.

FIG. 1.

Effect of RBS on the growth of P. furiosus. The concentrations of RBS are as follows: 0 μM (▴), 0.5 μM (▪), 1.0 μM (⧫), 2.0 μM (Δ), 3.0 μM (○), and 5.0 μM (□). The arrow indicates when RBS was added.

FIG. 2.

Effect of RBS on P. furiosus exposed to S⁰. Symbols are as follows: •, no S⁰; ▪, S⁰ added but no RBS; Δ, no S⁰ plus 2 μM RBS; ▴, S⁰ added plus 2 μM RBS. The arrow indicates when RBS was added.

FIG. 3.

Effect of RBS on P. furiosus after incubation with S⁰. Symbols are as follows: ▴, no S⁰ without RBS Δ, S⁰ and 2 μM RBS introduced to the culture at the same time; ⋄, 2 μM RBS added after culture was incubated with S⁰ for 10 min; ○, 2 μM RBS added after culture was incubated with S⁰ for 20 min; and ▪, 2 μM RBS added after culture was incubated with S⁰ for 30 min. The arrow indicates when RBS was added.

FIG. 4.

Toxicity of RBS at 4°C. P. furiosus cultures were transferred from 98°C to 4°C after 4 h (indicated by the first arrow) except the control which was maintained at the optimal growth temperature of 98°C. RBS was added to a culture once equilibrated to 4°C at the 6-h mark (indicated by the second arrow). Symbols are as follows: ▴, culture maintained at 98°C and no RBS added; □, culture transferred to 4°C but no RBS added; Δ, culture transferred to 4°C and 2.0 μM RBS added.

FIG. 5.

SEM images of P. furiosus exposed to 1 μM RBS. Images were collected before (A) and 60 s after (B) the addition of 1 μM RBS. Magnification, ×10,000.

FIG. 6.

Transfer of RBS toxicity in P. furiosus membrane fractions. Membranes from cultures treated with 20 μM RBS were added to fresh P. furiosus cultures with increasing injection volumes (μl). Each culture was monitored by cell count after a 30-min incubation with the RBS/membrane preparation. Symbols are as follows: ▪, no membranes added; •, RBS-treated membranes added; ⧫, RBS-treated membranes added after prior incubation with S⁰ (∼4 mM) at 98°C for 30 min; ▴, RBS-treated membranes added after prior incubation with polysulfide (4 mM) at 98°C for 30 min. Cultures with cell densities below 1 × 10⁶ cells/ml were considered not viable.

FIG. 7.

Fluorescent Live/Dead assays on P. furiosus cells treated with RBS. The cell concentrations were initially 5.0 × 10⁷ cells/ml. RBS (2 μM) was injected approximately 20 s after the beginning of the recording of the fluorescent scan. Dotted line, P. furiosus cells grown without S⁰; dashed line, P. furiosus cells grown with S⁰; solid line, control without cells.