Cysteamine, an Endogenous Aminothiol, and Cystamine, the Disulfide Product of Oxidation, Increase Pseudomonas aeruginosa Sensitivity to Reactive Oxygen and Nitrogen Species and Potentiate Therapeutic Antibiotics against Bacterial Infection

Abstract

Cysteamine is an endogenous aminothiol produced in mammalian cells as a consequence of coenzyme A metabolism through the activity of the vanin family of pantetheinase ectoenzymes. It is known to have a biological role in oxidative stress, inflammation, and cell migration. There have been several reports demonstrating anti-infective properties targeting viruses, bacteria, and even the malarial parasite. We and others have previously described broad-spectrum antimicrobial and antibiofilm activities of cysteamine. Here, we go further to demonstrate redox-dependent mechanisms of action for the compound and how its antimicrobial effects are, at least in part, due to undermining bacterial defenses against oxidative and nitrosative challenges. We demonstrate the therapeutic potentiation of antibiotic therapy against Pseudomonas aeruginosa in mouse models of infection. We also demonstrate potentiation of many different classes of antibiotics against a selection of priority antibiotic-resistant pathogens, including colistin (often considered an antibiotic of last resort), and we discuss how this endogenous antimicrobial component of innate immunity has a role in infectious disease that is beginning to be explored and is not yet fully understood.

Keywords: Pseudomonas aeruginosa; antibiotic resistance; antimicrobial agents; azithromycin; colistin; cysteamine; innate immunity; nitric oxide; reactive oxygen species; vanin-1.

FIG 1

CYS and NaNO₂ react to form a new product with absorbance peaks at 333 and 545 nm, typical of the S-nitrosothiol S-nitrosocysteamine. (A) The reaction favors acidic conditions, as shown by differences in absorbance at 333 nm above background, where 1 mg/ml CYS was reacted with 1 mg/ml NaNO₂ at 37°C in the dark for 1 h in phosphate-buffered saline across the pH range 6 to 7.4. The disulfide CTM did not react with NaNO₂ under the same conditions at the same concentrations. (B) CTM is rapidly converted into thiol by P. aeruginosa PAO1 when 1 × 10⁹ CFU/ml were challenged with 3 mM and grown in glucose minimal medium over time, as detected in the culture media by DTNB. The addition of 10 μM CCCP did not prevent conversion of CTM to thiol or export from the cell.

FIG 2

ROS production in P. aeruginosa PAO1 as detected by H2DCFDA fluorescence over time in response to CTM (A) and after 2-h challenge with CTM only (B), ciprofloxacin only (C), or selected combinations of both (D). (D) Bars 1 to 3 show CTM only at 64, 256, and 512 μg/ml. Bars 4 to 6 show ciprofloxacin only at 0.5, 1, and 2 μg/ml. Bars 7 to 9 show CTM at 64 μg/ml with ciprofloxacin at 0.5, 1, and 2 μg/ml. Bars 10 to 12 show CTM at 256 μg/ml with ciprofloxacin at 0.5, 1, and 2 μg/ml. Bars 13 to 15 show CTM at 512 μg/ml with ciprofloxacin at 0.5, 1, and 2 μg/ml (n = 3). One-way analysis of variance (ANOVA) with Tukey's post hoc analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 3

(A) Effects of ciprofloxacin and CYS on the microbial load (expressed as log₁₀ CFU per gram of tissue) from the neutropenic mouse thigh model experimentally infected with P. aeruginosa LES431 and treated with vehicle control (lane 1), colistin (5 mg/kg) (lane 2), CYS (1.25 mg/kg) (lane 3), ciprofloxacin (15 mg/kg) (lane 4), and ciprofloxacin plus CYS (lane 5) (n = 5 animals per treatment group). (B) Effects of d.p.i.-administered tobramycin and CYS (expressed as log₁₀ CFU per gram of tissue) in the neutropenic acute lung model of infection with P. aeruginosa ATCC 27853 treated with vehicle only (3 mg mannitol) (lane 1), 3 mg of 5% CYS (lane 2), 3 mg 10% CYS (lane 3), 0.188 mg tobramycin in lactose (4.5 mg total) (lane 4), 5% CYS plus tobramycin (lane 5), and 10% CYS plus tobramycin (lane 6) (n = 8 animals per treatment group). The horizontal lines denote the mean values. One-way ANOVA with Tukey's post hoc analysis (ns, not significant; **, P < 0.01; ***, P < 0.001).

FIG 4

(A) The incorporation of 512 μg/ml CTM in CA MHA removes a subpopulation of resistant cells of S. aureus DSM 11729 cultured with azithromycin Etest strips (control plate [left]). (B) An increased zone of clearance of N. gonorrhoeae strain NB04916 can be observed surrounding azithromycin Etest strips on plates with increasing concentrations of cysteamine (1, 0 μg/ml; 2, 128 μg/ml; 3, 256 μg/ml; 4, 512 μg/ml) incorporated into GC (plus Vitox supplement) agar, whereas there was no apparent difference for strain NB03916.

FIG 5

Redox-dependent anti-infective mechanisms of action for cysteamine/cystamine. (Step 1) Cysteamine can be supplied therapeutically or produced endogenously through the action of vanin-1 pantetheinase. (Steps 2 and 3) Cysteamine itself does not pass the bacterial cytoplasmic membranes of rapidly dividing cells (step 2) but is reported to have impacts upon host immunity to infection and autophagy (step 3). (Steps 4 and 5) Cysteamine can react (reversibly) with susceptible cysteine residues in a process termed cysteaminylation (step 4), and we demonstrated that it can form adducts with reactive nitrogen species, possibly forming S-nitrosocysteamine in mildly acidic environments (step 5). (Steps 6 and 7) Cysteamine readily forms the disulfide cystamine (and water) in the presence of oxygen (step 6) in a temperature-dependent manner and at millimolar concentrations rapidly generates ROS in the presence of transition metal ions (step 7). (Steps 8 and 9) Cystamine itself may interact with unknown periplasmic targets in Gram-negative cells and can enter the bacterial cell (step 8) via an unknown mechanism, where it generates ROS and interacts with susceptible intracellular targets, leading to dysregulation of small thiol pools and metabolism (step 9), disrupting pigment production or export. (Step 10) Reduced thiols, probably including cysteamine, are exported via an unknown mechanism.