Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance

Abstract

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H₂S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H₂S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H₂S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.

Fig. 1.. bCSE is a predominant source of H₂S in S. aureus and P. aeruginosa.

(A) Genomic organization of the cbs/cse operon in S. aureus (USA300) and P. aeruginosa (PAO1) and genetic tools used to selectively disrupt cbs and cse genes. A bracket shows gene replacement/deletion and arrows indicate transposon insertions (gene disruption) (see also Figs. S1, S2, and Table S1). (B) Quantitation of H₂S produced by wild type (wt) and cbs/cse(−) S. aureus USA300 (MRSA isolate JE2) and P. aeruginosa (PAO1). Top: Representative fluorescence images demonstrate H₂S production by live wt and mutant S. aureus (USA300) and P. aeruginosa (PAO1) cells treated with the TICT-based fluorescent H₂S probe. Bottom left: Quantification of images. Values are means ± SD (n = 3), ****P < 0.0001 (Student’s t-test; equal variance). Bottom right: Representative Pb-acetate-soaked paper strips show a brown stain of PbS as a result of the reaction with gaseous H₂S exiting liquid bacterial cultures. Numbers show H₂S production relative to wt cells. (C) Representative growth curves of wt and cbs/cse(–) S. aureus (USA300) and P. aeruginosa (PAO1) in the presence of gentamycin (Gm, 1 and 2 µg/ml, respectively), ampicillin (Amp, 0.1 µg/ml), or norfloxacin (Nor, 1 µg/ml), without or with the H₂S donor NaHS (0.2 and 0.4 mM, respectively). The data points are averages of optical density (OD) at 600 nm wavelength with a margin of error of less than 5%. See also Figs. S1 and S2.

Fig. 2.. Target validation of selected bCSE inhibitors.

(A) Chemical structures of selected bCSE inhibitors, hCSE inhibitor (PAG), and a negative control (NL1F₃). Id, indole; Bt, benzothiophene; Pz, pyrazole. IC₅₀ measurements were conducted with 1,000× and 100× excess of substrate Cys and coenzyme PLP, respectively, over SaCSE (mean ± SE, n=2–4). NI, no inhibition. S. aureus RN4220 was used for representative gentamycin (Gm) MIC potentiation by 10 µM of each inhibitor. (B) Inhibition of cellular H₂S production by the NL1–3 compounds. Representative fluorescence images and the bar plots demonstrate H₂S production by NL1-treated and untreated S. aureus (RN4220) and P. aeruginosa (PA14) cells using TICT-based fluorescent H₂S probe. Values are means ± SD (n=3). (C) Representative growth curves of wt and cbs/cse S. aureus RN4220 and P. aeruginosa PA14 in the presence of Gm and NL1. Cells were grown in triplicate at 37 °C with aeration using a Bioscreen C automated growth analysis system. The curves represent averaged values with a margin of error of less than 5%. (D) Representative growth curves and efficiencies of colony formation of S. aureus RN4220 and P. aeruginosa PA14 in the presence of Gm, NL1, and NaHS.

Fig. 3.. Co-crystal structures of SaCSE bound to bCSE inhibitors.

(A) Interactions of NL1 (violet) with the SaCSE monomer. Carbon, nitrogen and oxygen atoms are in green, blue and red, respectively. Gray dashed lines depict putative hydrogen bonds. Red dashed line indicates CH-π interactions. (B) View of NL1 (sticks) in the CSE binding pocket (surface representation). (C) Details of NL2 (brown) binding. (D) Details of NL3 (light blue) binding. Benzothiophene (Bt), indole (Id) and pyrazole (Pz) moieties of NL3 are indicated. (E) Bound leads (same colors) after all-atom superposition of the lead-bound structures shown with Y103 from the NL1-, NL2-, and NL3-bound structures (green, cyan, and light blue, respectively). (F) Representative data for the H₂S generating activity of SaCSE mutants evaluated by the fluorescence assay.

Fig. 4.. bCSE inhibition synergizes gentamycin in murine models of infection.

(A) Acute S. aureus-induced bacteremia model. Treatment with Gm or NL1 alone did not increase survival of mice after lethal infection with S. aureus. Treatment with NL1+Gm improved survival to 50%. Study design is shown on top. (B) P. aeruginosa intranasal lung infection model. Treatment with the combination of Gm and NL1 significantly decreased bacteria burden in lungs. Graph represents the mean ± SE, *P<0.05.

Fig. 5.. bCSE inhibition interferes with persistence and biofilm formation.

(A) Dynamics of persister viability in wt, bCSE-deficient, or NL1-treated S. aureus USA300 and P. aeruginosa PA14. The graphs show changes in the viable cell fraction of exponentially growing cultures challenged with 10 µg/ml (10× MIC) ciprofloxacin. Data points are mean ± SE (n=3). (B) The bar plots demonstrate H₂S production by S. aureus USA300 and P. aeruginosa PA14 cells before (black) and after (gray) 3 h incubation with 10 µg/ml ciprofloxacin. Cells were treated with the TICT-based fluorescent H₂S probe and the fluorescence signal was normalized to CFU. Data are mean ± SE (P<0.01) (n=3). (C) Inactivation of bCSE depletes P. aeruginosa of the reduced pyocyanin toxin. Pyocyanin (1-hydroxy-5-methyl-phenazine) chemical structure is shown on top. Oxidized pyocyanin gives the P. aeruginosa culture blue/green color. The tubes show stationary phase P. aeruginosa PA14 cultures (OD₆₀₀~1.5): untreated wt, untreated cbs/cse(−) mutant, and wt treated with NL1 (180 µM) for 18 h. (D) bCSE modulates colony morphology of P. aeruginosa PA14. wt and cse/cbs(−) cultures were spotted onto agar plates containing Congo Red and Coomassie Blue, and incubated with or without NL1 for 3 days prior to taking pictures. (E) Genetic or chemical inactivation of bCSE compromises P. aeruginosa PA14 biofilm formation, as determined by the crystal violet assay. Data are mean ± SE (n=3).