Tomatidine Is a Lead Antibiotic Molecule That Targets Staphylococcus aureus ATP Synthase Subunit C

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti-Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro-generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10⁵-fold for FC04-100.

Keywords: ATP synthase; Staphylococcus aureus; new target; small-colony variant; tomatidine.

FIG 1

Structures of TO and analogs used in this study. (A) TO is characterized by 6 rings, 12 stereogenic centers, a 3 β-hydroxyl group, and spiro-fused rings in the form of an aminoketal. (B) FC04-100 contains a diamine in position 3. The two epimers in position 3 were separated as major (M) and minor (m) although due to the complexity of nuclear magnetic resonance signals, their respective structures could not be unambiguously assigned. (C) TO analog FC02-190 shows an α-hydroxyl group in position 3. (D) Analog FC04-116 shows an open spiroaminoketal moiety.

FIG 2

Amino acid sequence alignments of the ATP synthase subunit C for selected species. First, the alignments for several species of Bacillales present a consensus sequence, highlighted in green. Amino acids additionally identical to those of S. aureus are highlighted in yellow. Amino acids mutated in TO/FC04-100-resistant mutants are in red and bold characters in the S. aureus NCTC 8325 sequence. Also shown below the Bacillales consensus sequence are the alignments for some bacterial species not targeted by TO. The changes in amino acids found in TO/FC04-100-resistant S. aureus (SaR1 to SaR6) are indicated below the alignments. The essential ion-binding glutamate (aspartate in E. coli) is indicated in bold black. Changes in amino acids reported for the bedaquiline-resistant strains of Mycobacterium tuberculosis or Mycobacterium smegmatis (MyR denotes a mixture of these two species) are also indicated below the alignments (40).

FIG 3

Monomeric and multimeric models of ATP synthase subunit C built, respectively, on homology with templates of PDB accession numbers 1WU0 and 3ZO6, using the SWISS-MODEL server. (A) Position of amino acids (in white) implicated in high-level TO resistance in the wild-type polypeptide. Essential amino acid Glu54 is in yellow. (B) Position of Ser17 mutation in SaR1. (C) Position of Cys18 mutation in SaR5. (D) Position of Leu26 mutation in SaR2, SaR3, and SaR4. (E) Position of Leu47 mutation in SaR6. (F) Overview of the multimeric assembly of ATP synthase subunit C. (G) Position of amino acids (Ala17, red; Gly18, green; Ser26, cyan; Phe47, magenta) implicated in resistance in the wild-type multimeric assembly. Glu54 is also shown in yellow. (H) Position of the Ser17 mutation in the multimeric assembly in SaR1. (I) Position of the Cys18 mutation in the multimeric assembly in SaR5. (J) Position of the Leu26 mutation in the multimeric assembly in SaR2, SaR3, and SaR4. (K) Position of the Leu47 mutation in the multimeric assembly in SaR6. The models were drawn using PyMOL software (version 1.8.7.0; DeLano Scientific, San Francisco, CA).

FIG 4

Effect of TO and analogs on the ATP synthase activity of S. aureus. (A) Relative ATP synthase activity of the SCV ΔhemB strain in the presence of various inhibitors. The control (CTRL) represents the maximal ATP production in the absence of ATP synthase inhibitor whereas the assay performed without addition of NADH represents the minimal value. nd, not determined. (B) Relative ATP synthase activity of the SCV ΔhemB atpE mutants (SaR1, SaR4, SaR5, and SaR6) in the presence of TO. The effects of TO on the parental ΔhemB strain and the prototypical strain Newbould (WT) and on human mitochondria are also shown for comparison. (C) Correlation between TO and analogs (FcM, Fcm, FC02-190, and FC04-116). Log₂ MICs and the log₁₀ IC₅₀s were determined in the ATP synthase assay using S. aureusΔhemB membrane vesicles. (D) ATP production (relative light units, RLU) by membrane vesicles prepared from the SCV ΔhemB atpE mutants. ATP production by the parental ΔhemB strain and the prototypical strain Newbould (WT) is also shown. In panel D, letters shared between or among the groups indicate no significant difference.