Article Figures & Data
Figures
- 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.
Tables
- TABLE 1
Susceptibility profile of the studied S. aureus strains and TO-resistant mutants selected after serial passage in the presence of TO or FC04-100
Strain or clonesa MIC (μg/ml)b TO FcM Fcm GEN S. aureus Newbould >128 2 8 0.25 S. aureus Newbould ΔhemB 0.06 0.06 2 8 P07intR-1, -2, and -3* 0.25 0.5–1 4 8 P11intR-1, -2, and -3* 0.25–0.5 0.5–1 4 8 P20intR-1, -2, and -3* 0.25–1 1 4 8 SaR1-1, -2, and -3# >64 2 8 8 SaR2-1, -2, and -3§ >64 2 8 8 SaR3-1, -2, and -3# >64 2 8 8 SaR4-1, -2, and -3# >64 2 8 8 SaR5-1, -2, and -3† >64 2 8–16 8–16 SaR6-1, -2, and -3† >64 2 8 8 ↵a Clones of S. aureus Newbould ΔhemB are indicated as follows: *, clones selected after 7, 11, or 20 passages, respectively, in the presence of TO; #, clones selected after 30 passages in the presence of TO; §, clones from a clone of S. aureus Newbould ΔhemB with low-level of resistance to TO (P07intR-1) after an additional 30 passages in the presence of TO; †, clones selected after 30 passages in the presence of FC04-100.
↵b TO, tomatidine; FcM, FC04-100 major stereoisomer; Fcm, FC04-100 minor stereoisomer; GEN, gentamicin. For the MIC, the “>” sign denotes the highest concentration tested.
- TABLE 2
Susceptibility profile of genetically manipulated S. aureus and B. subtilis strains
Strain MIC (μg/ml)a TO FcM TO (+HQNO)b S. aureus Newbould >64 2 0.12 S. aureus Newbould ΔhemB/empty vector 0.06 0.06 0.06 S. aureus Newbould ΔhemB atpE >64 8 ND S. aureus Newbould ΔhemB SaR5 >64 2 >64 B. subtilis 168 >64 ND 0.12 B. subtilis 168 with SaR5 atpE mutation >64 ND >64 - TABLE 3
MIC and inhibition of ATP synthesis (IC50) by TO, TO analogs, and reference compounds for different bacterial strains and mitochondriaa
Compound ΔhemB strain Newbould E. coli IC50 (μg/ml) for mitochondria MIC (μg/ml) IC50 (μg/ml) MIC (μg/ml) IC50 (μg/ml) MIC (μg/ml) IC50 (μg/ml) TO 0.06 18.5 ± 1.9 >128 95.5 ± 13.7 >128 264.73 ± 30.8 >1,024 FcM 0.06 18.9 ± 3.6 2 40.2 ± 13.8 32 102.42 ± 16.6 >1,024 Fcm 2 47.0 ± 9.6 8 111.0 ± 11.3 64 223.09 ± 45.9 >1,024 FC02-190 8 85.1 ± 7.0 >128 >1,024 >128 >1,024 >1,024 FC04-116 >128 >1,024 >128 >1,024 >128 >1,024 >1,024 DCCD NDb 1.44 ± 0.54 ND 1.32 ± 0.32 ND 1.62 ± 0.54 0.82 ± 0.17 CCCP ND 1.46 ± 0.27 ND 6.11 ± 1.1 ND 2.80 ± 1.01 7.19 ± 2.23 Oligomycin ND 8.67 ± 1.9 ND 9.41 ± 1.5 ND 5.63 ± 1.23 5.91 ± 0.82 Tomatine >128 >1,024 >128 >1,024 >128 >1,024 >1,024 Bedaquiline >128 >1,024 >128 >1,024 >128 >1,024 >128 Gentamicin 8 >1,024 0.25 >1,024 1 >1,024 >1,024 Levofloxacin 0.25 >1,024 0.25 >1,024 0.03 >1,024 >1,024 - TABLE 4
MIC and selective inhibition of ATP synthesis (IC50) by TO and TO analogs for different TO-resistant mutantsa
Compound SaR1-1 SaR4-1 SaR5-1 SaR6-1 MIC (μg/ml) IC50 (μg/ml) MIC (μg/ml) IC50 (μg/ml) MIC (μg/ml) IC50 (μg/ml) MIC (μg/ml) IC50 (μg/ml) TO >64 >512 >64 >512 >64 >512 >64 >512 FC02-190 >64 >512 >64 >512 >64 >512 >64 >512 FcM 2 238 ± 22.1 2 >512 2 110 ± 9.8 2 >512 Fcm 8 >512 8 >512 8 >512 8 >512 ↵a The “>” sign denotes the highest concentration tested.
Supplemental material
- Supplemental file 1 -
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