Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. B. burgdorferi Culture
2.3. Substrates and Inhibitors
2.4. Expression and Purification of Recombinant MTANs
2.5. Enzyme Activity and Kinetics
2.6. Luciferase Assay for B. burgdorferi Survival
2.7. Assessment of Nucleosides/Nucleoside Analogs as Antimicrobials
2.8. Assessment of Bacteriostatic Versus Bactericidal Activities of Nucleosides and Nucleoside Analogs
3. Results
3.1. MTAN is a Critical Enzyme in the Salvage Pathways of Auxotrophic Bacteria
3.2. Kinetic Analysis of Purified Recombinant MTANs with Nucleosides and Nucleoside Analogs
3.3. Determine Efficacy of Luciferase Activity to Measure B. burgdorferi Viability
3.4. Effect of Ampicillin and Selected Inhibitors of MTANs on the Growth of B. burgdorferi
3.5. Bacteriostatic or Bactericidal Activities of MTAN Inhibitors Against B. burgdorferi N40
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Specific Activity (U/mg) * | % Max Specific Activity ** | |||||
---|---|---|---|---|---|---|
Enzyme | Enzyme | |||||
Nucleoside | Bgp | MtnN | Pfs | Bgp | MtnN | Pfs |
MTA | 1.00 | 1.00 | 1.70 | 100 | 100 | 100 |
MSeA | 1.79 | 1.50 | 0.27 | 179 | 150 | 16 |
IADO | 0.04 | 0.03 | 0.11 | 4 | 3 | 6 |
PurTA | 0.14 | 0.04 | 0.04 | 14 | 4 | 2 |
FSBA | 0.12 | 0.02 | 0.02 | 12 | 2 | 1 |
Enzyme | |||
---|---|---|---|
Nucleoside | Bgp | MtnN | Pfs |
MTA * | Km = 0.49 ± 0.10 µM | Km = 9.09 ± 1.62 µM | Km = 0.61 ± 0.16 µM |
MSeA | Km = 1.62 ± 0.10 µM | Km = 1.50 ± 0.14 µM | Km = 2.12 ± 0.24 µM |
IADO | Ki = 0.63 ± 0.12 µM | Ki = 2.47 ± 0.46 µM | Ki = 0.90 ± 0.07 µM |
PurTA | Ki = 1.31 ± 0.18 µM | Ki = 2.05 ± 0.18 µM | Ki = 0.69 ± 0.09 µM |
FSBA | Ki = 0.34 ± 0.03 µM | Ki = 2.04 ± 0.13 µM | Ki = 0.39 ± 0.07 µM |
dEt-ImmA * | Ki = 2.48 ± 0.38 nM, Ki’ = 0.35 ± 0.02 nM | Ki = 1.48 ± 0.17 nM Ki’ = 0.58 ± 0.05 nM | Ki = 10.49 ± 2.01 nM Ki’ = 0.53 ± 0.15 nM |
B. burgdorferi/mL | Equation | R2 |
---|---|---|
1.0 × 108 | −0.5296x + 39.133 | 0.9188 |
1.5 × 108 | −0.7453x + 27.088 | 0.9237 |
2.0 × 108 | −0.7379x + 101.38 | 0.9834 |
Nucleoside Inhibitor | Inhibitor Activity | Dose |
---|---|---|
MSeA | Bacteriostatic | All doses |
IADO | Bactericidal | 2 µM, 10 µM |
PurTA | Bacteriostatic | All doses |
FSBA | Bacteriostatic | All doses |
dEt-ImmA | Bactericidal | 2 µM, 10 µM |
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Chakraborti, M.; Schlachter, S.; Primus, S.; Wagner, J.; Sweet, B.; Carr, Z.; Cornell, K.A.; Parveen, N. Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi. Pathogens 2020, 9, 678. https://doi.org/10.3390/pathogens9090678
Chakraborti M, Schlachter S, Primus S, Wagner J, Sweet B, Carr Z, Cornell KA, Parveen N. Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi. Pathogens. 2020; 9(9):678. https://doi.org/10.3390/pathogens9090678
Chicago/Turabian StyleChakraborti, Monideep, Samantha Schlachter, Shekerah Primus, Julie Wagner, Brandi Sweet, Zoey Carr, Kenneth A. Cornell, and Nikhat Parveen. 2020. "Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi" Pathogens 9, no. 9: 678. https://doi.org/10.3390/pathogens9090678