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A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

Forbes L, Ebsworth-Mojica K, DiDone L, Li SG, Freundlich JS, Connell N, Dunman PM, Krysan DJ - PLoS ONE (2015)

Bottom Line: Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed.We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules.Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.

ABSTRACT
Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

No MeSH data available.


Related in: MedlinePlus

Bactericidal but not bacteriostatic drugs induce adenylate kinase release from M. smegmatis.A. Exponential phase M. smegmatis was treated with a dilution series of streptomycin (MIC 0.5 μg/mL). The adenylate kinase activity of the supernatants and the number of viable cells for each concentration were determined after 6 hours of exposure as described in Materials and Methods. B. The adenylate kinase release induced by streptomycin is compared to that with the bacteriostatic drug ethambutol.
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pone.0129234.g001: Bactericidal but not bacteriostatic drugs induce adenylate kinase release from M. smegmatis.A. Exponential phase M. smegmatis was treated with a dilution series of streptomycin (MIC 0.5 μg/mL). The adenylate kinase activity of the supernatants and the number of viable cells for each concentration were determined after 6 hours of exposure as described in Materials and Methods. B. The adenylate kinase release induced by streptomycin is compared to that with the bacteriostatic drug ethambutol.

Mentions: Fig 1A shows a dose response for AK release induced by a streptomycin dilution series (0.125 μg/mL to 2 μg/mL) that ranges from 4-fold below to 4-fold above the MIC of streptomycin toward mc2155 (0.5 μg/mL). A smooth dose-response curve relating streptomycin concentration to AK activity in the culture medium was generated. Cell viability (CFU/mL) at each streptomycin concentration was also determined by plating the contents of the well. For these optimized conditions, we observed a two-fold increase in AK release relative to untreated cultures at 0.5XMIC, a concentration where there was no detectable decrease in culture viability. This suggests that AK release is a more sensitive measure of bactericidal antimicrobial activity than conventional growth-based assays. This high sensitivity is consistent with previously reported data with fungi and other bacterial pathogens [9, 10], indicating that the AK assay can detect as few as 500 lysed cells in a sample of 106 cells and emphasizes the greater sensitivity of the AK assay relative to growth-based assays.


A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

Forbes L, Ebsworth-Mojica K, DiDone L, Li SG, Freundlich JS, Connell N, Dunman PM, Krysan DJ - PLoS ONE (2015)

Bactericidal but not bacteriostatic drugs induce adenylate kinase release from M. smegmatis.A. Exponential phase M. smegmatis was treated with a dilution series of streptomycin (MIC 0.5 μg/mL). The adenylate kinase activity of the supernatants and the number of viable cells for each concentration were determined after 6 hours of exposure as described in Materials and Methods. B. The adenylate kinase release induced by streptomycin is compared to that with the bacteriostatic drug ethambutol.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476654&req=5

pone.0129234.g001: Bactericidal but not bacteriostatic drugs induce adenylate kinase release from M. smegmatis.A. Exponential phase M. smegmatis was treated with a dilution series of streptomycin (MIC 0.5 μg/mL). The adenylate kinase activity of the supernatants and the number of viable cells for each concentration were determined after 6 hours of exposure as described in Materials and Methods. B. The adenylate kinase release induced by streptomycin is compared to that with the bacteriostatic drug ethambutol.
Mentions: Fig 1A shows a dose response for AK release induced by a streptomycin dilution series (0.125 μg/mL to 2 μg/mL) that ranges from 4-fold below to 4-fold above the MIC of streptomycin toward mc2155 (0.5 μg/mL). A smooth dose-response curve relating streptomycin concentration to AK activity in the culture medium was generated. Cell viability (CFU/mL) at each streptomycin concentration was also determined by plating the contents of the well. For these optimized conditions, we observed a two-fold increase in AK release relative to untreated cultures at 0.5XMIC, a concentration where there was no detectable decrease in culture viability. This suggests that AK release is a more sensitive measure of bactericidal antimicrobial activity than conventional growth-based assays. This high sensitivity is consistent with previously reported data with fungi and other bacterial pathogens [9, 10], indicating that the AK assay can detect as few as 500 lysed cells in a sample of 106 cells and emphasizes the greater sensitivity of the AK assay relative to growth-based assays.

Bottom Line: Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed.We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules.Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.

ABSTRACT
Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

No MeSH data available.


Related in: MedlinePlus