TY - JOUR
T1 - A new screen for tuberculosis drug candidates utilizing a luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren
AU - Ozeki, Yuriko
AU - Igarashi, Masayuki
AU - Doe, Matsumi
AU - Tamaru, Aki
AU - Kinoshita, Naoko
AU - Ogura, Yoshitoshi
AU - Iwamoto, Tomotada
AU - Sawa, Ryuichi
AU - Umekita, Maya
AU - Enany, Shymaa
AU - Nishiuchi, Yukiko
AU - Osada-Oka, Mayuko
AU - Hayashi, Tetsuya
AU - Niki, Mamiko
AU - Tateishi, Yoshitaka
AU - Hatano, Masaki
AU - Matsumoto, Sohkichi
N1 - Funding Information:
This work was supported by Research on Emerging and Reemerging Infectious Diseases from the Ministry of Health, Labour and Welfare of Japan http://www.mhlw.go.jp/stf/seisakunitsuite/bunya/ hokabunya/kenkyujigyou/index.html, H25-Shikoippan- 005 (S Matsumoto); and Scientific Research (B) and (C) from the Japan Society for the Promotion of Science http://www.mext.go.jp/a_menu/shinkou/ hojyo/main5_a5.htm, 24390106 (S Matsumoto), 24501015 (Y Ozeki).
Publisher Copyright:
© 2015 Ozeki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2015/11/1
Y1 - 2015/11/1
N2 - Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multidrug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 ug/ml, 0.5 ug/ml, and 2.0-7.5 ug/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.
AB - Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multidrug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 ug/ml, 0.5 ug/ml, and 2.0-7.5 ug/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.
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U2 - 10.1371/journal.pone.0141658
DO - 10.1371/journal.pone.0141658
M3 - Article
C2 - 26571296
AN - SCOPUS:84956885277
SN - 1932-6203
VL - 10
JO - PloS one
JF - PloS one
IS - 11
M1 - e0141658
ER -
