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Mechanisms of Action: Physiological Effects

Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid

Feng Wang, Paras Jain, Gulcin Gulten, Zhen Liu, Yicheng Feng, Krishna Ganesula, Alifiya S. Motiwala, Thomas R. Ioerger, David Alland, Catherine Vilchèze, William R. Jacobs, Jr., James C. Sacchettini
Feng Wang
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128
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Paras Jain
Department of Microbiology and Immunology and the Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York 10461
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Gulcin Gulten
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128
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Zhen Liu
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128
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Yicheng Feng
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128
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Krishna Ganesula
Department of Computer Science and Engineering, Texas A&M University, College Station, Texas 77843-3112
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Alifiya S. Motiwala
Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
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Thomas R. Ioerger
Department of Computer Science and Engineering, Texas A&M University, College Station, Texas 77843-3112
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  • For correspondence: ioerger@cs.tamu.edu
David Alland
Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
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Catherine Vilchèze
Department of Microbiology and Immunology and the Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York 10461
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William R. Jacobs
Department of Microbiology and Immunology and the Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York 10461
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James C. Sacchettini
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128
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DOI: 10.1128/AAC.00453-10
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  • FIG. 1.
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    FIG. 1.

    Overexpression of the proteins in M. smegmatis confirmed by SDS-PAGE analysis. Lanes: M, marker; 1, vector; 2, Rv0155 (PntAA, 40 kDa); 3, Rv1484 (InhA, 29.5 kDa); 4, Rv2763c (DfrA, 17.4 kDa); 5, Rv1996 (34.9 kDa); 6, Rv2623 (32.7 kDa); 7, Rv2766c (FabG5, 30.8 kDa); 8, Rv2858 (AldC, 50 kDa); 9, uninduced Rv2763c (DfrA); 10, induced Rv2763c (DfrA).

  • FIG. 2.
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    FIG. 2.

    Growth of the wild-type M. smegmatis strain and transformed strains overexpressing DHFR or InhA on media containing the known DHFR inhibitor trimethoprim (50 or 150 μg/ml) or INH (10 or 120 μg/ml).

  • FIG. 3.
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    FIG. 3.

    Growth of M. tuberculosis H37Rv strains overexpressing DHFR or InhA on media containing INH (0.2 μg/ml) or kanamycin (100 μg/ml). Spots: A, pMV261::inhA (InhA overexpresser); B, pMV261 (plasmid only); C to F, four independent transformants of pMV261::dfrA (DHFR overexpresser).

Tables

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  • TABLE 1.

    MICs determined in 7H9 broth by serial dilution method after overexpressing the INH-NAD binding proteins from M. tuberculosis H37Rv in M. smegmatis

    Overexpressed proteinMIC (μg/ml)a
    INHETH
    None (mc24517 parental strain)4.79.4
    Rv1484 (InhA)300150
    Rv2763c (DfrA)4.79.4
    Rv3248c (SahH)4.79.4
    Rv0753c (MmsA)4.79.4
    Rv1187 (RocA)4.79.4
    Rv0155 (PntAA)4.79.4
    Rv2623 (universal stress protein)4.79.4
    Rv1996 (universal stress protein)4.79.4
    Rv0468 (FadB2)4.79.4
    Rv2691 (CeoB/TrkA)4.79.4
    Rv0091 (Mtn/Sah)NANA
    Rv2858c (AldC)4.79.4
    Rv1059 (unknown)4.79.4
    Rv0926c (unknown)4.79.4
    Rv3777 (probable oxidoreductase)4.79.4
    Rv2971 (probable oxidoreductase)NANA
    Rv2766 (FabG5)4.79.4
    Rv2671 (RibD)4.79.4
    • ↵a NA, not available (overexpression was not successful).

  • TABLE 2.

    Mutations in genes identified as potential INH-NAD(P) bindersa

    ProteinMutation in strain:
    507150725297532453585400M. bovisBCG
    Rv2763c/dfrANoneNoneNoneNoneNoneNoneNone
    Rv3248c/sahHNoneNoneNoneNoneNoneNoneNone
    Rv0753c/mmsANoneNoneNoneNoneNoneNoneNone
    Rv1187/rocANoneNoneNoneNoneNoneNoneNone
    Rv0155/pntAaA274ANoneNoneNoneNoneNoneNone
    Rv2623NoneNoneNoneNoneNoneNoneNone
    Rv1996NoneNoneNoneNoneNoneNoneNone
    Rv0468/fadB2NoneNoneNoneNoneL87LNoneL87L
    Rv2691/ceoBT117AT117AT117AT117AT117AT117AT117A
    Rv0091/mtnNoneA155PNoneQ174QNone931-bp deletionNone
    Rv2858c/aldCNoneNoneT21ANoneP77PNoneP77P
    Rv1059a-64cb1-bp deletion in P99NoneNoneNoneNoneNone
    Rv3777NoneNoneP101ANoneNoneNoneNone
    Rv3777V160AV160AV160AV160AV160AV160AV160A
    Rv3777L63LL63LL63LL63LL63LL63LL63L
    Rv0926cNoneNoneNoneNoneNoneNoneNone
    Rv2766c/fabG5NoneNoneNoneNoneNoneNoneNone
    Rv2671/ribDNoneNoneNoneNoneNoneNoneNone
    Rv2971NoneNoneNoneNoneN152HNoneN152H
    • ↵a Argyrou et al. (1). Mutations are indicated relative to the amino acid in the H37Rv reference sequence. Mutations that are shared with M. bovis BCG are assumed not to cause isoniazid resistance.

    • ↵b Single nucleotide polymorphism in promoter region.

  • TABLE 3.

    Mutations in genes traditionally associated with INH resistancea

    ProteinMutation(s) in strain:
    507150725297532453585400M. bovisBCG
    Rv1484/inhANoneNoneNoneNoneNoneNoneNone
    Rv1908c/katGNoneNoneNoneG285VR463LF129SR463L
    Rv1909c/furANoneNoneNoneNoneA46VNoneA46V
    Rv1854c/ndhNoneNoneNoneNoneG313RNoneG313R
    Rv0342/iniANoneNoneNoneNoneN88S, H481QNoneN88S, H481Q
    Rv0341/iniBNoneNoneNoneNoneNoneNoneNone
    Rv0343/iniCNoneNoneNoneNoneNoneNoneNone
    Rv3139/fadE24NoneNoneNoneNoneNoneNoneNone
    Rv2245/kasANoneNoneNoneNoneNoneNoneNone
    Rv2246/kasBNoneNoneNoneNoneNoneNoneNone
    Rv2428/ahpCNoneNoneNoneNoneNoneNoneNone
    Rv2242NoneNoneNoneNoneA363TNoneA363T
    Rv0340NoneNoneNoneNoneNoneNoneNone
    Rv1592cI322VI322VI322VI322VI322VI322VI322V
    Rv1772NoneNoneNoneNoneDeletionNoneDeletion
    Rv2846c/efpANoneNoneNoneQ513RT15RNoneT15R
    Rv0486/mshAN111SN111SNoneN111SNoneN111SNone
    • ↵a Mutations are indicated relative to the amino acid in the H37Rv reference sequence. Synonymous mutations are excluded.

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Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid
Feng Wang, Paras Jain, Gulcin Gulten, Zhen Liu, Yicheng Feng, Krishna Ganesula, Alifiya S. Motiwala, Thomas R. Ioerger, David Alland, Catherine Vilchèze, William R. Jacobs Jr., James C. Sacchettini
Antimicrobial Agents and Chemotherapy Aug 2010, 54 (9) 3776-3782; DOI: 10.1128/AAC.00453-10

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Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid
Feng Wang, Paras Jain, Gulcin Gulten, Zhen Liu, Yicheng Feng, Krishna Ganesula, Alifiya S. Motiwala, Thomas R. Ioerger, David Alland, Catherine Vilchèze, William R. Jacobs Jr., James C. Sacchettini
Antimicrobial Agents and Chemotherapy Aug 2010, 54 (9) 3776-3782; DOI: 10.1128/AAC.00453-10
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