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Mechanisms of Action: Physiological Effects

Signature Gene Expression Profiles Discriminate between Isoniazid-, Thiolactomycin-, and Triclosan-Treated Mycobacterium tuberculosis

Joanna C. Betts, Alistair McLaren, Mark G. Lennon, Fiona M. Kelly, Pauline T. Lukey, Steve J. Blakemore, Ken Duncan
Joanna C. Betts
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
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  • For correspondence: joanna.c.betts@gsk.com
Alistair McLaren
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
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Mark G. Lennon
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
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Fiona M. Kelly
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
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Pauline T. Lukey
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
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Steve J. Blakemore
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
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Ken Duncan
GlaxoSmithKline Research and Development, Stevenage, Hertfordshire, SG1 2NY, United Kingdom
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DOI: 10.1128/AAC.47.9.2903-2913.2003
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  • FIG. 1.
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    FIG. 1.

    Overlap of genes regulated by isoniazid, thiolactomycin, or triclosan treatment of M. tuberculosis. Numbers within the sectors indicate the total numbers of genes regulated uniquely or in common by either 1× or 5× MIC treatment of each drug at either 2 or 6 h (P < 0.001). INH, isoniazid; TLM, thiolactomycin; TRC, triclosan.

  • FIG. 2.
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    FIG. 2.

    Two-dimensional cluster analysis of the drug-treated expression profiles. Two-dimensional agglomerative clustering was performed on the 877 genes significantly regulated in response to any of the drug treatments (P < 0.001). The individual genes are represented on the x axis and the different samples are indicated on the y axis. Red, upregulation; green, downregulation; black, no change relative to the time zero control. INH, isoniazid; TLM, thiolactomycin; TRC, triclosan.

  • FIG. 3.
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    FIG. 3.

    Response of the kas operon to isoniazid, thiolactomycin, or triclosan treatment as measured by QRT-PCR. (A) Schematic representation of the kas operon in the M. tuberculosis H37Rv genome. Ratio between the number of cDNA copies detected in each sample relative to the time zero control by QRT-PCR at 2 h (B) and 6 h (C) is represented. Each value is the average of two biological replicates, each analyzed in duplicate. INH, isoniazid; TLM, thiolactomycin; TRC, triclosan.

  • FIG. 4.
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    FIG. 4.

    Genes induced by triclosan treatment of M. tuberculosis as measured by QRT-PCR. Organization of Rv1685c to Rv1687c (A) and Rv3160c to Rv3161c (C) in the M. tuberculosis H37Rv genome. Ratio between the number of cDNA copies detected in each sample relative to the time zero control by QRT-PCR for Rv1685c to Rv1687c (B) and Rv3160c to Rv3161c (D). Each value is the average of two biological replicates, each analyzed in duplicate. TRC, triclosan.

  • FIG. 5.
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    FIG. 5.

    PCA of isoniazid, thiolactomycin, triclosan, and control expression profiles. The largest source of variance is explained on the x axis and the second largest on the y axis. Each hybridization is represented by a single point. Isoniazid treatments, circles; thiolactomycin treatments, triangles; triclosan treatments, squares; vehicle control treatments, diamonds. Two-hour treatments, open shapes; 6-h treatments, closed shapes. 1× MIC, black; 5× MIC, red; vehicle control, blue.

Tables

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  • TABLE 1.

    Primers used for the QRT-PCR

    GeneForward primerReverse primer
    sigA TTCGCGCCTACCTCAAACAGGCTAGCTCGACCTCTTCCTCG
    fabD ACCCTGGTCTCCCAGCTCACAGTTCGCGTTTGGCGATACC
    acpM CAAGTACGGCGTCAAGATCCCACTTGGACTCGGCCTCAAGC
    kasA TGCTCATCGAGACGGAGGAGCTACCGGCACGAACACCATC
    kasB CCGTGCAGAAGTACATGCCCACCGCTTCGATCCTGGTCTC
    accD6 CACACCGTTCGACGAGTTCCTCTGCGCTTTCGGAGTTCAG
    Rv1685cACTTCGGCACCAAACAGCAGAACCGAGTTCCTCGACAGGC
    Rv1686cTGATGTTTGTGATCACGGCGGAACCAGAACGCCACAATGC
    Rv1687cGGTCGGGCAAGACAACACTGAGAGTTCGGCGAAGTAGCGG
    Rv3160cCTGGACCACCTGCATGCTTCCCATCTCCGCGACTAGCTCC
    Rv3161cCAGGGTTCCCTTCACCGTTCGCGGAATAAAGCCGAACCAC
  • TABLE 2.

    Numbers of genes regulated in M. tuberculosis by each drug's indicated MIC, according to functional class, at 2 and 6 ha

    Functional classbTotal no. on array2 h6 h
    1×-MIC INH5×-MIC INH1×-MIC TLM5×-MIC TLM1×-MIC TRC5×-MIC TRC1×-MIC INH5×-MIC INH1×-MIC TLM5×-MIC TLM1×-MIC TRC5×-MIC TRC
    UpDownUpDownUpDownUpDownUpDownUpDownUpDownUpDownUpDownUpDownUpDownUpDown
    0: Virulence, detoxification, adaptation981010101220940214110001913
    1: Lipid metabolism23141511415031111610548263131327
    2: Information pathways224200215300268491011021008660
    3: Cell wall and cell processes6994131144411414424211443446905133
    5: Insertion sequences and phages14101000300201210010000120101
    6: PE/PPE142000000000000000000000010
    7: Intermediary metabolism and respiration889774217615214542322550412605457
    8: Unknown265040036112210700100000003211
    9: Regulatory proteins18602120111231721012010110254
    10: Conserved hypothetical proteins1,0057106214915117424223942141708756
    Total3,88025262010783314155924207168983824141516142612288262
    • ↵ a INH, isoniazid; TLM, thiolactomycin; TRC, triclosan; Up and Down, up- and downregulated.

    • ↵ b According to TubercuList (http://genolist.pasteur.fr/TubercuList/ ).

  • TABLE 3.

    Genes commonly regulated by the drug treatments

    ORFGene nameDescriptionFunctional classa
    Induced by isoniazid, thiolactomycin, and triclosan
        Rv0040c mtc28 Secreted proline-rich protein3
        Rv1987Possible chitinase3
        Rv3354Conserved hypothetical protein10
    Downregulated by isoniazid, thiolactomycin, and triclosan
        Rv0352 dnaJ1 Probable chaperone protein0
        Rv0790cHypothetical protein8
        Rv0823cPossible transcriptional regulatory protein9
        Rv0824c desA1 Probable acyl-[ACP] desaturase1
        Rv1094 desA2 Possible acyl-[ACP] desaturase1
        Rv2840cConserved hypothetical protein10
    Induced by isoniazid and thiolactomycin
        Rv0179c lprO Possible lipoprotein3
        Rv0207cConserved hypothetical protein10
        Rv0312Conserved hypothetical proline- and threonine-rich protein10
        Rv0342 iniA Isoniazid-inducible gene protein3
        Rv0359Probable conserved integral membrane protein3
        Rv0569Conserved hypothetical protein10
        Rv0951 sucC Probable succinyl coenzyme A synthetase (beta chain)7
        Rv1592cConserved hypothetical protein10
        Rv1736c narX Probable nitrate reductase7
        Rv2007c fdxA Probable ferredoxin7
        Rv2193 ctaE Probable cytochrome c oxidase (subunit III)7
        Rv2243 fabD Malonyl coenzyme A-acyl carrier protein transacylase1
        Rv2244 acpM Meromycolate extension acyl carrier protein1
        Rv2245 kasA β-Ketoacyl-[ACP] synthase 11
        Rv2246 kasB β-Ketoacyl-[ACP] synthase 21
        Rv2428 ahpC Alkyl hydroperoxide reductase C0
        Rv2721cPossible conserved transmembrane alanine- and glycine-rich protein3
        Rv2846c efpA Possible integral membrane efflux protein3
        Rv3134cConserved hypothetical protein10
        Rv3456c rplQ Probable 50S ribosomal protein L172
        Rv3524Probable conserved membrane protein3
    • ↵ a Functional classes are listed in Table 1.

  • TABLE 4.

    Differential response of the kas operon to isoniazid, thiolactomycin, or triclosan treatment

    Treatmenta and timefabDacpMkasAkasBaccD6
    RatiobP valueRatioP valueRatioP valueRatioP valueRatioP value
    5×-MIC INH, 2 h1.533.06 × 10−43.15<1.00 × 10−462.812.78 × 10−292.781.49 × 10−28NS
    5×-MIC INH, 6 h2.27<1.00 × 10−464.53<1.00 × 10−464.401.87 × 10−313.54<1.00 × 10−46NS
    1×-MIC TLM, 2 h2.261.54 × 10−44.30<1.00 × 10−463.794.77 × 10−153.421.09 × 10−181.691.12 × 10−11
    5×-MIC TLM, 2 hNS3.70<1.00 × 10−462.947.92 × 10−202.081.43 × 10−61.418.06 × 10−5
    1×-MIC TLM, 6 h1.889.48 × 10−63.01<1.00 × 10−462.875.33 × 10−392.108.86 × 10−101.418.36 × 10−4
    5×-MIC TLM, 6 h1.795.01 × 10−64.00<1.00 × 10−462.934.49 × 10−372.411.61 × 10−14NS
    5×-MIC TRC, 2 hNS0.243.30 × 10−240.308.29 × 10−150.366.27 × 10−9NS
    5×-MIC TRC, 6 hNS0.13<1.00 × 10−460.20<1.00 × 10−460.345.37 × 10−25NS
    • ↵ a INH, isoniazid; TLM, thiolactomycin; TRC, triclosan.

    • ↵ b Average expression ratio (drug treated/t = 0 control) calculated from a total of four hybridizations of two biological replicates. NS, not significant (P < 0.001).

  • TABLE 5.

    Genes induced in M. tuberculosis by all triclosan (TRC) treatments

    ORFGene nameDescriptionFunctional classaMIC and exposure time
    1×-MIC TRC, 2 h5×-MIC TRC, 2 h1×-MIC TRC, 6 h5×-MIC TRC, 6 h
    RatiobP valueRatioP valueRatioP valueRatioP value
    Rv0077cProbable oxidoreductase71.881.05 × 10−65.463.33 × 10−81.601.87 × 10−113.577.01 × 10−45
    Rv0711 atsA Possible arylsulfatase72.644.48 × 10−173.16<1.00 × 10−461.654.40 × 10−71.921.86 × 10−29
    Rv1557 mmpL6 Probable conserved transmembrane transport protein32.165.31 × 10−161.672.71 × 10−121.419.25 × 10−81.662.59 × 10−19
    Rv1685cConserved hypothetical protein1017.89<1.00 × 10−4621.203.06 × 10−2613.15<1.00 × 10−4621.17<1.00 × 10−46
    Rv1686cProbable conserved integral membrane protein, ABC transporter314.99<1.00 × 10−4616.58<1.00 × 10−4611.02<1.00 × 10−4621.30<1.00 × 10−46
    Rv1687cProbable conserved ATP-binding protein, ABC transporter36.953.49 × 10−2612.951.26 × 10−175.06<1.00 × 10−4611.16<1.00 × 10−46
    Rv3160cPossible transcriptional regulatory protein (probably TetR family)92.404.62 × 10−69.512.34 × 10−182.013.35 × 10−98.63<1.00 × 10−46
    Rv3161cPossible dioxygenase74.771.03 × 10−3918.92<1.00 × 10−464.40<1.00 × 10−4618.42<1.00 × 10−46
    • ↵ a Functional classes are listed in Table 1.

    • ↵ b Average expression ratio (drug treated/t = 0 control) calculated from a total of four hybridizations of two biological replicates.

  • TABLE 6.

    The genes and discriminant function required for classification of isoniazid (INH), thiolactomycin (TLM), triclosan (TRC), or control groups

    ORFGene nameDescriptionAvg squared canonical correlationaINH [D2INH (x)]bTLM [D2TLM (x)]TRC [D2TRC (x)]Control [D2Control (x)]
    Constant−14,987,826−167,097,220−79,450,033−5,513,637
    Rv1686cProbable conserved integral membrane protein ABC transporter0.3325−19,099,766−100,023,236−36,942,559−19,032,909
    Rv2244 acpM Meromycolate extension acyl carrier protein0.6111110,774,262408,156,465243,639,84072,779,372
    Rv0677c mmpS5 Possible conserved membrane protein0.8533121,600,815512,108,617316,180,09794,776,642
    Rv2190cConserved hypothetical protein0.8904−68,508,665−286,328,959−181,659,649−52,963,842
    Rv3250c rubB Probable rubredoxin0.8954−10,150,989−44,990,430−22,707,705−8,353,423
    Rv2276 cyp121 Cytochrome P450 1210.9040−36,833,310−220,327,670−143,773,133−43,835,221
    Rv3049cProbable monooxygenase0.9491−114,909,397−452,847,559−265,224,702−82,194,446
    Rv0208cHypothetical methyltransferase0.9762−178,128,313−821,139,430−524,976,195−155,439,247
    Rv2745cPossible transcriptional regulatory protein0.9817−77,131,242−323,842,646−200,033,735−59,890,920
    Rv2253Possible secreted unknown protein0.9834−49,162,506−201,531,571−124,495,122−37,049,967
    Rv1072Probable conserved transmembrane protein0.984364,061,474246,735,357152,824,15044,629,671
    Rv0298Hypothetical protein0.986547,211,509172,163,109103,076,69130,611,076
    Rv3341 metA Probable homoserine O-acetyltransferase0.98671,400,025−26,153,983−29,413,890−6,482,707
    Rv0001 dnaA Chromosomal replication initiator protein0.9932−23,069,217−86,660,290−56,244,876−15,604,890
    Rv3457c rpoA Probable DNA-directed RNA polymerase (α chain)0.9942−82,964,894−232,795,471−118,734,295−37,450,414
    Rv1737c narK2 Possible nitrate/nitrite transporter0.9946−22,730,982−62,077,538−26,268,313−9,791,891
    Rv1871cConserved hypothetical protein0.996716,814,61346,910,79221,021,7727,485,856
    Rv3404cConserved hypothetical protein0.997146,788,129124,430,28361,274,97919,520,565
    Rv3486Conserved hypothetical protein0.9972−63,601,803−175,065,603−83,869,339−27,853,590
    Rv3310Possible acid phosphatase0.999835,921,00297,302,39647,325,50015,382,806
    Rv0283Possible conserved membrane protein1.000022,910,89662,309,71230,137,4649,865,584
    • ↵ a Cumulative canonical correlation showing the increasing amount of variance accounted for by each gene in the discriminant model.

    • ↵ b To classify an observation (x) into one of the groups, the discriminant score [D2y (x)] is calculated for each group (y) using the above functions. These scores are inversely related to the distance that observation is from the middle of each group. The observation belongs to the group whose function gives the highest discriminant score. The probability of an observation (x) belonging to a group (y) is calculated as eDy2(x)/[eDControl2(x)+eDINH2(x)+eDTLM2(x)+eDTRC2(x)].

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Signature Gene Expression Profiles Discriminate between Isoniazid-, Thiolactomycin-, and Triclosan-Treated Mycobacterium tuberculosis
Joanna C. Betts, Alistair McLaren, Mark G. Lennon, Fiona M. Kelly, Pauline T. Lukey, Steve J. Blakemore, Ken Duncan
Antimicrobial Agents and Chemotherapy Aug 2003, 47 (9) 2903-2913; DOI: 10.1128/AAC.47.9.2903-2913.2003

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Signature Gene Expression Profiles Discriminate between Isoniazid-, Thiolactomycin-, and Triclosan-Treated Mycobacterium tuberculosis
Joanna C. Betts, Alistair McLaren, Mark G. Lennon, Fiona M. Kelly, Pauline T. Lukey, Steve J. Blakemore, Ken Duncan
Antimicrobial Agents and Chemotherapy Aug 2003, 47 (9) 2903-2913; DOI: 10.1128/AAC.47.9.2903-2913.2003
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KEYWORDS

Anti-Bacterial Agents
Anti-Infective Agents, Local
antitubercular agents
Gene Expression Regulation, Bacterial
isoniazid
Mycobacterium tuberculosis
Thiophenes
triclosan

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