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Mechanisms of Resistance

Resistance of Streptococcus pneumoniaeto Deformylase Inhibitors Is Due to Mutations indefB

Peter Margolis, Corinne Hackbarth, Sara Lopez, Mita Maniar, Wen Wang, Zhengyu Yuan, Richard White, Joaquim Trias
Peter Margolis
Versicor, Inc., Fremont, California 94555
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Corinne Hackbarth
Versicor, Inc., Fremont, California 94555
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Sara Lopez
Versicor, Inc., Fremont, California 94555
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Mita Maniar
Versicor, Inc., Fremont, California 94555
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Wen Wang
Versicor, Inc., Fremont, California 94555
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Zhengyu Yuan
Versicor, Inc., Fremont, California 94555
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Richard White
Versicor, Inc., Fremont, California 94555
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Joaquim Trias
Versicor, Inc., Fremont, California 94555
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DOI: 10.1128/AAC.45.9.2432-2435.2001
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    Fig. 1.

    Alignment of conserved domains of deformylase proteins. Partial sequences of the predicted products of the defA anddefB homologs of S. aureus and S. pneumoniae are shown aligned with consensus PDF domains (13). Residues that diverge from the consensus are highlighted. Modifications in the deformylase enzyme in the resistantS. pneumoniae mutants VSPN6501, VSPN6503, and VSPN6504 are indicated by arrows. The positions of the motifs in the S. pneumoniae DefB are as follows: box 1, G69 to Q76; box 2, A123 to S132; and box 3, Q172 to G182. The sequences of the S. pneumoniae def homologs from strain R6x have been submitted to GenBank (accession numbers: defA, AY014508; defB,AY014509). Sequences of the S. pneumoniae def andfmt homologs from S. pneumoniae ATCC 49619 were also submitted to GenBank (accession numbers: defA,AY014510; defB, AY014511; fmt, AY014512).

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  • Table 1.

    Strains and plasmids used in this work

    Strain or plasmidRelevant characteristicsSource or reference
    Strains
     E. coli
      VECO2068F−araD139 Δ(ara, leu)7697 galE15 galK16Δ(lac)X74 rpsL hsdR2 (rk−mk+) mcrA mcrB1ΔtolCΔPdef::PBAD= def; PBAD-regulated defgene13
      BL21(DE3)/pLysSF−ompT hsdSB (rB−mB−) dcm gal λ(DE3) (pLysS); expression strain for PT7-regulated gene expressionNovagen, Madison, Wis.
     S. pneumoniae
      R6xUnencapsulated strain for laboratory genetics24
      ATCC 49619American Type Culture Collection, Manassas, Va.
      VSPN6501ATCC 49619defB(Q172K); actinonin-resistantThis study
      VSPN6503ATCC 49619 defB(A123D); actinonin-resistantThis study
      VSPN6504ATCC 49619defB(A123D); actinonin-resistantThis study
      VSPN7011R6x defA::pR326defA; defA disruptionThis study
      VSPN7035R6xdefB+::pPV302-7; expresses wild-typedefBThis study
      VSPN7036R6xdefB::pPV303-7; expresses defB(Q172K)This study
      VSPN7037R6xdefB::pPV304-5; expresses defB(A123D)This study
    Plasmids
     pET20b(+)PT7; overexpression vectorNovagen, Madison, Wis.
     pET20bSpn defBopt (C-His)pET20b(+) carrying Spn defB with optimized codon usage; encodes C-terminally His-tagged PDF (wild type)This study
     pET20bSpn defBopt (C-His, Q172K)pET20bSpn defBopt (C-His) mutagenized to introduce Q172K mutationThis study
     pET20bSpn defBopt(C-His, A123D)pET20bSpn defBopt (C-His) mutagenized to introduce A123D mutationThis study
     pGEX-5X-3Ptac; overexpression vector, GST fusion proteinAmersham Pharmacia, Piscataway, N.J.
     pGEXSpn defApGEX-5X-3::defA; Ptac-gst-defA fusionThis study
     pGEXSpn defBpGEX-5X-3::defB; Ptac-gst-defB fusionThis study
     pR326cat; E. coli, S. pneumoniae shuttle vector6
     pR326rafEpR326::'rafE'a23
     pR326defApR326::'defA'This study
     pR326defBpR326::'defB'This study
     pR326fmtpR326::'fmt'This study
     pPV302-7pR326::'defB+; downstream end of wild-type defB ORFThis study
     pPV303-7pR326::'defB(Q172K); downstream end of mutated defB ORFThis study
     pPV304-5pR326::'defB(A123D); downstream end of mutated defB ORFThis study
    • ↵a Apostrophes indicate the truncation of the gene at the upstream and/or downstream end, as indicated.

  • Table 2.

    Genotypes and susceptibilities to PDF inhibitors, and doubling times of S. pneumoniae actinonin-resistant and -susceptible strainsa

    StraindefBMean T2in min (SD)MIC (μg/ml)
    ActVRC3375b
    ATCC49619wt51 (1.4)3216
    VSPN6501Q172K60 (2.9)128256
    VSPN6503A123D64 (2.6)64128
    VSPN6504A123D66 (2.3)128128
    • ↵a wt, wild type;T2, doubling time; Act, actinonin

    • ↵b PDF inhibitor (D. Chen, C. Hackbarth, Z. J. Ni, W. Wang, C. Wu, D. Young, R. J. White, J. Trias, D. V. Patel, and Z. Yuan, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2175, 2000).

  • Table 3.

    Susceptibilities to actinonin of S. pneumoniaeR6x strains transformed with wild-type or mutated defB and inhibition of purified His-tagged PDF enzymes by actinonin

    S. pneumoniae strainExpressed defBMIC (μg/ml)EnzymeIC50 (nM)
    VSPN7035defB16Wild type53 ± 6
    VSPN7036defB (Q172K)64Q172K70 ± 8
    VSPN7037defB(A123D)≥64A123D136 ± 16
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Resistance of Streptococcus pneumoniaeto Deformylase Inhibitors Is Due to Mutations indefB
Peter Margolis, Corinne Hackbarth, Sara Lopez, Mita Maniar, Wen Wang, Zhengyu Yuan, Richard White, Joaquim Trias
Antimicrobial Agents and Chemotherapy Sep 2001, 45 (9) 2432-2435; DOI: 10.1128/AAC.45.9.2432-2435.2001

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Resistance of Streptococcus pneumoniaeto Deformylase Inhibitors Is Due to Mutations indefB
Peter Margolis, Corinne Hackbarth, Sara Lopez, Mita Maniar, Wen Wang, Zhengyu Yuan, Richard White, Joaquim Trias
Antimicrobial Agents and Chemotherapy Sep 2001, 45 (9) 2432-2435; DOI: 10.1128/AAC.45.9.2432-2435.2001
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KEYWORDS

Amidohydrolases
Aminopeptidases
Anti-Bacterial Agents
Enzyme Inhibitors
Hydroxamic Acids
Streptococcus pneumoniae

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