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Research Article

MexY-Promoted Aminoglycoside Resistance in Pseudomonas aeruginosa: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition

Calvin Ho-Fung Lau, Daniel Hughes, Keith Poole
Gerard D. Wright, Editor
Calvin Ho-Fung Lau
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Daniel Hughes
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Keith Poole
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Gerard D. Wright
McMaster University
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DOI: 10.1128/mBio.01068-14
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  • FIG 1 
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    FIG 1 

    Expression of mutant MexY proteins in P. aeruginosa K3315. Whole-cell extracts of P. aeruginosa ΔmexY strain K3315 expressing plasmid pRK415-borne wild-type (WT) or mutant mexY genes (amino acid substitutions in MexY are indicated) were electrophoretically separated by SDS-PAGE, electroblotted, and developed with antibodies directed against the MexY protein. The MexY proteins, absent in strain K3315 harboring pRK415 without a cloned mexY gene (pRK415), are indicated by arrowheads. The migration positions of molecular mass markers are shown at left.

  • FIG 2 
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    FIG 2 

    Mapping mutations impacting MexY-mediated antibiotic resistance on a 3-dimensional homology model of MexY. (A) An asymmetric trimer model of MexY constructed by homology modeling on the crystal structure of E. coli AcrB (PDB code 2HRT). Individual monomers are shown in space-fill formatting and are colored salmon red (loose/access conformation), gray (tight/binding conformation), and blue (open/extrusion conformation; mostly hidden behind the other 2 monomers). Structural details of the tight/binding monomer are shown in order to highlight the characteristic N- and C-terminal regions/subdomains of the docking domain (DN [yellow] and DC [purple], respectively), the porter domain (2 N-terminal [PN1, orange]/[PN2, pink] and 2 C-terminal [PC1, green]/[PC2, red] subdomains), and the transmembrane domain (transmembrane segments TM1-6 [light blue] and TM7-12 [dark blue], respectively) of RND transporters. The positions of the intermonomer vestibule and intramonomer cleft are also indicated. (B) Locations in the tight/binding MexY monomer of mutations (shown in space-fill formatting) that compromise MexY-mediated drug resistance. Relevant subdomains (as described for panel A) are highlighted in the corresponding colors. Structural models at left and right were rotated 50° counterclockwise and 100° clockwise, respectively, relative to the middle model in order to better illustrate the positions of various mutations. The dashed lines define the inner-membrane boundary. A cleft/opening, within which the putative proximal binding pocket occurs, is clearly seen in the structural model at left. A vestibule drug entry pathway is also highlighted in the leftmost structural model.

  • FIG 3 
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    FIG 3 

    Segments of amino acid sequence alignment of MexY and selected RND homologues in P. aeruginosa and other Gram-negative bacteria. The amino acid sequences of MexY and the indicated RND homologues were aligned using the multiple-sequence-alignment program T-coffee (55). Only those aligned sequences that encompassed the MexY residues whose mutation compromised drug resistance (indicated with an arrowhead and labeled) are shown. Residues that are conserved in other RND homologues are highlighted in bold lettering. Amino acid sequences were obtained from the NCBI Protein database (GenBank accession no. NP_250708 [MexY_P. aeruginosa PAO1], EGP45231 [AxyY_Achromobacter xylosoxidans AXX-A], YP_108402 [AmrB_Burkholderia pseudomallei K96243], CAJ77844 [AdeB_Acinetobacter baumannii AYE], YP_001972001 [SmeZ_Stenotrophomonas maltophilia K279a], NP_416965 [AcrD_Escherichia coli K-12 MG1655], NP_253288 [MexD_P. aeruginosa PAO1], NP_249117 [MexB_P. aeruginosa PAO1], NP_414995 [AcrB_E. coli K-12 MG1655], and YP_006275722 [BpeB_B. pseudomallei 1026b]). MexY, AxyY, AmrB, AdeB, SmeZ, and AcrD have been linked to aminoglycoside resistance.

Tables

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  • TABLE 1 

    Impact of MexY mutations on antimicrobial susceptibility of P. aeruginosaa

    StrainPlasmidMexY mutationMIC (μg/ml)b
    STRPARNEOAMIERYSPCCAMNORcCEFd
    K767NoneWT3225632251251264NDeND
    K3315NoneNone2840.5646464NDND
    K3315pRK415None21640.56464320.51
    K3315pCL10WT864815125123224
    K3315pCL14G216D216NDND6464320.51
    K3315pCL13R184H432NDND25625632NDND
    K3315pCL11S16F432NDND25625632NDND
    K3315pCL17A960T416NDND25625632NDND
    K3315pCL15V339M4168f15122563221
    K3315pCL12R166C432NDND2565123224
    K3315pCL16P562S832NDND25651232NDND
    K3315pCL19D133A41640.5256102432NDND
    K3315pCL20D133S41640.5256102432NDND
    K3315pCL18K79A81288g1512102432NDND
    K3315pCL21Y613A41640.55122563222
    • ↵a The antimicrobial susceptibility of P. aeruginosa ΔmexY strain K3315 carrying the indicated plasmids expressing wild-type (WT) MexY or MexY derivatives with the indicated amino acid substitutions is reported. Results for WT strain K767 and plasmid-free K3315 are provided for comparison purposes.

    • ↵b MIC values lower than that measured for pCL10-carrying K3315 expressing WT MexY are bolded. MIC values higher than that measured for pCL10-carrying K3315 are italicized. STR, streptomycin; PAR, paromomycin; NEO, neomycin; AMI, amikacin; ERY, erythromycin; SPC, spectinomycin; CAM, chloramphenicol; NOR, norfloxacin; CEF, cefepime.

    • ↵c NOR MICs were determined in the presence of 8 µg/ml chloramphenicol. The NOR MIC for all strains in the absence of chloramphenicol was 0.5 µg/ml.

    • ↵d CEF MICs were determined in the presence of 8 µg/ml chloramphenicol. The CEF MIC determined in the absence of chloramphenicol was 1 (for pRK415 control and MexYG216D) or 2 (for MexYWT and its V339M, R166C, and Y613A variants) μg/ml.

    • ↵e ND, not determined.

    • ↵f At half the NEO MIC (4 µg/ml), MexYV339M-expressing K3315 grew reproducibly slower than K3315 expressing MexYWT (data not shown), indicating that the V339M mutation compromises neomycin resistance.

    • ↵g At half the NEO MIC (4 µg/ml), MexYK79A-expressing K3315 grew reproducibly faster than K3315 expressing MexYWT (data not shown), indicating that the K79A mutation enhances neomycin resistance.

  • TABLE 2 

    Bacterial strains and plasmids used in this study

    Strain or plasmidDescriptionaReference or source
    E. coli strains
        DH5αφ80dlacZΔM15 endA1 recA1 hsdR17 (rK− mK+) supE44 thi-1 gyrA96 relA1 F− Δ(lacZYA-argF) U16949
        S17-1thi pro hsdR recA Tra+50
    P. aeruginosa strains
        K767PAO1 prototroph wild type51
        K1525K767ΔmexXY52
        K3315K767ΔmexYThis study
    Plasmids
        pEX18TcBroad-host-range gene replacement vector; sacB Tcr53
        pCL8pEX18Tc::ΔmexYThis study
        pCL9pEX18Tc::mexYThis study
        pRK415P. aeruginosa-E. coli shuttle cloning vector; Tcr54
        pCL10pRK415::mexYWTThis study
        pCL11pRK415::mexYS16FThis study
        pCL12pRK415::mexYR166CThis study
        pCL13pRK415::mexYR184HThis study
        pCL14pRK415::mexYG216DThis study
        pCL15pRK415::mexYV339MThis study
        pCL16pRK415::mexYP562SThis study
        pCL17pRK415::mexYA960TThis study
        pCL18pRK415::mexYK79AThis study
        pCL19pRK415::mexYD133AThis study
        pCL20pRK415::mexYD133SThis study
        pCL21pRK415::mexYY613AThis study
    • ↵a Tcr, tetracycline resistance; WT, wild type.

  • TABLE 3 

    Oligonucleotides used in this study

    PrimerOligonucleotide sequence (5′ → 3′)aSource
    mexYUP-FATTAAGAGCTCTACCGCCAGGCTGThis study
    mexYUP-RGCTGCATCTAGATCGTAGCGTTCTCThis study
    mexYDN-FAGTCGATCTAGATGCCCCTAGCGAAACThis study
    mexYDN-RGCAGACAAGCTTCTGGCCGACTATCThis study
    mexYscreen-FTGTTCCGCAATCCGCATCThis study
    mexYscreen-RGCGTAGCCGATCATCTGTCThis study
    mexY-3138-FGGCTCGAAGCTTATGGCTCGTTTCTTCThis study
    mexY-3138-RATCTAGGATCCTCAGGCTTGCTCCGTGThis study
    mexY-58-FTTATACAAGCTTCGACGCCCCCTCACCGCTThis study
    mexY-58-RTGGCGGTCATTTGGTTGACCThis study
    mexYseq-F1GCCTGAAGATCGTCGAGTCThis study
    mexYseq-F2CAAGCTGACCTCGATGAACCTThis study
    mexYseq-F3CGATCAACGTGCTGACCATGTThis study
    mexYseq-R4CAGTCCCTGCATCAATTGCTThis study
    mexYseq-R5ATCTCGTCCATGCTCACGCThis study
    mexY-K79A-FGGCCTGCTCTACACCGCGGCCACCAGCAGCACThis study
    mexY-K79A-RGTGCTGCTGGTGGCCGCGGTGTAGAGCAGGCCThis study
    mexY-D133A-FTGGAGAAGGCGGCGGCCAGCATCCAGCTGATThis study
    mexY-D133A-RATCAGCTGGATGCTGGCCGCCGCCTTCTCCAThis study
    mexY-D133S-FTGGAGAAGGCGGCGTCCAGCATCCAGCTGATThis study
    mexY-D133S-RATCAGCTGGATGCTGGACGCCGCCTTCTCCAThis study
    mexY-Y613A-FGGCGGCTTCAGCCTGGCCGGCGACGGCACCAGThis study
    mexY-Y613A-RCTGGTGCCGTCGCCGGCCAGGCTGAAGCCGCCThis study
    • ↵a Restriction endonuclease cleavage sites are underlined. Site-directed mutations are bolded and italicized.

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MexY-Promoted Aminoglycoside Resistance in Pseudomonas aeruginosa: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
Calvin Ho-Fung Lau, Daniel Hughes, Keith Poole
mBio Apr 2014, 5 (2) e01068-14; DOI: 10.1128/mBio.01068-14

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MexY-Promoted Aminoglycoside Resistance in Pseudomonas aeruginosa: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
Calvin Ho-Fung Lau, Daniel Hughes, Keith Poole
mBio Apr 2014, 5 (2) e01068-14; DOI: 10.1128/mBio.01068-14
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