Article Figures & Data
Figures
- 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
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
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
- TABLE 1
Impact of MexY mutations on antimicrobial susceptibility of P. aeruginosaa
Strain Plasmid MexY mutation MIC (μg/ml)b STR PAR NEO AMI ERY SPC CAM NORc CEFd K767 None WT 32 256 32 2 512 512 64 NDe ND K3315 None None 2 8 4 0.5 64 64 64 ND ND K3315 pRK415 None 2 16 4 0.5 64 64 32 0.5 1 K3315 pCL10 WT 8 64 8 1 512 512 32 2 4 K3315 pCL14 G216D 2 16 ND ND 64 64 32 0.5 1 K3315 pCL13 R184H 4 32 ND ND 256 256 32 ND ND K3315 pCL11 S16F 4 32 ND ND 256 256 32 ND ND K3315 pCL17 A960T 4 16 ND ND 256 256 32 ND ND K3315 pCL15 V339M 4 16 8f 1 512 256 32 2 1 K3315 pCL12 R166C 4 32 ND ND 256 512 32 2 4 K3315 pCL16 P562S 8 32 ND ND 256 512 32 ND ND K3315 pCL19 D133A 4 16 4 0.5 256 1024 32 ND ND K3315 pCL20 D133S 4 16 4 0.5 256 1024 32 ND ND K3315 pCL18 K79A 8 128 8g 1 512 1024 32 ND ND K3315 pCL21 Y613A 4 16 4 0.5 512 256 32 2 2 ↵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 plasmid Descriptiona Reference or source E. coli strains DH5α φ80dlacZΔM15 endA1 recA1 hsdR17 (rK− mK+) supE44 thi-1 gyrA96 relA1 F− Δ(lacZYA-argF) U169 49 S17-1 thi pro hsdR recA Tra+ 50 P. aeruginosa strains K767 PAO1 prototroph wild type 51 K1525 K767ΔmexXY 52 K3315 K767ΔmexY This study Plasmids pEX18Tc Broad-host-range gene replacement vector; sacB Tcr 53 pCL8 pEX18Tc::ΔmexY This study pCL9 pEX18Tc::mexY This study pRK415 P. aeruginosa-E. coli shuttle cloning vector; Tcr 54 pCL10 pRK415::mexYWT This study pCL11 pRK415::mexYS16F This study pCL12 pRK415::mexYR166C This study pCL13 pRK415::mexYR184H This study pCL14 pRK415::mexYG216D This study pCL15 pRK415::mexYV339M This study pCL16 pRK415::mexYP562S This study pCL17 pRK415::mexYA960T This study pCL18 pRK415::mexYK79A This study pCL19 pRK415::mexYD133A This study pCL20 pRK415::mexYD133S This study pCL21 pRK415::mexYY613A This study ↵a Tcr, tetracycline resistance; WT, wild type.
- TABLE 3
Oligonucleotides used in this study
Primer Oligonucleotide sequence (5′ → 3′)a Source mexYUP-F ATTAAGAGCTCTACCGCCAGGCTG This study mexYUP-R GCTGCATCTAGATCGTAGCGTTCTC This study mexYDN-F AGTCGATCTAGATGCCCCTAGCGAAAC This study mexYDN-R GCAGACAAGCTTCTGGCCGACTATC This study mexYscreen-F TGTTCCGCAATCCGCATC This study mexYscreen-R GCGTAGCCGATCATCTGTC This study mexY-3138-F GGCTCGAAGCTTATGGCTCGTTTCTTC This study mexY-3138-R ATCTAGGATCCTCAGGCTTGCTCCGTG This study mexY-58-F TTATACAAGCTTCGACGCCCCCTCACCGCT This study mexY-58-R TGGCGGTCATTTGGTTGACC This study mexYseq-F1 GCCTGAAGATCGTCGAGTC This study mexYseq-F2 CAAGCTGACCTCGATGAACCT This study mexYseq-F3 CGATCAACGTGCTGACCATGT This study mexYseq-R4 CAGTCCCTGCATCAATTGCT This study mexYseq-R5 ATCTCGTCCATGCTCACGC This study mexY-K79A-F GGCCTGCTCTACACCGCGGCCACCAGCAGCAC This study mexY-K79A-R GTGCTGCTGGTGGCCGCGGTGTAGAGCAGGCC This study mexY-D133A-F TGGAGAAGGCGGCGGCCAGCATCCAGCTGAT This study mexY-D133A-R ATCAGCTGGATGCTGGCCGCCGCCTTCTCCA This study mexY-D133S-F TGGAGAAGGCGGCGTCCAGCATCCAGCTGAT This study mexY-D133S-R ATCAGCTGGATGCTGGACGCCGCCTTCTCCA This study mexY-Y613A-F GGCGGCTTCAGCCTGGCCGGCGACGGCACCAG This study mexY-Y613A-R CTGGTGCCGTCGCCGGCCAGGCTGAAGCCGCC This study ↵a Restriction endonuclease cleavage sites are underlined. Site-directed mutations are bolded and italicized.
