Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.
The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound-ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.
Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori, Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan. kzst129os@yahoo.co.jp.
Kazuki
K
Sato
Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori, Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan. kenichi.tonpei@gmail.com.
Kenichi
K
Ushioda
Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori, Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan. pyanda@sea.plala.or.jp.
Keiji
K
Akiba
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. yoshimi@sanken.osaka-u.ac.jp.
Yoshimi
Y
Matsumoto
Department of Biological Science and Technology, Faculty of Engineering, 2-1 Minamijyousanjima-cho, Tokushima 770-8605, Japan. maseda@bio.tokushima-u.ac.jp.
Hideaki
H
Maseda
Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori, Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan. tasuke@bios.tohoku.ac.jp.
Tasuke
T
Ando
Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori, Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan. emiko@bios.tohoku.ac.jp.
Emiko
E
Isogai
Laboratory for Antimicrobial Agents, Kitasato University, 1-15-1 Kitasato, Sagamihara 228-8555, Japan. nakae-tj@insti.kitasato-u.ac.jp.
Taiji
T
Nakae
Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori, Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan. yoneyama@bios.tohoku.ac.jp.
Hiroshi
H
Yoneyama
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001398:Aztreonam / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001692:Biological Transport / Q000235:genetics; D002701:Chloramphenicol / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects; D005839:Gentamicins / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D007502:Iron Chelating Agents / Q000494:pharmacology*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009842:Oligopeptides / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D005839:Gentamicins; D007502:Iron Chelating Agents; D026901:Membrane Transport Proteins; D009842:Oligopeptides; C402263:OprM protein, Pseudomonas aeruginosa; D002701:Chloramphenicol; C042453:pyoverdin; D007501:Iron; D001398:Aztreonam