Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of
            <i>Pseudomonas aeruginosa</i>

Aires, Julio; Pechère, J.-C.; Delden, Christian van; Köhler, Thilo

doi:10.1128/aac.46.7.2169-2173.2002

Cited by 25 publications

(13 citation statements)

References 31 publications

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“…8). Although charged residues within the TM helices are known to be crucial in drug efflux (1,10), these results suggest that such residues outside the TM helices may also play significant roles in the efflux process, presumably in ligand-binding step(s). Et accumulation assay.…”

Section: Vol 187 2005mentioning

confidence: 80%

A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study

et al. 2005

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The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-␤-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specificity. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.Multidrug transporters cause serious problems in the chemotherapy of cancer as well as in the antibiotic treatment of bacterial infections. These membrane proteins recognize many structurally dissimilar toxic compounds and actively extrude them from cells. The Escherichia coli AcrB transporter (17, 18), which is a member of the resistance-nodulation-division (RND) family transporters (42) and is responsible for most of the intrinsic drug resistance of this organism (28,29,37), is one of the best-studied multidrug pumps. It occurs as a multiprotein complex (40,41,50) with the outer membrane channel TolC (9, 15) and a periplasmic linker protein, AcrA (17) (the structure of an AcrA homolog, MexA, was solved recently [2, 11]), and this complex structure allows the direct export of drugs to the external medium (28). The structural work of Murakami et al. (23) revealed that the unliganded AcrB is a homotrimer, where each subunit contains 12 transmembrane (TM) helices and two large periplasmic domains (each exceeding 300 residues) between TM helices TM1 and TM2 and between TM7 and TM8. The periplasmic domains of AcrB form a funnel-like structure at the top which is thought (23) to associate with the end of the periplasmic tunnel of TolC (15) and a connected "pore" at the center (Fig. 1A). The pore leads down to the large central cavity, with a diameter of 35 Å, formed by the TM domains of the three protomers. The central cavity is connected also to the periplasm through three vestibules located at subunit interfaces (...

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Section: Vol 187 2005mentioning

confidence: 80%

A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study

et al. 2005

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“…Eleven mutations affected the transmembrane domains 1, 3, 4, 5, 9, 11, and 12, 19 mutations were assigned to the cytoplasmic space, and 37 mutations were located in the periplasm, of which 13 and 20 amino acid variants were located in the large periplasmic loops L1/2 and L7/8, respectively (55). None of the functionally characterized mutations were present in our strain panel (54,56,57).…”

Section: Resultsmentioning

confidence: 99%

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

Greipel

Fischer

Klockgether

et al. 2016

Antimicrob Agents Chemother

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bThe chronic airway infections with Pseudomonas aeruginosa in people with cystic fibrosis (CF) are treated with aerosolized antibiotics, oral fluoroquinolones, and/or intravenous combination therapy with aminoglycosides and ␤-lactam antibiotics. An international strain collection of 361 P. aeruginosa isolates from 258 CF patients seen at 30 CF clinics was examined for mutations in 17 antimicrobial susceptibility and resistance loci that had been identified as hot spots of mutation by genome sequencing of serial isolates from a single CF clinic. Combinatorial amplicon sequencing of pooled PCR products identified 1,112 sequence variants that were not present in the genomes of representative strains of the 20 most common clones of the global P. aeruginosa population. A high frequency of singular coding variants was seen in spuE, mexA, gyrA, rpoB, fusA1, mexZ, mexY, oprD, ampD, parR, parS, and envZ (amgS), reflecting the pressure upon P. aeruginosa in lungs of CF patients to generate novel protein variants. The proportion of nonneutral amino acid exchanges was high. Of the 17 loci, mexA, mexZ, and pagL were most frequently affected by independent stop mutations. Private and de novo mutations seem to play a pivotal role in the response of P. aeruginosa populations to the antimicrobial load and the individual CF host. C ystic fibrosis (CF) is a severe autosomal recessive trait that is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) (1). CFTR dysfunction affects many organs, but lung disease is responsible for the vast majority of morbidity and mortality in people with CF. The basic defect in CF predisposes to viral and bacterial airway infections (2). The most prevalent airway pathogen in adolescents and adults with CF is Pseudomonas aeruginosa (3, 4). The chronic airway colonization with P. aeruginosa has been associated with a rapid decline in lung function, heightened lung inflammation, and worse prognosis (1).Antipseudomonal chemotherapy in CF comprises intravenous combination therapy with ␤-lactam antibiotics and aminoglycosides, oral fluoroquinolones, immunomodulatory treatment with azithromycin and aerosolized antibiotics such as tobramycin, aztreonam, or colistin (4-8). The high antimicrobial pressure exerted on the microorganism, which can persist and diversify in the patient's airways for decades, drives the emergence of drug resistance phenotypes (9, 10). Typical targets are elements of replication, transcription, and translation, the cell wall, and transport and its regulation (9-11).During ongoing studies on the long-term microevolution of P. aeruginosa in lungs of 12 CF patients, we have identified 17 loci to be repetitively hit by mutations. These loci are known from the literature to be associated with the emergence of drug resistance in P. aeruginosa (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Hence, we became interested to know if and to what extent the global P. aeruginosa population in lungs of patients (CF lungs) carries mutations in these ge...

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“…Di¡erent motifs in transmembrane segment IV of RND proteins probably determine substrate entry a Binding motifs were derived from Goldberg et al[57], Guan and Nakae[79] and Aires et al[80].…”

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confidence: 99%

Escherichia colimechanisms of copper homeostasis in a changing environment

2003

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Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions. The Cu(I)-translocating P-type ATPase CopA, the central component in copper homeostasis, is responsible for removing excess Cu(I) from the cytoplasm. The multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appear to safeguard the periplasmic space from copper-induced toxicity. Some strains of E. coli can survive in copper-rich environments that would normally overwhelm the chromosomally encoded copper homeostatic systems. Such strains possess additional plasmid-encoded genes that confer copper resistance. The pco determinant encodes genes that detoxify copper in the periplasm, although the mechanism is still unknown. Genes involved in copper homeostasis are regulated by MerR-like activators responsive to cytoplasmic Cu(I) or two-component systems sensing periplasmic Cu(I). Pathways of copper uptake and intracellular copper handling are still not identified in E. coli.

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Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa

Cited by 25 publications

References 31 publications

A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study

A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study

Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients

Escherichia colimechanisms of copper homeostasis in a changing environment

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