Hiroshi Yoneyama

Assignment of the Substrate-Selective Subunits of the MexEF-OprN Multidrug Efflux Pump of Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa expresses a low level of the MexAB-OprM efflux pump and shows natural resistance to many structurally and functionally diverse antibiotics. The mutation that has been referred to previously as nfxC expresses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluoroquinolones, imipenem, and chloramphenicol and hypersusceptibility to β-lactam antibiotics. To address the antibiotic specificity of the MexEF-OprN efflux pump, we introduced a plasmid carrying themexEF-oprN operon into P. aeruginosa lacking the mexAB-oprM operon. The transformants exhibited resistance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show β-lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEF-OprN pump subunit(s) responsible for substrate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacking MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/ΔMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to β-lactam antibiotics compared with the host strain. The MexAB-OprN/ΔOprM, MexAF-OprM/ΔMexB, and MexEB-OprM/ΔMexA mutants exhibited antibiotic susceptibility indistinguishable from that in the mutant lacking both types of efflux pumps. The results imply that the MexEF-OprM pump selects substrates by a MexEF functional unit. Interestingly, OprN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.

Use of fluorescence probes to monitor function of the subunit proteins of the MexA-MexB-oprM drug extrusion machinery in Pseudomonas aeruginosa.

The MexA-MexB-OprM efflux pump ofPseudomonas aeruginosa consists of two inner membrane proteins, MexA and MexB, and one outer membrane protein, OprM. We investigated the role of the components of this drug extrusion system by evaluating the repercussions of deleting these subunit components on the accumulation of several fluorescent probes. Fluorescence intensities of positively charged 2-(4-dimethylaminostyryl)-1ethylpyridinium and unchargedN-phenyl-1-naphtylamine were 7 and 4 times higher, respectively, in the mutant lacking OprM and 4 and 1.7 times higher, respectively, in the mutants lacking MexA or MexB than in the wild type strain. This order of fluorescence intensity was fully consistent with a previously reported minimum inhibitory concentration of antibiotics such as tetracycline, chloramphenicol, and fluoroquinolones. Ethidium bromide accumulation in all the Mex mutants proceeded at about 5 times faster than the rate in the wild type cells. This result is in accord with the minimum inhibitory concentration of β-lactam antibiotics. These results suggest that the fluorescence probes could be successfully used in real time monitoring of the function of the drug extrusion machinery in Gram-negative bacteria. The downhill extrusion kinetics of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, which orients perpendicular to the inner leaflet of the cytoplasmic membrane, from preloaded cells lacking the extrusion pump was preceded by a slow increase in fluorescence intensity, whereas the wild type cell immediately released the dye. This observation was explained by a slow trans-cytoplasmic membrane crossing of intracellular dye in the mutants. These results reflected higher accumulation of the probe in the cytoplasmic membrane in the mutants and strengthened the hypothesis that extrusion of hydrophobic substrate mediated by MexA-MexB-OprM mainly takes place from the interior of the cytoplasmic membrane.

Membrane Topology of the Xenobiotic-exporting Subunit, MexB, of the MexA,B-OprM Extrusion Pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly ofPseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.

Localization of the Outer Membrane Subunit OprM of Resistance-Nodulation-Cell Division Family Multicomponent Efflux Pump in Pseudomonas aeruginosa

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa. [3H]Palmitate uptake experiments revealed that OprM is a lipoprotein. The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space. (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.

Multiantibiotic resistance caused by active drug extrusion in hospital pathogens

All living organisms from bacteria to mammals extrude noxious compounds to the external medium. When exposed to antibiotics, bacteria actively extrude intracellular antibiotic and develop resistance to the drug. NosocomialStaphylococcus aureaus, Pseudomonas aeruginosa and other bacteria are resistant to a broad range of antibiotics and to structurally and functionally diverse chemotherapeutic agents and disinfectants. For this reason nosocomial infections are especially hard to treat in immunocompromised patients who may be infected by low-virulence bacteria.Extrusion-related antibiotic resistance inP. aeruginosa arises by the expression of Mex-extrusion pumps, including genetically distinctmexA-mexB-oprM, mexC-mexD-oprj, andmexE-mexF-oprN systems, each encoding two inner membrane proteins and one outer membrane protein.S. aureus becomes resistant to fluoroquinolone by expressing NorA extrusion proteins and to disinfectants by expressing Qac extrusion proteins. The drug extrusion machinery may be classified into several categories according to the number of transmembrane segments it exhibits. The proteins that belong to a major facilitator super family have 12 or 14 transmembrane segments. The extrusion proteins with molecular weight of 12,000 to 15,000 span the membrane 4 times and are collectively called small multidrug resistance proteins. The extrusion proteins that transport substrate across the inner and outer membrane of gram-negative bacteria are in the resistance nodulation cell division family.

The outer membrane of Pseudomonasaeruginosa is a barrier against the penetration of disaccharides

The outer membrane of Pseudomonas aeruginosa acted as a barrier against the penetration of di- (Mr, 342), tri- (Mr, 504) and tetrasaccharides (Mr, 666), whereas the membrane allowed the penetration of pentose (Mr, 150) and methylhexoses (Mr, 194) into the periplasm. When the intact cells of P. aeruginosa were treated with 600 mosM saccharides of various sizes and observed under an electron microscope, saccharides of Mr larger than 342 caused the extensive shrinking of the outer membrane. Whereas the cells treated with the saccharides of Mr less than 194 or with sucrose in the presence of EDTA showed plasmolysis. Determination of the extent of saccharide penetration into the periplasm of the cells treated with 600 mosM sodium chloride or with 600 mosM saccharides of various sizes showed that only pentose and hexoses, so far examined, were penetrable but di-, tri- and tetrasaccharides were impenetrable.

Nucleotide sequence of the protein D2 gene of Pseudomonas aeruginosa.

Protein D2 of the outer membrane of Pseudomonas aeruginosa was shown to form the imipenem-permeable pore. We cloned and sequenced the protein D2 gene. The protein D2 gene encodes a polypeptide with 443 amino acids consisting of 23 and 420 amino acid residues for the signal peptide and mature polypeptide (M(r), 46,010), respectively. Protein D2 contains the highest molar ratio of glycine and no cysteine. The polar amino acids are scattered throughout the sequence. Images

Cloning of the protein D2 gene of Pseudomonas aeruginosa and its functional expression in the imipenem-resistant host

Protein D2 forms the water‐filled pore across the outer membrane of Pseudomonas aeruginosa and allows the penetration of imipenem. We cloned the protein D2 gene by the antibody screening technique. When the imipenem‐resistant mutant lacking protein D2 harbored the plasmid with the cloned D2 gene, the mutant overproduced protein D2 in the outer membrane. These transformants exhibited fully‐restored imipenem susceptibility. The results prove unequivocally that protein D2 forms the imipenem‐permeable pore in the P. aeruginosa outer membrane.

Aminoglycoside resistance in Pseudomonas aeruginosa due to outer membrane stabilization

Pseudomonas aeruginosa PAO1 released a significant amount of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, in the presence of aminoglycoside and lysozyme. The extent of the enzyme release was inversely related to the MICs of the aminoglycoside. However, the aminoglycoside-resistant strain F3721, treated in the same way; released a less enzyme. The F3721 LPS was extracted in the phenol phase instead of the water phase in which PAO1 LPS was easily extracted. Electrophoretic analysis of the F3721 LPS showed the ladder bands at the high Mr position, suggesting that the LPS of the aminoglycoside-resistant cells has a structural modification(s) which somehow protects the outer membrane from aminoglycoside-mediated damage.

A simple method for determining the outer membrane permeability of gram-negative bacteria in intact cells

Abstract The permeability of the intact outer membrane of gram-negative bacteria was determined by weighing the centrifuged pellets of cells treated with hypertonic solutes [10]. The present study refined the existing procedures by focusing on the factors that may affect the results. The study revealed the following findinds: ( i ) The weight of cells treated with outer membrane-impermeable hypertonic saccharide was significantly reduced and the reduction was proportional to the solute osmolarity. On the other hand, the weight of cells treated with outer membrane-permeable hypertonic saccharide was unchanges. ( ii ) The centrifugal force up to 165,000 × g for 60 s or NaCl up to 150 mM produced no detectable influence on the assay. ( iii ) It was possible to measure the outer membrane permeability even in the hypotonic test solutes using plasmolyzed cells. ( iv ) This method proved equally applicable for measuring the outer membrane permeabilities of both smooth and rough strains. ( v ) Application of this technique to several species of gram-negative bacteria confirmed the previously reported exclusion limits. All these data show that the weighing of centrifuged pellets of hypertonic solute-treated cells is a useful method for determining the outer membrane permeability of gram-negative bacteria.