MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition

ABSTRACT The resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system,...

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Autores principales:	Calvin Ho-Fung Lau, Daniel Hughes, Keith Poole
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/5deec9456dac451bbf3c9a741f94382b
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spelling	oai:doaj.org-article:5deec9456dac451bbf3c9a741f94382b2021-11-15T15:45:12ZMexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition10.1128/mBio.01068-142150-7511https://doaj.org/article/5deec9456dac451bbf3c9a741f94382b2014-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01068-14https://doaj.org/toc/2150-7511ABSTRACT The resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system, have not been elucidated. To identify regions/residues of MexY important for aminoglycoside resistance, plasmid-borne mexY was mutagenized and mutations that impaired MexY-promoted aminoglycoside (streptomycin) resistance were identified in a ΔmexY strain of P. aeruginosa. Sixty-one streptomycin-sensitive mexY mutants were recovered; among these, 7 unique mutations that yielded wild-type levels of MexY expression were identified. These mutations compromised resistance to additional aminoglycosides and to other antimicrobials and occurred in both the transmembrane and periplasmic regions of the protein. Mapping of the mutated residues onto a 3-dimensional structure of MexY modeled on Escherichia coli AcrB revealed that these tended to occur in regions implicated in general pump operation (transmembrane domain) and MexY trimer assembly (docking domain) and, thus, did not provide insights into aminoglycoside recognition. A region corresponding to a proximal binding pocket connected to a periplasm-linked cleft, part of a drug export pathway of AcrB, was identified in MexY and proposed to play a role in aminoglycoside recognition. To test this, selected residues (K79, D133, and Y613) within this pocket were mutagenized and the impact on aminoglycoside resistance was assessed. Mutations of D133 and Y613 compromised aminoglycoside resistance, while, surprisingly, the K79 mutation enhanced aminoglycoside resistance, confirming a role for this putative proximal binding pocket in aminoglycoside recognition and export. IMPORTANCE Bacterial RND pumps do not typically accommodate highly hydrophilic agents such as aminoglycosides, and it is unclear how those, such as MexY, which accommodate these unique substrates, do so. The results presented here indicate that aminoglycosides are likely not captured and exported by this RND pump component in a unique manner but rather utilize a previously defined export pathway that involves a proximal drug-binding pocket that is also implicated in the export of nonaminoglycosides. The observation, too, that a mutation in this pocket enhances MexY-mediated aminoglycoside resistance (K79A), an indication that it is not optimally designed to accommodate these agents, lends further support to earlier proposals that antimicrobials are not the intended pump substrates.Calvin Ho-Fung LauDaniel HughesKeith PooleAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 2 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Calvin Ho-Fung Lau Daniel Hughes Keith Poole MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
description	ABSTRACT The resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system, have not been elucidated. To identify regions/residues of MexY important for aminoglycoside resistance, plasmid-borne mexY was mutagenized and mutations that impaired MexY-promoted aminoglycoside (streptomycin) resistance were identified in a ΔmexY strain of P. aeruginosa. Sixty-one streptomycin-sensitive mexY mutants were recovered; among these, 7 unique mutations that yielded wild-type levels of MexY expression were identified. These mutations compromised resistance to additional aminoglycosides and to other antimicrobials and occurred in both the transmembrane and periplasmic regions of the protein. Mapping of the mutated residues onto a 3-dimensional structure of MexY modeled on Escherichia coli AcrB revealed that these tended to occur in regions implicated in general pump operation (transmembrane domain) and MexY trimer assembly (docking domain) and, thus, did not provide insights into aminoglycoside recognition. A region corresponding to a proximal binding pocket connected to a periplasm-linked cleft, part of a drug export pathway of AcrB, was identified in MexY and proposed to play a role in aminoglycoside recognition. To test this, selected residues (K79, D133, and Y613) within this pocket were mutagenized and the impact on aminoglycoside resistance was assessed. Mutations of D133 and Y613 compromised aminoglycoside resistance, while, surprisingly, the K79 mutation enhanced aminoglycoside resistance, confirming a role for this putative proximal binding pocket in aminoglycoside recognition and export. IMPORTANCE Bacterial RND pumps do not typically accommodate highly hydrophilic agents such as aminoglycosides, and it is unclear how those, such as MexY, which accommodate these unique substrates, do so. The results presented here indicate that aminoglycosides are likely not captured and exported by this RND pump component in a unique manner but rather utilize a previously defined export pathway that involves a proximal drug-binding pocket that is also implicated in the export of nonaminoglycosides. The observation, too, that a mutation in this pocket enhances MexY-mediated aminoglycoside resistance (K79A), an indication that it is not optimally designed to accommodate these agents, lends further support to earlier proposals that antimicrobials are not the intended pump substrates.
format	article
author	Calvin Ho-Fung Lau Daniel Hughes Keith Poole
author_facet	Calvin Ho-Fung Lau Daniel Hughes Keith Poole
author_sort	Calvin Ho-Fung Lau
title	MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
title_short	MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
title_full	MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
title_fullStr	MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
title_full_unstemmed	MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition
title_sort	mexy-promoted aminoglycoside resistance in <named-content content-type="genus-species">pseudomonas aeruginosa</named-content>: involvement of a putative proximal binding pocket in aminoglycoside recognition
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/5deec9456dac451bbf3c9a741f94382b
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MexY-Promoted Aminoglycoside Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition

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