Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>

ABSTRACT Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is abse...

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Autores principales:	Andrei Rajkovic, Sarah Erickson, Anne Witzky, Owen E. Branson, Jin Seo, Philip R. Gafken, Michael A. Frietas, Julian P. Whitelegge, Kym F. Faull, William Navarre, Andrew J. Darwin, Michael Ibba
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d1cabc8d922f4855984231bd81128d84
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spelling	oai:doaj.org-article:d1cabc8d922f4855984231bd81128d842021-11-15T15:49:02ZCyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>10.1128/mBio.00823-152150-7511https://doaj.org/article/d1cabc8d922f4855984231bd81128d842015-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00823-15https://doaj.org/toc/2150-7511ABSTRACT Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival. IMPORTANCE Infections with pathogenic Salmonella, E. coli, and Pseudomonas isolates can all lead to infectious disease with potentially fatal sequelae. EF-P proteins contribute to the pathogenicity of the causative agents of these and other diseases by controlling the translation of proteins critical for modulating antibiotic resistance, motility, and other traits that play key roles in establishing virulence. In Salmonella spp. and E. coli, the attachment of β-Lys is required for EF-P activity, but the proteins required for this posttranslational modification pathway are absent from many organisms. Instead, bacteria such as P. aeruginosa activate EF-P by posttranslational modification with rhamnose, revealing a new role for protein glycosylation that may also prove useful as a target for the development of novel antibiotics.Andrei RajkovicSarah EricksonAnne WitzkyOwen E. BransonJin SeoPhilip R. GafkenMichael A. FrietasJulian P. WhiteleggeKym F. FaullWilliam NavarreAndrew J. DarwinMichael IbbaAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 3 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Andrei Rajkovic Sarah Erickson Anne Witzky Owen E. Branson Jin Seo Philip R. Gafken Michael A. Frietas Julian P. Whitelegge Kym F. Faull William Navarre Andrew J. Darwin Michael Ibba Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>
description	ABSTRACT Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival. IMPORTANCE Infections with pathogenic Salmonella, E. coli, and Pseudomonas isolates can all lead to infectious disease with potentially fatal sequelae. EF-P proteins contribute to the pathogenicity of the causative agents of these and other diseases by controlling the translation of proteins critical for modulating antibiotic resistance, motility, and other traits that play key roles in establishing virulence. In Salmonella spp. and E. coli, the attachment of β-Lys is required for EF-P activity, but the proteins required for this posttranslational modification pathway are absent from many organisms. Instead, bacteria such as P. aeruginosa activate EF-P by posttranslational modification with rhamnose, revealing a new role for protein glycosylation that may also prove useful as a target for the development of novel antibiotics.
format	article
author	Andrei Rajkovic Sarah Erickson Anne Witzky Owen E. Branson Jin Seo Philip R. Gafken Michael A. Frietas Julian P. Whitelegge Kym F. Faull William Navarre Andrew J. Darwin Michael Ibba
author_facet	Andrei Rajkovic Sarah Erickson Anne Witzky Owen E. Branson Jin Seo Philip R. Gafken Michael A. Frietas Julian P. Whitelegge Kym F. Faull William Navarre Andrew J. Darwin Michael Ibba
author_sort	Andrei Rajkovic
title	Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>
title_short	Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>
title_full	Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>
title_fullStr	Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>
title_full_unstemmed	Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>
title_sort	cyclic rhamnosylated elongation factor p establishes antibiotic resistance in <named-content content-type="genus-species">pseudomonas aeruginosa</named-content>
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/d1cabc8d922f4855984231bd81128d84
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Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>

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