title: Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in Pseudomonas aeruginosa.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Andrei
authors.initials: A
authors.lastname: Rajkovic
authors.affiliation: Department of Chemistry, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Erickson
authors.affiliation: Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Witzky
authors.affiliation: Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Owen E
authors.initials: OE
authors.lastname: Branson
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, New York, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Seo
authors.affiliation: Fred Hutchinson Cancer Research Center, Proteomics Facility, Seattle, Washington, USA.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Gafken
authors.forename: Michael A
authors.initials: MA
authors.lastname: Frietas
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, Pasarow Mass Spectrometry Laboratory, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, Pasarow Mass Spectrometry Laboratory, Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Kym F
authors.initials: KF
authors.lastname: Faull
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: William
authors.initials: W
authors.lastname: Navarre
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, New York, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Darwin
authors.affiliation: Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA ibba.1@osu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Ibba
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D017353:Gene Deletion; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000378:metabolism*; D012210:Rhamnose / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D010445:Peptide Elongation Factors; C020746:factor EF-P; D006602:Hexosyltransferases; D012210:Rhamnose