title: Membrane localization and topology of the DnpA protein control fluoroquinolone tolerance in Pseudomonas aeruginosa.
abstract: DnpA, a putative de-N-acetylase of the PIG-L superfamily, is required for antibiotic tolerance in Pseudomonas aeruginosa Exactly how dnpA (gene locus PA5002) directs the formation of antibiotic-tolerant persister cells is currently unknown. Previous research provided evidence for a role in surface-associated process(es), possibly in lipopolysaccharide biosynthesis. In silico sequence analysis of DnpA predicts a single transmembrane domain and Nin/Cout orientation of DnpA. In contrast, we here show that DnpA is an integral inner membrane protein containing two transmembrane domains, with the major C-terminal part located at the cytoplasmic face. Correct insertion into the inner membrane is necessary for DnpA to promote fluoroquinolone tolerance. The membrane localization of DnpA further supports its role in cell envelope-associated process(es). In addition to shedding light on the biological role of DnpA, this study highlights the risks of overreliance on the predictive value of bioinformatics tools and the importance of rigorous experimental validation of in silico predictions.
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, 3001 Leuven, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Liebens
authors.affiliation: Department of Sciences, Roma Tre University, Rome 00146, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Frangipani
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, 3001 Leuven, Belgium.
authors.forename: Annelies
authors.initials: A
authors.lastname: Van der Leyden
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, 3001 Leuven, Belgium imec, Smart Systems and Emerging Technologies Unit, Department of Life Science Technologies, 3001 Leuven, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Fauvart
authors.affiliation: Department of Sciences, Roma Tre University, Rome 00146, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Visca
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, 3001 Leuven, Belgium jan.michiels@kuleuven.be.
authors.forename: Jan
authors.initials: J
authors.lastname: Michiels
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003198:Computer Simulation; D024841:Fluoroquinolones / Q000378:metabolism / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D024841:Fluoroquinolones; D008070:Lipopolysaccharides; D008565:Membrane Proteins
keywords: LPS; antibiotic tolerance; cytoplasmic membrane; de-N-acetylase; persistence