Membrane localization and topology of the DnpA protein control fluoroquinolone tolerance in Pseudomonas aeruginosa

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.status: publishe

Membrane localization and topology of the DnpA protein control fluoroquinolone tolerance in Pseudomonas aeruginosa

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

A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance in Pseudomonas aeruginosa

A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P.aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P.aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P.aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P.aeruginosa infections