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

Abstract

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.

Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii

“…The number of CFU was then determined in each case. The persister fraction is defined as the number of surviving cells after treatment with ofloxacin divided by the number of cells after control treatment (18). Our results show that light effectively increases the persister fraction of this strain by about 80% compared to values obtained for cultures kept in the dark (Fig.…”

“…Journal of Bacteriology dnpA gene) is involved in noninherited fluoroquinolone tolerance in Pseudomonas aeruginosa (18). This phenomenon refers to the presence of antibiotic-insensitive persister cells, i.e., phenotypic variants of the wild type that are transiently capable of surviving prolonged periods of antibiotic treatment, without having developed or acquired resistance mechanisms to the drug (18).…”

“…This phenomenon refers to the presence of antibiotic-insensitive persister cells, i.e., phenotypic variants of the wild type that are transiently capable of surviving prolonged periods of antibiotic treatment, without having developed or acquired resistance mechanisms to the drug (18). De-N-acetylases of the PIG-L superfamily typically display low overall sequence identity (ϳ25%) but share a catalytic site containing a number of conserved residues that are essential for enzymatic activity (18).…”

“…The sequence of the putative enzyme present in A. baumannii ATCC 19606, encoded by DJ41_1943 and shown in this work to be induced by light (Fig. 7A), contains those conserved residues in the active site (18). We next analyzed whether tolerance to fluoroquinolones was affected by light by comparing the persister fraction of A. baumannii wild-type cells used in this study under blue light or in the dark.…”

Pseudolysogeny and sequential mutations build multiresistance to virulent bacteriophages in Pseudomonas aeruginosa

“…The wzy, wzz2 and wbpL genes are members of the heteropolymeric O-specific antigen biosynthesis cluster in PAO1 (Lam et al, 2011). Gene migA encodes a rhamnosyltransferase involved in LPS core capping (Poon et al, 2008), whereas wapH and dnpA are known to be involved in the synthesis of LPS (Hansen et al, 2007;Liebens et al, 2014), and pgi encodes a glycosyltransferase (Rocchetta et al, 1999). Mutations in algC affect the biosynthesis of alginate, LPS and rhamnolipids, biosurfactants necessary for bacterial swarming motility and biofilm formation (Olvera et al, 1999).…”