title: Systematic identification of genetic loci required for polymyxin resistance in Campylobacter jejuni using an efficient in vivo transposon mutagenesis system.
abstract: The aim of this study was to identify genetic loci required for polymyxin (PM) resistance in Campylobacter jejuni using an efficient in vivo random mutagenesis system. PM has been widely used as a model peptide to examine mechanisms of bacterial resistance to antimicrobial peptides (AMPs), the major effectors of host innate immunity and also candidates for a new generation of antibiotics. In this study, a commercially available transposon mutagenesis approach (EZ-Tn5 <KAN-2> Transposome; Epicentre, Madison, WI) was evaluated and used to systematically identify Campylobacter mutants with increased susceptibility to PM. This simple, yet efficient, transposon mutagenesis approach identified 12 mutants representing seven different genes of C. jejuni 81-176 involved in acquired PM resistance. Backcrossing of the transposon mutations into the parent strain confirmed that the PM-sensitive phenotype in each mutant was linked to the gene with a specific transposon insertion. The genes are identified as being involved in the synthesis of cell-surface carbohydrates, modification of intracellular targets, signal transduction, and modulation of transmembrane potential. The mutant with the highest susceptibility to PM contains a transposon insertion in a putative galU gene that is essential for production of uridine diphosphate glucose (UDP)-glucose, a precursor required for lipooligosaccharide (LOS) synthesis. LOS analysis by tricine SDSPAGE showed significant truncation of the LOS core structure in the galU mutant. Susceptibility assays also indicated that GalU contributed C. jejuni resistance to some natural AMPs. Complementation of the galU mutant in trans fully restored LOS synthesis and resistance to the levels of the parent strain. Together, these results define seven C. jejuni genetic loci that will be useful for characterizing the molecular basis of Campylobacter resistance to PM and natural AMPs, and also highlight the usefulness of the in vivo mutagenesis approach for systematic characterization of functionally important Campylobacter genes.
authors.affiliation: Department of Animal Science, The University of Tennessee, Knoxville, 37996-4574, USA. jlin6@utk.edu
authors.forename: Jun
authors.initials: J
authors.lastname: Lin
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Ky Van
authors.initials: KV
authors.lastname: Hoang
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002169:Campylobacter Infections / Q000382:microbiology*; D016123:Campylobacter jejuni / Q000187:drug effects* / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011113:Polymyxins / Q000494:pharmacology*; D015398:Signal Transduction
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011113:Polymyxins