title: Genetic mechanisms involved in the repression of flagellar assembly by Pseudomonas aeruginosa in human mucus.
abstract: Pseudomonas aeruginosa downregulates flagellin transcription when it is grown in purulent mucus from patients with cystic fibrosis (CF) and non-CF bronchiectasis. This response possibly abrogates the potent inflammatory response mediated by the interaction of flagellin with Toll-like receptor 5. The molecular mechanisms involved are thus far unknown. Known flagellar transcriptional regulators were not involved, thus Tn5 mutagenesis was used to ascertain whether novel regulators existed. Five clones with independent Tn5 insertions in flgM showed derepression of flagellin synthesis, suggesting that FlgM was involved in this phenomenon. Furthermore, examination of mucus-grown bacteria showed FlgM accumulation and overexpression of fliA in mucus-grown bacteria reversed the repression of flagellin synthesis. A related study from our laboratory had identified neutrophil elastase in mucus as the molecule responsible for fliC repression, therefore we examined whether loss of the flagellar hook (FlgE), by proteolysis was involved, because the flagellar hook is required for FlgM export. Western immunoblot of membranes from mucus-grown bacteria showed the absence of FlgE, despite the fact that the protein is made and the operon encoding FlgE is upregulated in mucus. A model is proposed wherein neutrophil elastase in mucus proteolytically cleaves the flagellar hook, thus completion of the hook basal body is never sensed, resulting in FlgM accumulation within the cell, causing repression of flagellin synthesis. We speculate that the cyclical bouts of inflammation observed in CF patients may result from flagellin synthesis and its repression, caused by presence of neutrophils at the site of infection.
authors.affiliation: Department of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Jeevan
authors.initials: J
authors.lastname: Jyot
authors.forename: Avinash
authors.initials: A
authors.lastname: Sonawane
authors.forename: Weihui
authors.initials: W
authors.lastname: Wu
authors.forename: Reuben
authors.initials: R
authors.lastname: Ramphal
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D005407:Flagella / Q000235:genetics*; D005408:Flagellin / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism; D009093:Mucus / Q000382:microbiology*; D009154:Mutation; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000166:cytology* / Q000235:genetics*; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C097681:FlgE protein, Bacteria; C064971:FliA protein, Bacteria; D012808:Sigma Factor; C473503:Tn5 transposase; D005408:Flagellin; C071266:FlgM protein, Bacteria; D019895:Transposases; D019272:Leukocyte Elastase