title: AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle.
abstract: The facultative human pathogen Vibrio cholerae has to adapt to different environmental conditions along its lifecycle by means of transcriptional, translational and post-translational regulation. This study provides a first comprehensive analysis regarding the contribution of the cytoplasmic AAA+ proteases Lon, ClpP and HslV to distinct features of V. cholerae behaviour, including biofilm formation, motility, cholera toxin expression and colonization fitness in the mouse model. While absence of HslV did not yield to any altered phenotype compared to wildtype, absence of Lon or ClpP resulted in significantly reduced colonization in vivo. In addition, a Δlon deletion mutant showed altered biofilm formation and increased motility, which could be correlated with higher expression of V. cholerae flagella gene class IV. Concordantly, we could show by immunoblot analysis, that Lon is the main protease responsible for proteolytic control of FliA, which is required for class IV flagella gene transcription, but also downregulates virulence gene expression. FliA becomes highly sensitive to proteolytic degradation in absence of its anti-sigma factor FlgM, a scenario reported to occur during mucosal penetration due to FlgM secretion through the broken flagellum. Our results confirm that the high stability of FliA in the absence of Lon results in less cholera toxin and toxin corgulated pilus production under virulence gene inducing conditions and in the presence of a damaged flagellum. Thus, the data presented herein provide a molecular explanation on how V. cholerae can achieve full expression of virulence genes during early stages of colonization, despite FliA getting liberated from the anti-sigma factor FlgM.
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Pressler
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Dina
authors.initials: D
authors.lastname: Vorkapic
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Lichtenegger
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Gerald
authors.initials: G
authors.lastname: Malli
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Barilich
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Fatih
authors.initials: F
authors.lastname: Cakar
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Franz G
authors.initials: FG
authors.lastname: Zingl
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria.
authors.forename: Joachim
authors.initials: J
authors.lastname: Reidl
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria. Electronic address: stefan.schild@uni-graz.at.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schild
mesh_terms: D000818:Animals; D018441:Biofilms / Q000254:growth & development; D002772:Cholera Toxin / Q000378:metabolism; D049071:Endopeptidase Clp / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000382:microbiology; D008124:Locomotion; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism*; D060066:Protein Interaction Maps; D014734:Vibrio cholerae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*
chemical_list: D002772:Cholera Toxin; D010447:Peptide Hydrolases; D049071:Endopeptidase Clp
keywords: Biofilm; Cholera; Flagella; Motility; Mucosal penetration; Virulence