title: Homologs of the LapD-LapG c-di-GMP Effector System Control Biofilm Formation by Bordetella bronchiseptica.
abstract: Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica.
authors.affiliation: Instituto de Biotecnología y Biología Molecular (IBBM)-CCT-CONICET-La Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Ambrosis
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.
authors.forename: Chelsea D
authors.initials: CD
authors.lastname: Boyd
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.
authors.forename: George A
authors.initials: GA
authors.lastname: O Toole
authors.affiliation: Instituto de Biotecnología y Biología Molecular (IBBM)-CCT-CONICET-La Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
authors.forename: Julieta
authors.initials: J
authors.lastname: Fernández
authors.affiliation: Instituto de Biotecnología y Biología Molecular (IBBM)-CCT-CONICET-La Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
authors.forename: Federico
authors.initials: F
authors.lastname: Sisti
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development*; D015153:Blotting, Western; D001885:Bordetella Infections / Q000382:microbiology*; D016950:Bordetella bronchiseptica / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D008807:Mice, Inbred BALB C; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D009154:Mutation; D011551:Pseudomonas fluorescens / Q000235:genetics; D012141:Respiratory Tract Infections / Q000382:microbiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP