title: Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model.
abstract: Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFβ. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.
authors.affiliation: 1​ Department of Microbiology, College of Science, Oregon State University, Corvallis, OR, USA.|2​ Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA.
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Everman
authors.affiliation: 3​ Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.
authors.forename: Torsten M
authors.initials: TM
authors.lastname: Eckstein
authors.affiliation: 4​ Comparative Medicine and Integrative Biology Program, Michigan State University, East Lansing, MI, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Roussey
authors.affiliation: 4​ Comparative Medicine and Integrative Biology Program, Michigan State University, East Lansing, MI, USA.|5​ Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Coussens
authors.affiliation: 6​ National Animal Disease Center, USDA Agricultural Research Service, Ames, IA, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Bannantine
authors.affiliation: 2​ Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA.|1​ Department of Microbiology, College of Science, Oregon State University, Corvallis, OR, USA.
authors.forename: Luiz E
authors.initials: LE
authors.lastname: Bermudez
mesh_terms: D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D004705:Endocytosis; D004847:Epithelial Cells / Q000276:immunology / Q000382:microbiology*; D020869:Gene Expression Profiling; D007082:Ileum / Q000473:pathology; D008055:Lipids / Q000032:analysis; D008264:Macrophages / Q000276:immunology / Q000382:microbiology*; D008954:Models, Biological; D016927:Mycobacterium avium subsp. paratuberculosis / Q000276:immunology*; D010283:Paratuberculosis / Q000473:pathology*
chemical_list: D016207:Cytokines; D008055:Lipids