title: Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank.
abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska Lincoln, NE, USA.
authors.forename: Govardhan
authors.initials: G
authors.lastname: Rathnaiah
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St. Paul MN, USA.
authors.forename: Elise A
authors.initials: EA
authors.lastname: Lamont
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska Lincoln, NE, USA.
authors.forename: N Beth
authors.initials: NB
authors.lastname: Harris
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska Lincoln, NE, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fenton
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska Lincoln, NE, USA.
authors.forename: Denise K
authors.initials: DK
authors.lastname: Zinniel
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska Lincoln, NE, USA.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Liu
authors.affiliation: School of Veterinary Medicine, University of Wisconsin Madison, WI, USA.
authors.forename: Josh
authors.initials: J
authors.lastname: Sotos
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska Lincoln, NE, USA.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Feng
authors.affiliation: The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem Rehovot, Israel.
authors.forename: Ayala
authors.initials: A
authors.lastname: Livneh-Kol
authors.affiliation: The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem Rehovot, Israel.
authors.forename: Nahum Y
authors.initials: NY
authors.lastname: Shpigel
authors.affiliation: School of Veterinary Medicine, University of Wisconsin Madison, WI, USA.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Czuprynski
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St. Paul MN, USA.
authors.forename: Srinand
authors.initials: S
authors.lastname: Sreevatsan
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska Lincoln, NE, USA.
authors.forename: Raúl G
authors.initials: RG
authors.lastname: Barletta
mesh_terms: D000818:Animals; D018070:Biological Specimen Banks; D002417:Cattle; D003523:Cycloserine / Q000494:pharmacology; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000276:immunology; D016254:Mutagenesis, Insertional; D009154:Mutation; D016927:Mycobacterium avium subsp. paratuberculosis / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D010283:Paratuberculosis / Q000276:immunology / Q000382:microbiology*; D010641:Phenotype
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D003523:Cycloserine
keywords: Johne's Disease; Mycobacterium paratuberculosis; bovine macrophages; mutant bank; transposon