title: Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication.
abstract: Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella, indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host-pathogen interface to influence the intracellular fate of this pathogen.
authors.affiliation: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Audrey
authors.initials: A
authors.lastname: Chong
authors.forename: Robert
authors.initials: R
authors.lastname: Child
authors.forename: Tara D
authors.initials: TD
authors.lastname: Wehrly
authors.forename: Dedeke
authors.initials: D
authors.lastname: Rockx-Brouwer
authors.forename: Aiping
authors.initials: A
authors.lastname: Qin
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Mann
authors.forename: Jean
authors.initials: J
authors.lastname: Celli
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005604:Francisella tularensis / Q000254:growth & development*; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D013329:Structure-Activity Relationship; D014406:Tularemia / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D037521:Virulence Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C083436:FopA protein, Francisella tularensis; D037521:Virulence Factors