title: A Functional Core of IncA Is Required for Chlamydia trachomatis Inclusion Fusion.
abstract: Chlamydia trachomatis is an obligate intracellular pathogen that is the etiological agent of a variety of human diseases, including blinding trachoma and sexually transmitted infections. Chlamydiae replicate within a membrane-bound compartment, termed an inclusion, which they extensively modify by the insertion of type III secreted proteins called Inc proteins. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of nonfusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively, these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for identification of chlamydia with a method for complementation of site-specific mutants.
  
  
  OBJECTIVE
  Chlamydia trachomatis replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusions are nonfusogenic with vesicles in the endocytic pathway but, in multiply infected cells, fuse with each other to form a single large inclusion. This homotypic fusion is dependent upon the presence of a chlamydial inclusion membrane-localized protein, IncA. Specificity of membrane fusion in eukaryotic cells is regulated by SNARE (soluble N-ethylmaleimide sensitive factor attachment receptor) proteins on the cytosolic face of vesicles and target membranes. IncA contains two SNARE-like domains. Newly developed genetic tools for the complementation of targeted mutants in C. trachomatis are used to confirm the minimal requirement of SNARE-like motifs necessary to promote the homotypic fusion of inclusions.
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Mary M
authors.initials: MM
authors.lastname: Weber
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Noriea
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Laura D
authors.initials: LD
authors.lastname: Bauler
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Lam
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Sager
authors.affiliation: Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Wesolowski
authors.affiliation: Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Paumet
authors.affiliation: Host Parasite Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA ted_hackstadt@nih.gov.
authors.forename: Ted
authors.initials: T
authors.lastname: Hackstadt
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002692:Chlamydia trachomatis / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D050600:SNARE Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C460868:IncA protein, Chlamydia trachomatis; D008565:Membrane Proteins; D050600:SNARE Proteins