title: High resolution electron microscopy of the Helicobacter pylori Cag type IV secretion system pili produced in varying conditions of iron availability.
abstract: Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the "pilus". Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface(. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.
authors.affiliation: Department of Medicine - Division of Infectious Diseases, Vanderbilt University School of Medicine.
authors.forename: Kathryn Patricia
authors.initials: KP
authors.lastname: Haley
authors.affiliation: Department of Medicine - Division of Infectious Diseases, Vanderbilt University School of Medicine.
authors.forename: Eric Joshua
authors.initials: EJ
authors.lastname: Blanz
authors.affiliation: Department of Medicine - Division of Infectious Diseases, Vanderbilt University School of Medicine; Tennessee Valley Healthcare Systems, U. S. Dept. of Veterans Affairs; jennifer.a.gaddy@vanderbilt.edu.
authors.forename: Jennifer Angeline
authors.initials: JA
authors.lastname: Gaddy
mesh_terms: D004847:Epithelial Cells / Q000382:microbiology; D010861:Fimbriae, Bacterial / Q000378:metabolism* / Q000648:ultrastructure*; D005753:Gastric Mucosa / Q000166:cytology / Q000382:microbiology; D044404:Genomic Islands; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000378:metabolism* / Q000648:ultrastructure*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods; D015398:Signal Transduction; D000069319:Type IV Secretion Systems / Q000096:biosynthesis* / Q000378:metabolism / Q000648:ultrastructure*; D037521:Virulence Factors / Q000378:metabolism
chemical_list: D000069319:Type IV Secretion Systems; D037521:Virulence Factors; D007501:Iron