Jennifer Gaddy
Research Instructor
Last active: 9/13/2018

High resolution electron microscopy of the Helicobacter pylori Cag type IV secretion system pili produced in varying conditions of iron availability.

Haley KP, Blanz EJ, Gaddy JA
J Vis Exp. 2014 (93): e52122

PMID: 25489938 · PMCID: PMC4354248 · DOI:10.3791/52122

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.

MeSH Terms (12)

Epithelial Cells Fimbriae, Bacterial Gastric Mucosa Genomic Islands Helicobacter Infections Helicobacter pylori Humans Iron Microscopy, Electron Signal Transduction Type IV Secretion Systems Virulence Factors

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