High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Summary

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to 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^1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide³. 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^4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells^5,6. 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^5-8. 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”⁵. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface^9,10. 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.

Introduction

H. pylori infection is a significant risk factor for gastric cancer¹. However, disease outcomes vary and depend on numerous factors such as host genetics, genetic diversity of H. pylori strains, and environmental elements such as host diet¹¹. Previous reports have established that a correlation exists between H. pylori infection, iron deficiency (as measured by decreased blood ferritin and hemoglobin concentrations), and increased proinflammatory cytokine production, including IL-8 secretion, which ultimately leads to increased gastric disease progression¹². Acute H. pylori infection is also assoc....

Protocol

1. H. pylori Growth in Various Conditions of Iron Availability and Co-culture with Human Gastric Epithelial Cells

1. Select H. pylori strain PMSS1 for these studies because it has an intact cag pathogenicity island and expresses a functioning type IV secretion system. Also utilize an isogenic cagE mutant (PMSSI ΔcagE) as a negative control. Grow bacteria on TSA plates supplemented with 5% sheep blood (blood agar plates) for 24 hr at 37 °C in the presence of 5% CO₂.
   NOTE: Prepare reagents and assemble materials as outlined in the Materials and Equipment Table. Final concentratio....

Results

In this report, we have demonstrated that conditions of varying iron availability have the capacity to modulate H. pylori Cag-T4SS pilus biogenesis at the host pathogen interface. When cultured in medium alone, H. pylori forms an average of 3 pili/cell. When H. pylori is grown in iron deplete conditions (using the synthetic chelator dipyridyl) that are sub-inhibitory to bacterial growth (Figure 1), the bacteria produce numerous Cag-T4SS pili (~7 pili/cell) when co-cultured with.......

Discussion

Iron is an essential micronutrient for most forms of life, including bacterial pathogens. In an effort to restrict the viability of invading microorganisms, vertebrate hosts sequester nutrient iron in a process known as “nutritional immunity”¹⁸. In response to this, bacterial pathogens have evolved to use iron as a global signaling molecule to sense their surroundings and regulate the elaboration of virulence features such as iron acquisition systems, toxins, and toxin secretion machinery^19,20.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Modified brucella broth
Peptone from casein (10 g/L)	Sigma	70172
Peptic digest of animal tissue (10 g/L)	Sigma	70174
Yeast extract (2 g/L)	Sigma	92144
Dextrose (1 g/L)	Sigma	D9434
Sodium chloride (5 g/L)	Thermo Fisher	S271-10
Cholesterol (250X) (4 ml/L)	Life Technologies	12531018
Ferric chloride (100 or 250 uM)	Sigma	157740-100G
Dipyridyl (200 uM)	Sigma	D216305-100G
Modified RPMI
RPMI+HEPES (1X)	Life Technologies	22400-121
Fetal bovine serum (100 ml/L)	Life Technologies	10438-026
Electron Microscopy Preparation
Paraformaldehyde (2.0% aqueous)	Electron Microscopy Sciences	15713
Gluteraldehyde (2.5% aqueous)	Electron Microscopy Sciences	16220
Sodium cacodylate (0.05 M)	Electron Microscopy Sciences	12300
Osmium tetroxide (0.1% aqueous)	Electron Microscopy Sciences	19150
Ethanol (absolute)	Sigma	E7023
Colloidal silver paint	Electron Microscopy Sciences	12630
SEM sample stubs	Electron Microscopy Sciences	75220
Coverslips	Thermo Fisher	08-774-383
IL-8 Secretion Evaluation
Quantikine IL-8 ELISA kit	R&D Systems	D8000C