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GASTRIC Publication-derived

Chemodetection and Destruction of Host Urea Allows Helicobacter pylori to Locate the Epithelium

Source Huang et al., 2015 · Stanford University School of Medicine · 10.1016/j.chom.2015.07.002

👤 Julie Y. Huang, Emily Goers Sweeney, Michael Sigal, Hai C. Zhang, S. James Remington, Michael A. Cantrell, Calvin J. Kuo, Karen Guillemin, Manuel R. Amieva ⏱ 42 days 📋 12 phases 🧫 Human gastric epithelial cells (from surgical specimens)

Abstract

This protocol establishes a 3D human gastric organoid system to investigate how Helicobacter pylori detects and responds to host metabolites emanating from polarized gastric epithelium. The method combines gastric organoid culture, conditioned media preparation, and real-time microgradient chemotaxis assays to demonstrate that H. pylori is rapidly attracted to urea via the chemoreceptor TlpB, and that bacterial urease simultaneously destroys the signal to enable sensitive detection of nanomolar urea concentrations.

Cell source
Human gastric epithelial cells (from surgical specimens)
Application
Bacterial chemotaxis and host–pathogen interaction modeling

Protocol overview

60 steps across 12 phases

Human Gastric Organoid Culture and Preparation 1–14
  1. 1 Obtain and verify normal gastric tissue from surgical specimens
  2. 2 Isolate gastric glands from tissue
  3. 3 Embed isolated glands in Matrigel
  4. 4 Culture organoids until differentiation is complete
Preparation of Conditioned Media 1
  1. 1 Wash organoid cultures extensively
  2. 2 Incubate organoids in fresh DMEM to generate conditioned media
  3. 3 Collect conditioned media
Preparation of Alternative Epithelial Models (Optional) 1–2
  1. 1 Culture Caco-2 cells as polarized monolayers on Transwell filters
  2. 2 Wash Caco-2 monolayers
  3. 3 Incubate Caco-2 monolayer with fresh DMEM
  4. 4 Collect Caco-2 cell-conditioned media
  5. 5 Culture MDCK cells as polarized monolayers
  6. 6 Prepare MDCK cell-conditioned media
Urea Quantification and Urease Treatment 1
  1. 1 Quantify urea concentration in conditioned media
  2. 2 Prepare Jack Bean urease solution
  3. 3 Treat conditioned media with urease
  4. 4 Filter urease-treated media
Bacterial Strain Preparation 1
  1. 1 Culture H. pylori from overnight culture
  2. 2 Prepare subculture to mid-log phase
  3. 3 Collect bacterial suspension for microgradient assay
Microgradient Chemotaxis Assay Setup 1
  1. 1 Place bacterial suspension in glass-bottom dish
  2. 2 Mount dish on heated microscope stage
  3. 3 Fill micropipette with test solution
  4. 4 Mount micropipette on micromanipulator
  5. 5 Connect micropipette to pressure injection system
  6. 6 Set up video recording system
Microgradient Assay Execution 1
  1. 1 Record baseline bacterial motion (pre-injection)
  2. 2 Apply compensation pressure and generate gradient
  3. 3 Record bacterial response during gradient exposure (post-injection)
  4. 4 Remove micropipette and terminate assay
Urea Pulse Assay (Alternative Sensitive Detection Method) 1
  1. 1 Fill micropipette with urea solution for pulse delivery
  2. 2 Mount and align micropipette as in standard assay
  3. 3 Begin video recording
  4. 4 Deliver high-pressure urea pulse
  5. 5 Continue recording bacterial response
Analysis of Chemotactic Response 1
  1. 1 Extract video frames and generate motility tracings
  2. 2 Threshold images and define region of interest
  3. 3 Quantify pixel density over time
  4. 4 Generate response plots
  5. 5 Perform statistical analysis
Thermofluor Assay for TlpB-Ligand Binding (Optional) 1
  1. 1 Prepare TlpB periplasmic domain protein
  2. 2 Prepare ligand solutions
  3. 3 Prepare reaction mixtures for thermofluor assay
  4. 4 Cover plate and load onto thermal cycler
  5. 5 Perform thermal denaturation ramp
  6. 6 Calculate melting temperature (Tm)
Analysis of TlpB Crystal Structure and Ligand Binding Configurations (Optional) 1
  1. 1 Download crystal structure models from Protein Data Bank
  2. 2 Inspect ligand binding electron density maps
  3. 3 Model refinement and hydrogen bonding assessment
  4. 4 Compare binding configurations among ligands
Animal Infection Studies (In Vivo Validation) 14–42
  1. 1 Obtain and house laboratory mice
  2. 2 Prepare H. pylori inoculum
  3. 3 Orally infect mice
  4. 4 Sacrifice mice at 2 weeks post-infection
  5. 5 Harvest and process stomach tissue
  6. 6 Homogenize stomach tissue
  7. 7 Perform serial dilutions and plate counts
  8. 8 Count colonies and calculate CFU per gram stomach
  9. 9 Repeat at 4 and 6 weeks post-infection
  10. 10 Perform statistical analysis of colonization data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Huang et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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