Home › Cell Biology › Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Cell Biology JoVE (Open Access) Citable · DOI

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

DOI: 10.3791/51601-v
What you'll learn
  • ✓ Perform Shigella flexneri infection of cultured HeLa cells and apply pharmacological treatments to modulate autophagy-cytoskeleton responses
  • ✓ Prepare and inject S. flexneri into zebrafish larvae to assess bacterial infection and host defense mechanisms in vivo
  • ✓ Conduct live imaging and survival analysis of infected zebrafish to evaluate autophagy-cytoskeleton interactions during bacterial infection
Protocol

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Difficulty
advanced
Total time
~3–5 days (cell culture infection: ~4–6 hrs; zebrafish preparation and injection: ~2–3 hrs; live imaging and survival monitoring: 24–48 hrs)
Model organism
HeLa cells, Danio rerio (zebrafish larvae)
Biosafety
BSL-2

Steps

1
Infect cultured HeLa cells with Shigella flexneri

Prepare HeLa cell monolayers and expose them to Shigella flexneri at specified multiplicity of infection (MOI) to establish controlled bacterial infection. Allow infection to proceed for defined time periods to enable host autophagy and cytoskeletal responses.

▶ 01:58
2
Apply pharmacological treatments to infected HeLa cells

Treat infected cell cultures with pharmacological agents to modulate autophagy and cytoskeleton dynamics. Document responses to identify host and pathogen determinants involved in bacterial compartmentalization and autophagy induction.

▶ 05:06
3
Prepare Shigella and zebrafish larvae for injection

Grow and prepare Shigella flexneri inoculum at appropriate concentration. Anesthetize zebrafish larvae and position them for controlled microinjection into designated anatomical sites.

▶ 06:05
4
Inject Shigella into zebrafish larvae and monitor survival

Perform microinjection of S. flexneri into anesthetized larvae at standardized dose and location. Monitor injected larvae for survival, phenotypic changes, and behavioral responses over defined observation periods.

▶ 07:30
5
Perform live imaging of infected zebrafish larvae

Image infected zebrafish larvae using fluorescence or confocal microscopy to visualize bacterial localization, autophagy markers, and cytoskeletal dynamics in real time. Acquire temporal image series to characterize host-pathogen interactions in vivo.

▶ 09:17
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