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Effector Translocation Assay: Differential Solubilization.

Irina S Franco1, Sara V Pais1, Nuno Charro1

  • 1UCIBIO-REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa (FCT NOVA), Caparica, Portugal.

Methods in Molecular Biology (Clifton, N.J.)
|July 2, 2017
PubMed
Summary
This summary is machine-generated.

This study presents a novel method for identifying bacterial effector proteins in host cells. By using Triton X-100 detergent, researchers can isolate host cell proteins, aiding virulence factor research.

Keywords:
Bacterial protein secretion systemDetergent solubilizationEffectorImmunoblottingSDS-PAGETranslocationType III secretion

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Area of Science:

  • Microbiology
  • Cell Biology
  • Pathogen-Host Interactions

Background:

  • Bacterial pathogens utilize specialized nanomachines to inject effector proteins into host cells.
  • Understanding these effector proteins is crucial for deciphering pathogen virulence mechanisms.

Purpose of the Study:

  • To describe a novel method for isolating bacterial effector proteins from infected mammalian host cells.
  • To facilitate the study of virulence factors delivered by bacterial pathogens.

Main Methods:

  • Utilized mammalian tissue culture infection models.
  • Employed Triton X-100 detergent to selectively solubilize host cell membranes.
  • Isolated a bacterial-free, host-enriched protein fraction.
  • Probed the fraction for effector proteins using immunoblotting.

Main Results:

  • Successfully isolated a Triton-soluble fraction containing host cell proteins but lacking bacteria.
  • This fraction was enriched in proteins from the host cell cytoplasm and plasma membrane.
  • The method allows for the detection of translocated bacterial effector proteins.

Conclusions:

  • The described method provides an effective means to identify bacterial effector proteins in host cells.
  • This technique aids in the investigation of bacterial virulence and pathogen-host interactions.