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Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens
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Bacterial killing by complement requires membrane attack complex formation via surface-bound C5 convertases.

Dani Ac Heesterbeek1, Bart W Bardoel1, Edward S Parsons2

  • 1Department of Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

The EMBO Journal
|January 16, 2019
PubMed
Summary

The immune system uses membrane attack complexes (MACs) to kill bacteria. Local assembly of these complexes on bacterial surfaces is crucial for effective bacterial membrane permeabilization and killing.

Keywords:
Gram‐negative bacteriaatomic force microscopycomplement; convertasemembrane attack complex

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Area of Science:

  • Immunology
  • Microbiology
  • Molecular Biology

Background:

  • The immune system combats Gram-negative bacteria using membrane attack complexes (MACs).
  • The precise mechanism by which MACs disrupt the bacterial cell envelope remains unclear.
  • MAC formation is initiated by complement enzymes cleaving C5.

Purpose of the Study:

  • To elucidate the role of C5 convertase enzymes in MAC assembly beyond C5 cleavage.
  • To understand how MACs achieve bactericidal activity against Gram-negative bacteria.
  • To investigate the requirements for efficient MAC pore insertion into bacterial membranes.

Main Methods:

  • Utilized flow cytometry to analyze MAC formation and bacterial interaction.
  • Employed atomic force microscopy to visualize MAC pore insertion.
  • Investigated the bactericidal activity of purified MAC components versus locally assembled MACs.

Main Results:

  • Purified MACs can perforate artificial membranes and mammalian cells but lack bactericidal activity.
  • Local assembly of MACs by C5 convertase enzymes is essential for permeabilizing both bacterial membranes.
  • C5b6 loses its capacity to form bactericidal pores rapidly, necessitating immediate in situ conversion and insertion.

Conclusions:

  • Bacterial killing by the immune system requires in situ C5 conversion and rapid MAC assembly at the bacterial surface.
  • Local MAC assembly ensures efficient insertion of pores into bacterial membranes, leading to cell death.
  • These findings provide fundamental molecular insights into MAC assembly and bacterial defense mechanisms.