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Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy
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Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7.

Dennis J Doorduijn1, Bart W Bardoel1, Dani A C Heesterbeek1

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The Membrane Attack Complex (MAC) requires rapid C7 binding to C5b6 to kill bacteria. This anchoring prevents bacterial evasion of complement-dependent killing by blocking MAC pore formation.

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Area of Science:

  • Immunology
  • Microbiology
  • Molecular Biology

Background:

  • The human complement system's Membrane Attack Complex (MAC) directly kills Gram-negative bacteria.
  • MAC pores form when C5b initiates assembly with C6, C7, C8, and C9 on bacterial surfaces.
  • Previous work indicated bacterial killing by MAC pores necessitates local C5 conversion by surface-bound convertases.

Purpose of the Study:

  • To elucidate the molecular mechanisms underlying the essential requirement for local MAC pore assembly in bacterial killing.
  • To investigate why the spatial proximity of MAC component assembly is critical for effective bacterial lysis.

Main Methods:

  • Utilized binding experiments with fluorescently labeled C6 to assess C5b6 release.
  • Employed trypsin shaving and atomic force microscopy to evaluate C5b-7 anchoring to bacterial surfaces.
  • Analyzed complement-resistant clinical *Escherichia coli* strains to identify evasion strategies.

Main Results:

  • Demonstrated that rapid C7 interaction with C5b6 is crucial for forming bactericidal MAC pores on *E. coli*.
  • Showed that C7 binding to C5b6 prevents the dissociation of C5b6 from the bacterial surface.
  • Identified that complement-resistant *E. coli* strains interfere with C5b-7 anchoring, preventing stable MAC pore insertion and bacterial killing.

Conclusions:

  • The efficient anchoring of C5b-7 to the bacterial cell envelope, mediated by rapid C7 interaction with C5b6, is essential for stable MAC pore formation and bacterial lysis.
  • Bacterial pathogens can evade complement-mediated killing by disrupting the anchoring of MAC components, specifically C5b-7.
  • These findings offer fundamental molecular insights into MAC pore assembly and bacterial resistance mechanisms against complement attack.