Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing

Area of Science:

• Immunology
• Microbiology
• Molecular Biology

Background:

• The complement system plays a crucial role in innate immunity, mediating bacterial killing through the formation of the membrane attack complex (MAC).
• Previous studies indicated a requirement for multiple C9 molecules in complement-mediated bacterial killing, but the precise mechanism and threshold were not fully elucidated.

Purpose of the Study:

• To determine the minimum number of C9 molecules required for complement-mediated killing of Escherichia coli strain J5.
• To investigate the relationship between C9:C5b-8 ratio, membrane damage, and bacterial lysis.
• To identify the critical pore size of the C5b-9 complex necessary for releasing periplasmic and cytoplasmic markers.

Main Methods:

• Escherichia coli strain J5 was transformed to enhance beta-lactamase (BLA) production for periplasmic marker release studies.
• Varying ratios of C9 to C5b-8 were generated on the bacterial surface, and the C9:C5b-8 ratio was modulated.
• Release of periplasmic (BLA) and cytoplasmic (86Rb, beta-galactosidase) markers was measured to assess membrane permeability and pore formation.

Main Results:

• A minimum C9:C5b-8 ratio of 3:1 was required for the release of the periplasmic marker BLA and for bacterial killing, with sigmoidal dose-response curves observed.
• Release of the small cytoplasmic marker 86Rb also necessitated a C9:C5b-8 ratio of at least 3:1, correlating with killing.
• The large cytoplasmic marker beta-galactosidase was not released even at high C9:C5b-8 ratios, indicating intact peptidoglycan and specific outer membrane damage.

Conclusions:

• Complement-mediated killing of E. coli J5 requires sufficient C5b-9 channel formation, necessitating at least three C9 molecules per C5b-8 complex.
• The formation of a C5b-9 channel large enough to release periplasmic contents is a critical step for bactericidal activity.
• The data suggest that at lower C9:C5b-8 ratios, C5b-9 may not access the cytoplasmic space, or cellular transport mechanisms compensate for small channels.