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

E. F. Bloch, M. A. Schmetz, J. Foulds, C. H. Hammer, M. M. Frank, Keith A Joiner

Research output: Contribution to journalArticle

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Abstract

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker β-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp®). Multimeric C9 within C5b-9 (C9:C5b-8 >3) was required to release BLA (m.w. 28,900) from J5-Amp® regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker β-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.

Original languageEnglish (US)
Pages (from-to)842-848
Number of pages7
JournalJournal of Immunology
Volume138
Issue number3
StatePublished - 1987
Externally publishedYes

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Complement Membrane Attack Complex
Escherichia coli
Membranes
Muramidase
Galactosidases
complement C5b-8 complex
Peptidoglycan
Serum
Potassium

ASJC Scopus subject areas

  • Immunology

Cite this

Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing. / Bloch, E. F.; Schmetz, M. A.; Foulds, J.; Hammer, C. H.; Frank, M. M.; Joiner, Keith A.

In: Journal of Immunology, Vol. 138, No. 3, 1987, p. 842-848.

Research output: Contribution to journalArticle

Bloch, EF, Schmetz, MA, Foulds, J, Hammer, CH, Frank, MM & Joiner, KA 1987, 'Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing', Journal of Immunology, vol. 138, no. 3, pp. 842-848.
Bloch, E. F. ; Schmetz, M. A. ; Foulds, J. ; Hammer, C. H. ; Frank, M. M. ; Joiner, Keith A. / Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing. In: Journal of Immunology. 1987 ; Vol. 138, No. 3. pp. 842-848.
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abstract = "We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker β-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp{\circledR}). Multimeric C9 within C5b-9 (C9:C5b-8 >3) was required to release BLA (m.w. 28,900) from J5-Amp{\circledR} regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker β-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9{\%} killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.",
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AU - Bloch, E. F.

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AU - Hammer, C. H.

AU - Frank, M. M.

AU - Joiner, Keith A

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N2 - We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker β-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp®). Multimeric C9 within C5b-9 (C9:C5b-8 >3) was required to release BLA (m.w. 28,900) from J5-Amp® regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker β-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.

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