Studies on the mechanism of bacterial resistance to complement-mediated killing. IV. C5b-9 forms molecular weight complexes with bacterial outer membrane constituents on serum-resistant but not on serum-sensitive Neisseria gonorrhoeae

K. A. Joiner, K. A. Warren, E. J. Brown, J. Swanson, M. M. Frank

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66 Scopus citations


The mechanism of complement resistance and of Ab-mediated killing of serum-resistant Neisseria gonorrhoeae (GC) was studied. Consumption of C9 and deposition of C7 and C9 from pooled normal human serum (PNHS) or hypogammaglobulinemic serum (HγS) were examined on transparent, nonpiliated, serum-resistant strains from disseminated gonococcal infection (DGI). Results were compared with similar measurement on serum-sensitive isolates. Serum-resistant and serum-sensitive strains consumed equivalent amounts of C9 and bound similar numbers of C7 and C9 molecules when incubated in 10% PNHS. Presensitization of one resistant isolate (NRL 6305) with immune rabbit antisera resulted in 92 to 96% killing and increased C9 uptake twofold when this strain was subsequently incubated in 10% PNHS. When presensitized NRL 6305 incubated in HγS, C9 uptake and killing increased as the serum concentration was raised from 10 to 40%. No killing of nonpresensitized NRL 6305 occurred in HγS, even with deposition of 68,000 molecules of C9/organism, indicating that Ab-mediated killing was not simply a consequence of greater terminal component deposition. There was no difference between serum-sensitive and serum-resistant strains in the elution of 125I-C9 by low ionic strength or isotonic buffer, 1 M NaCl, 0.01 M EDTA, or 1% sodium deoxycholate (NaDOC), and results suggest that the terminal complex is attached by hydrophobic bonds. However, 53.7 ± 4.2% of 125I-C9 counts were released from serum-resistant bacteria by 0.1% trypsin, whereas only 25.9 ± 1.7% release was observed with serum-sensitive strains. Presensitization of NRL 6305 decreased trypsin release of 125I-C9 to the level seen with serum-sensitive bacteria. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) autoradiography of released and bound 125I-C9 after trypsin treatment showed identical cleavage fragments of C9 for serum-sensitive organisms and for serum-resistant organisms with and without presensitization. Sucrose density gradient ultracentrifugation of detergent-extracted C5b-9 complexes indicated that a) the major bactericidal form of C5b-9 on the surface of serum-sensitive or presensitized serum-resistant GC sediments as a 33-S Complex when extracted with SB12 or NaDOC; b) the nonbactericidal C5b-9 on serum-resistant NRL 6305 exists in larger 35-S and 43-S aggregates of C5b-9 or as C5b-9 complexes to bacterial outer membrane constituents; and c) a 13.5-S peak containing C9 is uniquely extracted with NaDOC or SB12 from serum-resistant NRL 6305, but this peak disappears when the organism is rendered serum sensitive by presensitization with bactericidal Ab. The possibility was investigated that complex formation occurred between C5b-9 and GC outer membrane constituents in the SB12 extracts. One-quarter of 125I-C9 in SB12 extracts from presensitized and nonpresensitized NRL 6305 was immunoprecipitable with antibodies directed against the whole organism. Threefold more 125I-GC was coprecipitable from the nonpresensitized than from the presensitized NRL 6305 when SB12 extracts were incubated with anti-C5 Sepharose. Double diffusion analysis of the same SB12 extracts with antibodies to C5, C9, and whole GC showed identity between C5b-9 and bacterial constituents for both the presensitized and nonpresensitized organism. In summary these experiments demonstrate that the molecular configuration of C5b-9 bound to serum-resistant GC is different from the configuration of C5b-9 bound to serum-sensitive GC or to the resistant strain rendered sensitive by presensitization with bactericidal Ab.

Original languageEnglish (US)
Pages (from-to)1443-1451
Number of pages9
JournalJournal of Immunology
Issue number3
StatePublished - 1983

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology


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