Kinetics of assembly and decay of complement components on Escherichia coli O111:B4 preparation of stable intermediates

Abstract

The preparation of bacterial intermediates bearing complement components at various steps of the complement sequence was investigated by suspending immunoglobulin M-opsonized Escherichia coli Oll1:B4 cells in complement-deficient sera at different temperatures and ionic strengths. The optimal conditions for the formation of the intermediates at T,n were found to be an ionic strength of 0.091 , and a temperature of 37°C, except for BAC142, which could be formed equally well at room temperature. In contrast to all the other intermediates, which, once formed at Tmax, were stable in the presence of the whole serum, BAC142 decayed with a half-life of 10 min due to the lability of bound C2. Washing with a buffer of either 0.091 or 0.046 ,u did not affect the bacterial intermediates, with the exception of BAC1-3 formed either in the presence of CS-deficient serum or with purified C3 added to BAC142. All the intermediates were found to be stable after incubation in 0.091-,u buffer for 30 min at 37°C.

Bactericidal-activities of human polymorphonuclear leukocyte proteins against Escherichia coli O111:B4 coated with C5 or C8

“…The reason 30 to 35% of BAC1-5 escaped killing by PNS or acetate extract could be explained neither by an intrinsic decay of BAC1-5 nor by an extrinsic decay induced by the soluble acetate extract. In fact, we previously showed that this BAC is rather stable (16), and the results presented in Fig. 3 indicate that its stability is not affected by incubation with the granule extract.…”

“…Buffers. Krebs-Ringer phosphate (KRP) buffer (pH 7.4; ionic strength, 0.091 pu) supplemented with 0.64 mM MgCI2 and 0.16 mM CaCl2 was prepared as previously described (16).…”

“…collection of the Institute of Microbiology, University of Trieste. The bacteria were grown in nutrient broth (Difco Laboratories, Detroit, Mich.) for 6 h at 37°C and then on nutrient agar (Difco) overnight as previously described (16). The standard bactericidal assays were performed by incubating the mixture of 5 x 105 bacteria and either complement components or PMN extract (final volume, 0.2 ml) at 37°C for 30 min.…”

“…nents. As previously reported (16), BACs were prepared by mixing 3 x 107 BA with optimal dilutions of complementdeficient sera at 0.91 ,. ionic strength to a total volume of 300 ,ul.…”