Functional microassay of complement activation by pneumococci

Abstract

A microassay to estimate complement (C) activation by pneumococci was developed. In this assay, heat-killed pneumococci are, for a short period, incubated with diluted normal human serum. During this incubation, the bacteria activate C and give rise to C5b6 formation. Subsequently, chicken erythrocytes (ChE) in EDTA are added and the mixtures are reincubated to allow reactive (C5b6-mediated) lysis of the innocent bystander erythrocytes. The ‘number of active sites per erythrocyte’ (Z) is used to express the C-activating ability of pneumococci. In this study, an unique pair of encapsulated S3 pneumococci and an unencapsulated S3 mutant obtained by transposon-insertion are used. The original, virulent S3 strain appeared to be a poor C activator, whereas the laboratory mutant strain activated C virtually well. Treatment of the pneumococci with enzymes and in particular with pepsin markedly increased C activation by S3 and only slightly C activation by the unencapsulated strain. As judged by ELISA, the capsule itself was resistant to enzyme treatment. Based on these data, we presume that the anticomplementary activity is a property of a cell wall-associated, pepsin-sensitive component, probably a protein. Since the anticomplementary component may be an important virulence factor, its identity needs further investigation.