Tn916[delta]e transposon mutagenesis and inactivation of group B streptococcus C5a-ASE

Group B Streptococcal infections are a major cause of sepsis, pneumonia and meningitis in human neonates. The inflammatory response to invasion of the body by these organisms is sparce, however. The majority of strains of group B streptococci rapidly inactive the major complement derived chemotactic factor, C5a. Inactivation is due to an enzyme with an apparent molecular weight of 120,000 which cleaves a heptapeptide from the carboxy terminus of the C5a molecule which is critical in binding of C5a to neutrophil receptors. Cleavage of this heptapeptide results in an approximate 650 Dalton decres in Mr by SDS-PAGE. We used the transposon Tn916 containing and erythromycin resistance gene in mutagenesis studies with a type III GBS C5a-ase positive strain. After screen approximately 3659 transposon-induced mutants of the parent strain containing the erythromycin resistance gene, a C5a-ase negative mutant was found. When incubated with recombinant human C5a (rhC5a), this strain failed to inhibit C5a induced neutrophil chemotaxis or adherence to gelatin coated surfaces. Furthermore, the mutant strain failed to alter the migration of rhC5a in SDS-PAGE. Transposon insertion into the GBS was confirmed by southern blot analysis. This transposon induced C5a-ase negative group B streptococcal strain may contribute greatly to our understanding of the role of GBS C5a-ase in the pathogenesis of disease due to these organisms.