Molecular aspects of complement-mediated bacterial killing - Periplasmic conversion of C9 from a protoxin to a toxin

As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coil cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts, This conversion is independ ent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a sign al sequence are toxic. Toxicity re quires disulfide linked C9 because expor t into the periplasm of cells defective in disulfide bond synthesis (dsbA a nd dsbB mutants) is not toxic unless N-acetylcysteine is added externally t o promote cystines, A N-terminal fragment, C9[1-144], is not toxic nor is c ytoplasmically expressed C9, even in trxB mutants that are able to form dis ulfide bonds in the cytoplasm, Importantly, expression of full-length C9 in complement-resist ant cells has no effect on their viability. Expression a nd translocation into the periplasm may provide a novel model to identify m olecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.