Complement resistance is an important virulence determinant in the pathogenesis of bacterial gram-negative infections. A more complete understanding of complement resistance determinants may lead to novel therapies in the treatment of disease caused by these organisms. We have used transposon mutagenesis (TnphoA) to randomly mutate a clinical blood isolate of Escherichia coli (CP9, 04/K54/H5) that we are using as a model pathogen for extra-intestinal disease. This strain possesses a group 2 capsule and is highly serum resistant, as is characteristic of clinical isolates. Isogenic mutants have been identified that have an increased sensitivity to serum. Some of these mutants have been identified as being capsule-minus. We have defined at least 3 physical loci that are involved in capsule production in our strain and they are linked. DNA sequence analysis has demonstrated that we have disrupted at least 5 separate genes and that one has homology with capsular transport genes of Escherichia coli serotype K1, Haemophilus influenza and Neisseria meningitidis. In vitro biologic studies have established that the K54 capsule protects against complement killing that is mediated via the alternative complement pathway. C3 binding is equal in capsule-positive and capsule- minus strains suggesting the mechanism of complement resistance conferred by the K54 capsule is different than that of the K1 capsule. Additional in vitro studies have demonstrated that the K54 capsule protects against killing by neutrophils. Three different animal models were used to test whether the K54 capsule conferred protection in vivo as well. In summary, these studies demonstrated that the possession of the K54 capsule resulted in a significant decrease of the LD50 in a mouse model of systemic infection, significantly decreased intra-vascular clearance and protected against killing in a local infection model. These studies also demonstrated that the K54 capsular polysaccharide may be one of the bacterial antigens responsible for inciting the cascade of events that leads to septic shock. They also demonstrated that other bacterial factors are induced in vivo that enhance survival and are important in the pathogenic process.
