The mechanism of complement-mediated killing of Gram-negative bacteria has remained elusive. The long-term objectives of this project are to uncover the molecular basis of the bactericidal activity of the membrane attack complex of complement (C5b-9) and the changes made by bacteria to become resistant. The immediate goals are to identify the regions in C9 required for toxicity, to understand the molecular requirements for translocation of C9 across the periplasmic space and its insertion into the inner (plasma) membrane, and to characterize bacterial mutations that render them resistant to its effects.
Three specific aims are proposed:
In Specific Aim 1, the minimal structural elements in C9 required for bacterial cytotoxicity will be determined. Various forms of recombinant C9 will be expressed and secreted into the periplasm of E. coli strains that lack enzymes which form disulfide bonds. The addition of redox reagents will effect disulfide pairing and folding and lead to C9-mediated cell death.
In Specific Aim 2, bacterial outer membrane or periplasmic constituents necessary for translocation of C9 across the cell wall will be identified. Native C9 will be introduced into the periplasm of intact cells. Binding of radiolabeled C9 followed by unlabeled molecules (or other permutations of this sequence) will be performed to understand why poly C9 is required for bacteriolysis.
In Specific Aim 3, complement-resistant E. coli strains resembling those isolated from adults will be generated so that mutated genes contributing to serum resistance can be characterized. A two-arm screening and selection mechanism will be employed to identify mutants that are resistant to the cytotoxic action of recombinant C9 exported into the periplasm and to C9 present in serum.
| Rossi, Veronique; Wang, Yunxia; Esser, Alfred F (2010) Topology of the membrane-bound form of complement protein C9 probed by glycosylation mapping, anti-peptide antibody binding, and disulfide modification. Mol Immunol 47:1553-60 |
