We hypothesize that modifications and/or reorganization of membrane components, induced by plasmid carriage, enable invasive bacterial pathogens to avoid the bactericidal effect of complement. To explore the basis for such plasmid mediated resistance we will utilize a) a genetically well defined E. coli K- 12, susceptible to serum killing by the classical pathway of complement system, b) a well-characterized plasmid which confers serum resistance on E. coli K-12 and c) a series of mutants of the plasmid which have lost one or both serum resistance genes (iss, traT). Mapping of traT and iss will be completed by gene cloning and transposon mutagenesis and plasmid DNA loci affecting serum resistance will be isolated from a genetically engineered mini pWS18 plasmid which carries both serum resistance genes. We will sequence small cloned DNA fragments, containing iss gene(s) or traT. Potential antigenic determinants of the protein(s) coded for by these fragment will be synthesized for preparation of antibodies. The mechanism(s) underlying resistance in hosts bearing altered plasmid genes will be examined using 1d and 2d gels as well as immunological techniques to quantitate gene products and localize their position in the membranes. Monoclonal antibodies will be prepared to unique components of the membranes of susceptible or resistant strains differing only in plasmid carriage. Immunochemical studies will provide information of differences in surface antigenic determinants available for reaction with bactericidal serum components. Effects of masking individual surface determinants on serum resistance will be examined with Fab fragments of monoclonal antibodies to avoid complications due to positioning of Fc on the outer bacterial membrane. Geographic relationships of outer membrane components may have equal or more relevance to serum resistance than composition data derived from electrophoretic studies. These geographic relationships, as well as localization of the membrane attack complex of the complement system, will be examined using gold labeled antibodies as probes in immunoelectron microscopy. In addition, heterobifunctional cross-linking agents will be used to probe the membrane environment adjacent to the membrane attack complex (C5b-C9) of the complement system in resistant and susceptible strains.
| Banerjee, A; Weber, P C; Palchaudhuri, S (1990) Comparison of the CopB systems of plasmids R1 and ColV2-K94: a single base alteration in CopB gene is responsible for the increased copy number of the low copy number plasmid ColV2-K94. Mol Gen Genet 220:320-4 |
| Chuba, P J; Leon, M A; Banerjee, A et al. (1989) Cloning and DNA sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda DNA. Mol Gen Genet 216:287-92 |
