Molecular mechanisms of enterobacterial resistance to complement

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Complement (C') comprises over thirty proteins located predominantly in the blood compartment that help defend the host against microbial invaders. C' is able to kill many Gram-negative bacteria (GNB), including antibiotic resistant strains that currently pose a severe health threat in hospitals and in the community. C' pathways are activated on bacterial surfaces, either by surface-bound antibodies or by recognition of the foreign nature of the bacterial surface, leading to the formation of multi-protein assemblages, termed C5b-9 complexes, that insert into the outermost layer of the bacterial cell wall, the outer membrane (OM). Stable insertion of C5b-9 complexes disrupts the integrity of the OM bilayer leading to perturbation of the inner, cytoplasmic membrane and bacterial cell death by mechanisms that are not completely understood. Unfortunately, many GNB have found a way to resist C' attack, either by preventing C' activation, by ensuring degradation of C' components prior to formation of C5b-9 complexes or by elaboration of an OM that prevents stable insertion of C5b-9 into lipid regions of the OM.Much of our knowledge of the basis of C' resistance comes from observations made over thirty years ago and focused on identifying the macromolecular structures at the bacterial surface, such as polysaccharide capsules and lipolysaccharide O-side chains, that contribute to resistance. These studies made little attempt to provide an integrated picture of bacterial surface topography that might explain why C5b-9 complexes do not insert in stable fashion into the bacterial OM. There has been no significant recent progress towards clarifying the basis of C' resistance in spite of new insights that have fundamentally changed our understanding of the organisation of the Gram-negative OM. We propose, for the first time, to employ state-of-the-art structural, biophysical and microscopy-based approaches to determine, in fine detail, the bacterial surface topography that defines C'-resistant pathogens (the so-called resistance phenotype). As GNB have evolved a variety of mechanisms that prevent effective C' attack, we will employ a number of clinical isolates of Escherichia coli and Klebsiella pneumoniae that express a variety of surface macromolecules known to affect C' activation and C5b-9 deposition in different ways. We will determine the bacterial components responsible for C' resistance by screening large libraries of mutants generated by a technique known as Transposon Directed Insertion Sequencing, or TraDIS, and then generate a range of mutants defective in the synthesis of candidate resistance determinants to define their contribution to the resistant phenotype. We will examine the capacity of the clinical strains and their mutants to prevent C' activation and to bind C' inhibitor proteins that might prevent C5b-9 formation. We will use molecular probes that have been tailored in-house, as well as commercially available antibodies, to visualise by fluorescence microscopy the principal components of the bacterial surface and compare their distribution and abundance with that of the mutants and C'-susceptible GNB. We will consolidate this data into contour maps to provide a detailed topography of the bacterial surface. Finally, the capacity of mutants lacking surface structures that we have identified as contributing to resistance will be examined in rodent models of bacterial infection that we have developed in-house; such in vivo work will define the contribution of major C' resistance determinants to the capacity of the bacteria to cause lethal infection.