Molecular mechanisms of enterobacterial resistance to complement

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.

Complement (C') resistance is a major virulence determinant of Gram-negative bacteria (GNB). Although the basis of C' resistance has historically been intensively investigated, many features of the resistant phenotype remain elusive and have not been addressed in recent years, even though new molecular and microscopic techniques have yielded novel insights into the organisation of the outer membrane (OM). We will employ cutting-edge molecular, structural, biophysical and microscopy-based approaches to determine bacterial surface topography that defines C'-resistant pathogens. Resistance results from the capacity of target GNB to avoid deposition of terminal C' (C5b-9) complexes onto the OM by preventing C' pathway activation, degradation of early C' components or elaboration of an OM surface preventing insertion of C5b-9 into the bilayer. We will prepare high density transposon mutant libraries of Escherichia coli and Klebsiella pneumoniae C'-resistant isolates by Transposon Directed Insertion Sequencing (TraDIS) to determine gene products that contribute to the resistance phenotype. Mutants and complemented derivatives will be generated as determined by outcomes of TraDIS screens, to examine the contribution of individual gene products to resistance. Activation of C' by wild type and mutant surfaces will be examined by quantification of binding of early and late C' components and of C' inhibitor proteins. Spatiotemporal patterning of OM surface constituents will be determined by microscopic imaging of protein assemblies with novel fluorescent ligands, lipopolysaccharide and capsules with antibodies, hydrophobic regions susceptible to C5b-9 attack with fluorescent probes and C5b-9 neoepitopes with antibodies. Contour maps will be constructed to provide a detailed topography of the GNB surface. Mutants defective in synthesis of key resistance determinants will be examined in rodent models of systemic infection to determine their contribution to pathogenesis.

The laboratory of the PI, along with others, is searching for novel agents and new therapeutic paradigms for infections caused by highly drug resistant bacteria. He is particularly interested in ways to inhibit or counter the expression of macromolecules at the bacterial surface that confer resistance to the host's innate and immune defences. A more detailed understanding of the nature of bacterial surfaces may provide insights into ways to modify pathogens at the site of infection to allow immune defences to overwhelm the pathogen and prevent progression of the disease process. He and others have accumulated evidence that interruption of capsule expression leads to rapid and early cessation of Gram-positive and Gram-negative infections. There is growing interest in both academic and commercial laboratories to exploit such modalities that disarm the pathogen rather than just kill it, a process that runs the risk of selecting for drug resistant variants.
Data generated within the lifetime of the study will enhance the scientific knowledge base and make a major contribution towards understanding the fine detail of the contours of the Gram-negative surface. The proposal provides an opportunity for postdoctoral training in an interdisciplinary project involving two centres of excellence in infectious disease research and molecular microbiology. Successful candidates will be exposed to state-of-the-art techniques in molecular biology, single-cell imaging, fluorescence assays, flow cytometry and development of robust animal models of bacterial infection. PDRAs will be embedded in a laboratory with a focus on bacterial infections but also heavily involved in other research areas impacting on novel approaches to bacterial chemotherapy. This will provide an opportunity to interact with successful scientists in-house and to spend periods in each other's laboratory in this highly cooperative project. Thus, opportunities for cross-fertilisation of ideas and technologies are very high.