The Functional Consequences and Therapeutic Potential of Iron Uptake-Associated Systems in Escherichia coli (2012)

The proliferation of antibiotic-resistant Gram-negative strains presents the challenge of untreatable bacterial infections, underscoring the need for therapeutic approaches with new microbial targets. This dissertation identifies such targets in the iron-acquisition pathways of Gram negative bacteria, and explores previously unappreciated mechanisms by which natural products associated with these pathways act as virulence determinants. To circumvent iron-limiting conditions within the mammalian host, bacteria have evolved high affinity iron-uptake systems consisting of iron-scavenging molecules called siderophores and their cognate receptors. Iron-siderophore transport through the cognate receptor requires energy transduction by the inner membrane-anchored adaptor protein TonB. Over five hundred different siderophore types spanning a range of chemical classes and physicochemical properties have been ascribed the singular function of iron chelation. The selective pressures driving this vast chemical diversity among siderophore types, and their relationship to disease, are unclear. Studies described in this thesis outline how pathogenic Escherichia coli elaborate their iron-acquisition machinery to promote infection. The thiazoline siderophore yersiniabactin has been specifically described among disease causing isolates of uropathogenic E. coli and other Enterobacteriaceae as a virulence determinant. A new mass spectrometric screen reported in this thesis reveals that yersiniabactin strongly binds urinary copper to form a novel cupric-yersiniabactin complex. Levels of this complex exceed those of ferric-yersiniabactin in mice and humans with E. coli urinary tract infection. A strong correlation between yersiniabactin expression and copper resistance in a clinical E. coli isolate collection suggests a protective function for this copper-binding activity. Chemical and culture-based experiments reveal that yersiniabactin promotes bacterial copper resistance by sequestering toxic copper (II) and preventing its catechol-mediated reduction to copper (I). Findings in this dissertation also demonstrate that cupric-yersiniabactin is a superoxide dismutase mimic, a catalytic property which protects these pathogens from killing by macrophages. Density functional theory (DFT) simulations support yersiniabactin as a facile copper ligand and provide a structure-activity based explanation for cupric-yersiniabactin's SOD activity. Yersiniabactin-expressing pathogens could be therapeutically targeted by exploiting its associated TonB-dependent transporter FyuA, which is upregulated during infection and is often virulence-associated. This dissertation additionally focuses on investigating FyuA and other outer membrane TonB-dependent proteins expressed by E. coli as rational candidates for the development of a new, virulence-targeted bactericidal agent. Using in silico methods, this dissertation identifies multiple compounds that exert TonB-dependent bactericidal effects in vitro against multiple Gram-negative strains and species. These first-generation compounds represent the first drug-like, TonB-dependent bactericidal small-molecules. A common feature of these chemical structures is a tetracyclic scaffold that can be optimized for the rational design of a bactericidal agent with a highly specific method of action. The continued emergence of antibiotic-resistant Gram-negative strains necessitates new biomarkers and therapeutic targets for treatment of infection. This dissertation advances our understanding of the multi-functional roles that could be fulfilled by pathogen-associated secondary compounds. Understanding the mechanisms by which siderophores neutralize host countermeasures may suggest additional infection prophylaxis strategies and biomarker candidates. This dissertation additionally yields an improved understanding of how an important microbial function, iron acquisition, can be exploited as target for a new generation of virulence-targeted therapeutics. Identifying a bactericidal agent with a new mechanism of action against multiple Gram-negative strains and species is an important step in the process of finding alternative therapeutic strategies active against antibiotic resistant infections. The general importance of siderophore expression and TonB-dependent transport to microbial infection pathophysiology suggests that discoveries outlined in this work may be broadly applicable to other infectious diseases, many of which are less amenable to translational studies in humans.