The prevalence of antibiotic resistance among pathogenic bacteria has become a major health concern and has spurred the search for novel antibiotic targets. A particularly promising target is the superfamily of bacterial ATP-binding cassette (ABC) transporters, which couple the hydrolysis of ATP to the transport of a wide variety of solutes across the cell membrane. Bacterial ABC transporters work in conjunction with a high affinity solute binding protein (SBP) that specifically binds substrate and delivers it to te transporter. In Salmonella enterica and Streptococcus pneumoniae among others, disruption of genes encoding ABC transporters and SBPs specific for Zn or Mn dramatically attenuates virulence in animal models, highlighting these systems as potent drug targets. In a number of bacteria, a fourth, uncharacterized gene is located adjacent to those of Zn or Mn ABC transporter systems. It is hypothesized that this protein is required for optimal import of metals, potentially by acting as a metal chaperone or adaptor between the ABC transporter and the solute binding protein. This project seeks to characterize such an ABC transporter system and its putative accessory protein in Paracoccus dentrificans as a model for highly homologous systems in the human pathogens Klebsiella pneumoniae and enterobacter aerogenes. These organisms are associated with broad-spectrum antimicrobial resistance and the causative agents of potentially deadly nosocomial infections. The accessory protein (Pden1598) as well as the putative solute binding protein (Pden1597) will be characterized in vitro for their metal binding and transfer properties. The stoichiometry and identity of bound metal will be assessed using ICP-MS and binding affinities for relevant metals will be determined using a spectroscopic assay or by isothermal titration calorimetry (ITC). Protein-protein interactions and metal transfer between proteins will be assessed through incubation of holo- and apo-proteins followed by chromatographic separation and determination of metal content. Alternatively, spectroscopic techniques such as extended X-ray absorption fine structure (EXAFS) may be used. Crystal structures of Pden1598, Pden1597 and any stable complex that forms between them will be solved, providing structural information on the metal binding environment and mechanisms of metal binding and transfer. Finally, the role of both proteins in metal import in vivo will be determined by generating knockouts in P. denitrificans and assessing bacterial growth in Zn- and Mn-limiting conditions. Such structural and functional information will yield new insight into the mechanisms of transition metal import in bacteria and potentially provide a basis for the rational design of metal uptake inhibitors as antibiotics.
The widespread emergence of antibiotic resistance in bacteria has placed a renewed emphasis on the identification of more effective antibiotics. ABC transporter systems, which enable pathogenic bacteria to acquire metals and other essential nutrients from the host, represent a new class of antibiotic target. This project aims to characterize one such system and its accessory factors, providing structural and functional information that may eventually lead to the production of effective remedies for antibiotic resistant infections.