The CDC recently released a report detailing antibiotic resistant threats in the US. Of particular emphasis in the CDC report is the increased prevalence of multidrug-resistant, Gram-negative bacteria (MDR- GNB) and the need to develop the next generation of antibiotics to combat them. All Gram-negative bacteria rely on a set of homologous, yet highly-specific, outer membrane TonB-dependent transporters (TBDTs) to import critical nutrients from their environment, especially metals like iron, which are bound by high-affinity, metal chelating compounds called siderophores. Recent antibiotic developments have shown that siderophore-antibiotic conjugates can be selectively targeted to specific bacteria, and that this delivery mechanism overcomes several key antibiotic resistance mechanisms. However, a significant limitation of this delivery system is the low expression levels of the TBDTs. The long-term objective of this proposal is to provide a mechanistic understanding of how Gram-negative bacteria transcriptionally regulate their TBDTs in order to manipulate this process to selectively up-regulate TBDT levels and enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. In this proposal we will elucidate the structural basis for protein interaction events that are responsible for up-regulatin the transcription of particular TBDTs. As a model system we are using the pseudobactin BN7/8 transport system from Psuedomonas putida that consists of the TBDT, PupB, the inner membrane ?-regulator, PupR, and the cytoplasmic ?-factor, PupI. To accomplish our objective we will pursue the following two specific aims: 1) delineate the mechanism by which the PupR:PupI interaction at the cytoplasmic face of the inner membrane is altered to allow transcriptional activation by PupI, and 2) establish the thermodynamics and atomic-level structural details of the interaction between the PupB and PupR. For the successful completion of our aims we will employ a multidisciplinary approach including NMR spectroscopy, X-ray crystallography, molecular biology, cellular assays, and biophysical techniques such as isothermal titration calorimetry. This research will provide the first structural information for a ?-regulator, explain how localization of a ?-factor to the inner membrane limits its activity, and th extent to which periplasmic interactions between the TBDT and ?-regulator lead to conformational changes that might be important for controlling transcriptional activation.
Gram-negative bacteria, especially multi-drug resistant Gram-negative bacteria, are one of the major modern threats to human health. These bacteria rely on a highly evolved set of transporters to import critical nutrients, especially metals like iron, fro their environment that can be exploited for drug delivery. This proposal will, for the first time, investigate atomic details of signal transduction mechanism by which these transporters self- regulate their own numbers, providing a clearer understanding of Gram-negative bacterial survival, and information that may be used in the future rational design of novel therapeutics.
