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. A significant limitation of this delivery system is the low expression levels of the TBDTs. However, a subset of these TBDTs controls their own expression through a cell-surface signaling (CSS) process that up-regulates their own expression. The long- term objective of this research is to understand the CSS regulatory process and manipulate TBDT expression to enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. Research outlined in this proposal will help elucidate the structural basis for CSS by a sigma-regulator. As a model system, the pseudobactin BN7/8 transport system of Psuedomonas putida, which consists of the TBDT, PupB, the inner membrane ?-regulator, PupR, and the cytoplasmic ?-factor, PupI, is being used. To accomplish this proposal's objective the following three specific aims will be pursued: 1) establish that PupR anti-?-factor domain dimerization influences transcriptional activation by PupI, 2) identify the structural determinants and delineate the role of the PupR:PupB periplasmic interactions on the stability of the PupR periplasmic C- terminal CSS domain (CCSSD), and 3) determine changes in the full-length PupB:PupR CCSSD complex in the presence and absence of its cognate siderophore, pseudobactin BN7/8.
These aims will be accomplished using a multidisciplinary approach; including X-ray crystallography, small-angle X-ray scattering, molecular biology, cellular assays, and biophysical techniques such as isothermal titration calorimetry and circular dichroism spectroscopy. This research will provide critical structural information about a ?-regulator; explain how it interacts with a ?-factor at the inner membrane, and the extent to which periplasmic conformational changes between the TBDT and ?-regulator lead to proteolytic degradation that is important for controlling transcriptional activation.

Public Health Relevance

Gram-negative bacteria, especially multi-drug resistant Gram-negative bacteria, are one of the most important modern threats to human health. These bacteria rely on a set of transporters that can be exploited for drug delivery to import critical nutrients, especially metals like iron, from their environment. This proposal will investigate atomic details of the signal transduction mechanism by which Gram-negative bacteria respond to stimuli outside the cell to regulate expression of these transporters, providing a clearer understanding of bacterial survival, and information that may be used in the future rational design of novel therapeutics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Sledjeski, Darren D
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North Dakota State University
Schools of Arts and Sciences
United States
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