Sigma-regulator recognition and activation of regulated intramembrane proteolysis by a site-1 protease during iron import cell surface signaling in Gram-negative bacteria. - PROJECT SUMMARY/ABSTRACT Gram-negative bacteria (GNB) play an important role in human health. GNB include species beneficial to humans as well as some of the most dangerous human pathogens, many of which have evolved to produce multi-drug resistant strains as emphasized in the 2019 Antibiotic Resistance Threats Report from the CDC. This underscores the need to better understand essential cellular processes in GNB in order to develop new, more potent and specific therapeutics. All GNB import critical nutrients from their environment using a set of homologous, yet highly specific, outer membrane TonB-dependent transporters (TBDTs). To import metals, especially iron, GNB secrete high-affinity, metal-chelating compounds called siderophores, and iron-laden siderophores are imported by specific TBDTs. A subset of these TBDTs transcriptionally up-regulate their own expression by a cell surface signaling (CSS) process. The objective of the research outlined in this proposal is to determine the structural basis by which a C-terminal processing protease activates CSS. The Pseudomonas capeferrum pseudobactin BN7/8 transport system is our model system. It consists of a cytoplasmic transcription factor or s-factor, PupI, which is localized to the inner membrane by binding to the inner membrane s-regulator, PupR, and a TBDT, PupB. The C-terminal processing protease, Prc, which is also known as a site-1 protease, activates CSS by proteolyzing PupR to release PupI from the membrane. The overall goal of this proposal is to understand the structural basis of this process. This will be achieved by successfully accomplishing three specific aims: 1) demonstrating that the PupB N-terminal signaling domain binds to the PupR C-terminal CSS domain (CCSSD) and prevents recognition by the site-1 protease, Prc, 2) elucidating the mechanism of Prc-mediated proteolysis of the PupR CCSSD, and 3) identifying structural determinants for CCSSD recognition and proteolysis by Prc. These aims will be accomplished using a multidisciplinary approach involving structural biology methods such as X-ray crystallography and small-angle X-ray scattering; biophysical methods such as mass spectrometry, microscale thermophoresis, and isothermal titration calorimetry; and biochemical and molecular biology techniques such as SDS-PAGE, site directed mutagenesis, and proteolysis assays. Thus, this research will provide critical structural information explaining how a s-regulator is recognized and degraded by a site-1 protease during activation of CSS. Ultimately, this information will be useful in designing therapeutics targeting these interactions to modulate TBDT expression. Recently developed siderophore-antibiotic conjugates have been shown to selectively target specific bacteria, thereby circumventing several key antibiotic resistance mechanisms. However, delivery of these antibiotic conjugates is significantly limited by the numbers of each TBDT expressed on the cell surface. Therefore, methods to up-regulate TBDT expression will enhance siderophore-antibiotic conjugate therapy for treatment of serious pathogens including many multidrug-resistant GNB infections.
