Elucidating the Mechanism of Heme Transport across the Outer Membrane of Gram-neg

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DESCRIPTION (provided by applicant): The proposed project is part of an ongoing effort to understand the roles played by heme uptake by and transport within bacterial pathogens. The long-term goal of this work is to elucidate the mechanistic parameters that govern the specificity and efficacy of heme transfer among the uptake and transport proteins. The relevance of this work to human health lies in its potential to spawn new strategies for treatment of bacterial infections. Inhibition of heme uptake and/or transport could provide alternative treatments for antibiotic-resistant infections. The next phase of this project will focus on elucidating the mechanistic details of two classes of outer membrane heme receptors. This will be accomplished through a combination of stopped flow spectrophotometry and freeze-quench resonance Raman (FQrR) spectroscopy in conjunction with site directed mutagenesis. The reason for this focus is that these proteins carry out the first step toward internalization of heme. Thus, if heme-dependent mechanisms for iron assimilation are to be inhibited via clinical intervention, these receptors would appear to be the best first targets as they reside at the cell surface. The receptors to be targeted in our mechanistic studies are the TonB- dependent ShuA and SmHasR receptors from Shigella dysenteriae and Serratia marcescense, respectively. The specific aims of the proposed project are: 1. to elucidate the mechanistic details of heme uptake into the extracellular binding pocket of ShuA. ShuA has been shown to take heme from hemoglobin and we have recently shown that it binds free heme. Rates and mechanisms of heme uptake by ShuA(WT) and its conserved His mutants from these two sources will be compared and contrasted in an effort to establish the order of axial ligand exchange steps and quantify the mechanistic importance of protein-protein complexation in the efficiency of loading the extracellular binding pocket. 2. to elucidate the mechanistic details of heme uptake into the extracellular binding pocket of HasR. Has R has been shown by ITC to take heme from its cognate secreted hemophore, HasA, and to bind free heme. In a fashion similar to that described for ShuA, the kinetics of these reactions will be compared and contrasted in order to shed light on the importance of the HasA-HasR association to the efficiency of this first step in heme assimilation. PUBLIC HEALTH RELEVANCE: Bacterial infections in human and animal hosts continue to undermine public health the world over, both directly and through their deleterious effects on the food supply. Among the deterministic factors for establishing infection in the host is the availability of iron. Many bacterial pathogens produce proteins that tap the huge pool of heme, the iron-containing pigment in the blood of the host, to acquire iron. This advantage is taken by excising heme from host proteins, internalizing it, and excising the iron. Importantly, these organisms can starve for iron if their heme assimilation system is compromised. As iron starvation undermines their ability to colonize the host, the proteins critical to their acquisition and intracellular transport of heme are potential targets for prophylactic and/or therapeutic treatments against bacterial infection. This study aims to lay a foundation for targeting these proteins by building a detailed understanding of how they function.