Chemical Biology of Nitroxyl (HNO) in Bacillus Subtilis

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PROJECT SUMMARY Many pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Bacillus anthracis and Mycobacterium tuberculosis, mobilize sulfur through redox conversion of sulfate (SO4-) to hydrogen sulfide (H2S) that supports cysteine biosynthesis for protein and low molecular weight (LMW) thiol production. These redox reactions are required for normal metabolic processes. The versatile chemistry of nitrogen and sulfur allows their participation in many biochemical redox pathways; as intermediary reactive nitrogen and sulfur species (RNS and RSS), they often act together with reactive oxygen species (ROS). Various lines of evidence indicate nitroxyl (HNO) sits at the intersection of RNS and RSS crosstalk and this intersection inspires the proposed work. The interplay of bacterial sulfur mobilization and human health remains poorly explored but presents an opportunity for understanding chemical redox biology and the exploitation of new antibiotic strategies. The goal of the proposed research consists of defining HNO’s role in RSS and RNS crosstalk and its significance in microbial physiology. The proposed mechanistic study combines several approaches to define the reactivity of the key chemical components—HNO, H2S, and bacillithiol, (BSH)—in vitro and in Bacillus subtilis, a model organism capable of endogenous nitric oxide (NO) and H2S generation that produces BSH as its predominant LMW thiol. In conjunction with these chemical investigations, biochemical studies will delineate HNO’s effect on phenotypic outcomes. This research goal is based on the hypothesis that “Bacteria lacking BSH demonstrate redox sensitivity to HNO”. This hypothesis will be probed using synthetic organic and analytical chemistry to define the reactivity of BSH and HNO both in vitro and in vivo by identifying the products of this reaction including the sulfinamide and the persulfide modified versions of BSH (Specific Aim 1). Microbiological growth curve experiments will show the effect of HNO on B. subtilis phenotype and analytical biochemistry will reveal the amount of oxidized thiol content. Specific Aim 2 builds on Specific Aim 1 to design and synthesize compounds that inhibit BSH biosynthesis as potential antibiotics activated in the presence of an HNO donor. This aim will be approached by organic synthesis, analytical and mechanistic biochemistry, and standard microbiological methods. Our team is well poised to address these questions and Wake Forest University provides an ideal setting for undergraduate training at the interface of a strong undergraduate college and small graduate program. Achieving a complete understanding of this fundamental chemistry will inform a wide range of physiological responses modulated by HNO-thiol reactions and potentially lead to the development of HNO-based antibiotics that target microbial sulfur metabolism/biochemistry not present in humans.