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Structural and Molecular Basis of Heme Acquisition by the Gram-positive Pathogens Staphylococcus aureus and Listeria monocytogenes


The work described in this dissertation examines how surface receptors in the Gram-positive pathogens Staphylococcus aureus and Listeria monocytogenes acquire heme from human hemoglobin (Hb). Specifically, it describes studies of conserved NEAr Transporter (NEAT) domains present in these Gram-positive pathogens that mediate heme and hemoglobin (Hb) binding. The majority of iron in the human body is located in heme (ferrous protoporphyrin IX) that is in turn, bound to human hemoglobin within erythrocytes. Since iron is an essential nutrient for microbial growth, both S. aureus and L. monocytogenes lyse red blood cells and acquire heme bound iron. Research described in chapters 2 and 3 investigated the structural basis through which S. aureus uses the IsdH protein to bind to Hb and extract its heme. We show that the receptor uses a bi-NEAT domain unit to efficiently extract heme and we define the molecular basis through which IsdH binds to Hb.

Chapter 4 in this thesis describes biochemical and structural studies of the heme acquisition system present in L. monocytogenes. This microbe expresses two poorly characterized NEAT containing proteins, Hbp1 and Hbp2, which have been previously implicated in heme acquisition. Our results indicate that Hbp2 functions as a hemophore that binds extracellular heme and then delivers it to Hbp1, a cell wall-associated hemoprotein. We demonstrate that Hbp2 scavenges heme using three NEAT domains that each bind heme with high affinity. Moreover, the mechanism of heme binding to the central NEAT domain in Hbp2 (Hbp2N2) is elucidated by determining the structure of Hbp2N2 and the Hbp2N2-heme complex using X-ray crystallography. The combined results of this work enable a model of the hemin acquisition mechanism of L. monocytogenes to be constructed and to reveal a novel mode of heme binding by a NEAT domain. The results of the structural and biochemical experiments presented in this thesis shed light onto the mechanism used by Gram-positive pathogens to plunder host heme and could facilitate the development of novel anti-infective agents that work by preventing heme uptake.

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