Molecular basis of heme scavenging by Gram-positive bacteria

项目总结

Project Summary Nearly all species of bacteria require iron to grow because it is an essential metal cofactor that is used by microbial enzymes to mediate cellular metabolism. During infections, bacterial pathogens forage iron from human hemoglobin (Hb) that is released from red blood cells, which contains ∼75–80% of the human body's total iron content in the form of heme (iron protoporphyrin IX). The basic science studies outlined in this proposal will determine how the human pathogen Corynebacterium diphtheriae acquires iron from Hb. This work will have a broad impact, as C. diphtheriae is a model organism within the Actinobacteria phylum, which contains several species of bacteria that are human pathogens, as well as microbes that are major components of the human gastrointestinal microbiome. Research will be performed by an established team of investigators that have complementary expertise in microbiology, proteomics, biochemistry and structural biology. We will determine how microbial receptors capture Hb and remove its heme, and how cell wall embedded proteins ferry released heme into the cell. In aim #1, we will determine how C. diphtheriae uses the HbpA receptor to capture Hb on the cell surface and test the hypothesis that the receptor works in concert with surface associated heme-receptors to distort Hb and trigger heme release. In aim #2, we will determine the molecular basis through which heme is passed across the cell wall by determining how widely distributed Conserved Region (CR) domains in Actinobacteria directly exchange heme by forming low-affinity transfer complexes. In aim #3, we will obtain a systems-level understanding of the heme uptake process by applying mass spectrometry proteomics methods to determine each component’s abundance and location, and by developing and applying a novel fluorogenic Hb-reporter to track heme removal from Hb in cell culture. These studies will enable us to quantitatively assess the importance of each system component in heme uptake, and to test the hypothesis that they form a molecular wire through which heme flows to the membrane. The results of these studies will provide fundamental insight into how C. diphtheriae and other Actinobacteria acquire heme-iron, and develop generalizable tools to study this process in live bacteria. Combined, the results of this research could lead to new therapeutics to treat infections caused by antibiotic resistant bacteria that work by disrupting heme import.