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Novel imaging modalities to study infectious diseasesDuring infection, the struggle for nutrient metal occurs at sites of interaction between host and pathogen. In the case of S. aureus, this infectious battleground often takes the form of a tissue abscess. Abscesses represent infectious foci that confine bacterial invaders and restrict the spread of infection to neighboring tissue. Abscesses are discrete sites of infection that are distinct from neighboring healthy tissue. We have taken advantage of this macroscopic difference between healthy and diseased tissue to identify metal-binding proteins that are expressed specifically in staphylococcal abscesses. To this end, we pioneered the application of imaging mass spectrometry to study host-pathogen interactions. Specifically, we used matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to image protein (MALDI-TOF-MS) and metal (LA-ICP-MS) distribution in animal models of infectious diseases. Current work is focused on combining these modalities with magnetic resonance imaging (MRI) in an effort to monitor protein expression within infected animals in three dimensions. The novel application of these cutting-edge technologies has provided an unprecedented view into the struggle for metal between bacterial pathogens and their vertebrate hosts. Published in Science, 2008 Feb 15;319(5865):965, (Figure 4). PMCID:2395006 ⇓ |
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Bacterial acquisition of iron from vertebrate bloodIron is required by virtually all bacterial pathogens. Vertebrate hosts have taken advantage of this requirement by sequestering iron in high-affinity binding proteins. Greater than 80% of sequestered iron is complexed as a component of heme, the co-factor of hemoglobin. We have found that S. aureus satisfies its nutrient iron requirement through the acquisition of heme from hemoglobin. In fact, we have shown that heme is the preferred source of iron during staphylococcal infections. In addition, we have identified the staphylococcal machinery responsible for heme-iron acquisition, which we have named the iron-regulated surface determinant system (Isd). The Isd system comprises a “molecular bucket brigade” that recognizes hemoglobin and removes heme (IsdAB), transports heme through the cell wall and inner membrane (IsdCDEF), and degrades heme in the cytoplasm to release free iron for use as a nutrient source (IsdGI). Current and future experiments are focused on testing this model for Isd-mediated heme acquisition. To date, the Isd system represents the only identified system devoted to the transport of substrates across the cell wall of gram positive bacteria. Thus, analysis of the Isd system provides a unique experimental platform for studying the fundamental process of transport across the gram positive cell wall. Considering the conservation of the Isd system throughout gram positive pathogens results obtained from these studies will be applicable across a range of disease-causing microorganisms, including S. aureus, B. anthracis, Listeria monocytogenes, and Clostridium tetani. ⇒ |
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The adaptive response of bacterial pathogens to metal as a component of bloodHeme represents a paradox to infecting bacteria due to its extreme reactivity. Although heme is a valuable nutrient iron source, the intracellular amassing of heme leads to cell death through the accumulation of reactive oxygen species and membrane damage. To cope with this liability, bacteria that utilize heme must have mechanisms for metabolizing excess heme and avoiding toxicity. In this regard, we have found that S. aureus adapts to heme-toxicity through the inducible expression of a novel transport system that we have named the heme regulated transporter (HrtAB). Expression of hrtAB is activated in response to heme by a novel signal transduction system that we have named the heme sensing system (HssRS). Staphylococcal strains lacking either hrtAB or hssRS are acutely sensitive to heme toxicity. The HrtAB and HssRS systems exist in a variety of gram positive pathogens, including S. aureus, B. anthracis, L. monocytogenes, and E. faecalis. Concordantly, we have found that B. anthracis is capable of adapting to heme-mediated toxicity and that this adaptation is entirely mediated by the B. anthracis HrtAB and HssRS systems. Taken together, these findings establish HssRS-mediated activation of HrtAB as a conserved mechanism by which a variety of gram positive pathogens cope with heme stress. Published in Cell Host and Microbe. 2007, Vol 1, p117 (Figure 7).← |
Metal chelation as a host defense against infectionIn order to protect against bacterial infection, vertebrates sequester nutrient metal in a process termed nutritional immunity. We have identified the neutrophil protein calprotectin as being expressed exclusively in abscessed tissue and responsible for inhibiting microbial growth through manganese and zinc chelation. Calprotectin is a heterodimeric protein that comprises 50% of the cytoplasmic protein compartment of the neutrophil. We have shown that the recruitment of calprotectin to the abscess is dependent on neutrophils. Once inside the abscess, neutrophil calprotectin inhibits staphylococcal growth through the chelation of nutrient manganese and zinc. Mice genetically deficient in calprotectin are more susceptible to staphylococcal infection as judged by increased bacterial burden and enhanced abscess formation in comparison to controls. Using LC-ICP-MS-based metal-imaging technologies, we have discovered that abscesses are devoid of detectable manganese and zinc, and calprotectin is required for binding and removal of these metals from the abscess. Current efforts are focused on determining the impact of calprotectin on the host-pathogen interaction and identifying additional host factors involved nutritional immunity.⇒ |
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