All bacterial pathogens must acquire nutrient metals within their hosts in order to colonize and cause disease. Vertebrates have taken advantage of this requirement by evolving high-affinity metal binding proteins that sequester metals and prevent bacterial growth in a process known as "nutritional immunity". To compete with nutritional immunity, bacteria sense alterations in metal levels and coordinate gene expression changes that enable adaptation to this environmental flux. Although the bacterial regulatory circuits that are activated in response to altered metal levels have been described in vitro, when and where bacteria experience metal stress during vertebrate infection is not known. In addition, the complete catalogue of host proteins that contribute to nutritional immunity has not been defined. In this application, we propose to fill these gaps in knowledge through the application of multi-modal imaging modalities to murine models of Staphylococcus aureus infection. S. aureus is chosen for these experiments because it is the leading cause of infection in the United States and our laboratory is experienced in murine models of staphylococcal systemic infection, osteomyelitis, and pneumonia. The diversity of these infection models will provide valuable information regarding the contribution of nutritional immunity to infection at a variety of distinc sites. To achieve a whole-animal three-dimensional image of the struggle for metal between host and pathogen, mice will be infected with S. aureus and sequentially subjected to a series of distinct imaging modalities. To observe anatomical changes that occur in whole animals following infection, we will employ magnetic resonance imaging (MRI) and computed tomography (CT). To define bacterial metal-dependent gene expression within infected animals we will use in vivo bioluminescence imaging (BLI). To study the impact of infection on protein and elemental abundance and distribution within whole animals, we will use imaging mass spectrometry (IMS) which we have recently pioneered for the study of infectious diseases. Data obtained from each of these imaging modalities will be co-registered into a single three-dimensional image using computational analysis tools that we have recently developed. Combined, these data will define the impact of infection on metalloprotein distribution and metal abundance and determine how bacteria respond to these changes. These data will lay the foundation for the rational design of therapeutics that target nutrient metal acquisition. In addition, the technologies developed as a result of these experiments will be applicable to all physiologically relevant processes that can be studied using animal models.
This proposal will lead to the development of new imaging modalities that will enable us to view the molecular components of infectious disease with unprecedented resolution. Results obtained as a result of these studies will uncover new targets for therapeutic intervention and antibiotic development. Moreover, the techniques developed as a result of this proposal will be broadly applicable to all physiologically relevant processes, profoundly impacting biomedical research.
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