The goal of the proposed research is to investigate how pathogenic organisms recognize and circumvent the host innate immune response, a mechanism crucial to the evolution of antimicrobial peptide (AMP) resistance. Non-typeable Haemophilus influenzae (NTHI) utilizes the ABC transport system, the Sap transporter (sensitivity to antimicrobial peptides), to shuttle host-derived antimicrobial peptides into the cell’s cytoplasm. Dr. Pinkett and her lab solved the structure of the SapA periplasmic binding protein (PBP), which delivers substrate to the Sap transporter. For this proposal, Dr. Pinkett and lab will continue structural and biochemical studies of the Sap transporter to define the mechanism of resistance. In aim 1, the Pinkett lab will conduct site-directed mutagenesis of the SapA binding pocket and identify which residues are involved in heme and/or AMP binding through gel-shift assays. The mutagenesis studies coupled with crystallization of SapA bound to the substrate heme will elucidate the selectivity mechanism of the Sap transporter. In Aim 2, Dr. Pinkett will express and purify components of the Sap transporter and define assembly. The transporter will be reconstituted into proteoliposomes for subsequent transport assays. This series of experiments will identify which components are necessary to shuttle heme or AMPs across the membrane and into the cytoplasm for subsequent degradation. This will allow us to understand how the transporter assembles and how this mechanism allows the organism to regulate transport of AMPs to circumvent the host innate immune response
|Effective start/end date||3/1/19 → 2/29/24|
- Research Institute at Nationwide Children's Hospital (700191-0222-00//5R01AI139519-03)
- National Institute of Allergy and Infectious Diseases (700191-0222-00//5R01AI139519-03)
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