Uncoupling Substrate Specificity for Heme and Antimicrobial Peptide Binding and Import by the Multifunctional SAP Transporter

Project: Research project

Project Details

Description

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
StatusActive
Effective start/end date3/1/192/29/24

Funding

  • Research Institute at Nationwide Children's Hospital (700191-0221-00//5R01AI139519-02)
  • National Institute of Allergy and Infectious Diseases (700191-0221-00//5R01AI139519-02)

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