Self-assembly of human defensin 6 and its role in innate immunity and siderophore-based strategies to target Gram-Negative bacteria

Abstract

The first part of this thesis focuses on studies of structural and functional properties of human defensin 6 (HD6). This peptide is produced and secreted by intestinal Paneth cells as a part of innate immune response to invading microbes. Instead of killing microbes, HD6 self-assembles into higher-order oligomers to entrap and prevent microbes from invading into the host cells. This activity of HD6 is unusual among defensins. Herein, we employed biophysical and biological techniques to decipher the molecular details of the unusual behavior of HD6. We demonstrate that the self-assembly of HD6 is driven by hydrophobicity and this work highlights how variable amino acid sequences among defensins afford different biological function. We further elucidated how HD6 is stored in Paneth cells such that its self-assembly is suppressed. Similar to zymogens, HD6 utilizes a pro region to control its self-assembly and upon secretion, trypsin cleaves the propeptide to unleash mature HD6 and trigger host-defense function. We also discovered that HD6 suppresses virulence traits of Candida albicans, an opportunistic fungal pathogen. This study expands the scope of the broad-spectrum function of HD6. In the second part of this thesis, we present two siderophore-based strategies that target Gram-negative bacteria. Siderophores are small-molecule iron chelators that bacteria employ to sequester iron from the environment. Our approaches focus on enterobactin (Ent) and its glycosylated derivatives (GIcEnt), which are virulence factors of certain enteric pathogens, such as Salmonella spp. The first approach relies on the use of GlcEnt-[beta]-lactam conjugates to target these pathogens. In addition to enhanced uptake efficiency of the drug into bacteria, GIcEnt specifically delivers the antibiotic to pathogens and leave commensal bacteria unaffected. In the second approach, we aim to use Ent/GlcEnt-specific antibodies to inhibit bacterial iron acquisition, and thereby prevent the bacteria from colonizing in the host. We demonstrate that antibodies against Ent/GlcEnt are produced in the mice immunized with a protein-siderophore conjugate. These mice exhibit reduced intestinal colonization, reduced systemic dissemination of S. Typhimurium, and increased resistance against the challenge of this pathogen.