Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1

Area of Science:

• Biochemistry
• Immunology
• Structural Biology

Background:

• Short palate lung nasal epithelial clone 1 (SPLUNC1) is a secreted innate immune protein in the respiratory tract.
• SPLUNC1 exhibits bacteriostatic, antibiofilm, lipopolysaccharide (LPS)-binding, and surfactant properties.

Purpose of the Study:

• To identify the structural elements responsible for the surfactant and antimicrobial functions of human SPLUNC1.
• To elucidate the role of specific structural motifs in SPLUNC1's interaction with LPS and bacterial pathogens.

Main Methods:

• Site-directed mutagenesis of a unique alpha-helix (α4) and disulfide bond formation to assess functional impact.
• Analysis of fluid-spreading surfactant activity, bacteriostatic effects against Gram-negative bacteria, and antibiofilm activity.
• Crystallography to confirm surface electrostatic mutations.

Main Results:

• The α4 helix is essential for SPLUNC1's bacteriostatic, surfactant, and LPS-binding activities.
• Mutations in α4 significantly impair fluid spreading and bacteriostatic action against Burkholderia cenocepacia and Pseudomonas aeruginosa.
• Conformational flexibility and specific surface electrostatic mutations (away from α4) also influence SPLUNC1's antimicrobial functions, including novel antibiofilm activity against Staphylococcus aureus.

Conclusions:

• Specific structural motifs, particularly the α4 helix, are crucial for SPLUNC1's multifaceted antimicrobial and surfactant roles.
• SPLUNC1's functional versatility extends to antibiofilm activity against Gram-positive bacteria through distinct structural elements.
• Understanding these structural-functional relationships enhances our knowledge of innate immunity in the respiratory tract.