title: Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1.
abstract: SPLUNC1 is an abundantly secreted innate immune protein in the mammalian respiratory tract that exerts bacteriostatic and antibiofilm effects, binds to lipopolysaccharide (LPS), and acts as a fluid-spreading surfactant. Here, we unravel the structural elements essential for the surfactant and antimicrobial functions of human SPLUNC1 (short palate lung nasal epithelial clone 1). A unique α-helix (α4) that extends from the body of SPLUNC1 is required for the bacteriostatic, surfactant, and LPS binding activities of this protein. Indeed, we find that mutation of just four leucine residues within this helical motif to alanine is sufficient to significantly inhibit the fluid spreading abilities of SPLUNC1, as well as its bacteriostatic actions against Gram-negative pathogens Burkholderia cenocepacia and Pseudomonas aeruginosa. Conformational flexibility in the body of SPLUNC1 is also involved in the bacteriostatic, surfactant, and LPS binding functions of the protein as revealed by disulfide mutants introduced into SPLUNC1. In addition, SPLUNC1 exerts antibiofilm effects against Gram-negative bacteria, although α4 is not involved in this activity. Interestingly, though, the introduction of surface electrostatic mutations away from α4 based on the unique dolphin SPLUNC1 sequence, and confirmed by crystal structure, is shown to impart antibiofilm activity against Staphylococcus aureus, the first SPLUNC1-dependent effect against a Gram-positive bacterium reported to date. Together, these data pinpoint SPLUNC1 structural motifs required for the antimicrobial and surfactant actions of this protective human protein.
authors.affiliation: Departments of Chemistry, Biochemistry, and Microbiology, University of North Carolina , 4350 Genome Sciences Building, Chapel Hill, North Carolina 27599-3290, United States.
authors.forename: William G
authors.initials: WG
authors.lastname: Walton
authors.affiliation: Marsico Lung Institute, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina , 7102 Marsico Hall, Chapel Hill, North Carolina 27599-7248, United States.
authors.forename: Saira
authors.initials: S
authors.lastname: Ahmad
authors.affiliation: Departments of Chemistry, Biochemistry, and Microbiology, University of North Carolina , 4350 Genome Sciences Building, Chapel Hill, North Carolina 27599-3290, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Little
authors.affiliation: Marsico Lung Institute, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina , 7102 Marsico Hall, Chapel Hill, North Carolina 27599-7248, United States.
authors.forename: Christine S K
authors.initials: CS
authors.lastname: Kim
authors.affiliation: Marsico Lung Institute, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina , 7102 Marsico Hall, Chapel Hill, North Carolina 27599-7248, United States.
authors.forename: Jean
authors.initials: J
authors.lastname: Tyrrell
authors.affiliation: Department of Environmental and Occupational Health, University of Pittsburgh , 331 Bridgeside Point Building, Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Lin
authors.affiliation: Department of Environmental and Occupational Health, University of Pittsburgh , 331 Bridgeside Point Building, Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Y Peter
authors.initials: YP
authors.lastname: Di
authors.affiliation: Marsico Lung Institute, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina , 7102 Marsico Hall, Chapel Hill, North Carolina 27599-7248, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Tarran
authors.affiliation: Departments of Chemistry, Biochemistry, and Microbiology, University of North Carolina , 4350 Genome Sciences Building, Chapel Hill, North Carolina 27599-3290, United States.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Redinbo
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects; D001980:Bronchi / Q000166:cytology / Q000187:drug effects*; D057508:Burkholderia cenocepacia / Q000187:drug effects* / Q000276:immunology; D002478:Cells, Cultured; D003460:Crystallization; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D008070:Lipopolysaccharides / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000276:immunology; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; C485599:BPIFA1 protein, human; D006023:Glycoproteins; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D011663:Pulmonary Surfactants