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 reduce the fluid spreading abilities of SPLUNC1, as well as its bacteriostatic actions against the Gram-negative pathogens Burkholderia cenocepacia and Pseudomonas aeruginosa. Conformational flexibility in the body of the 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, are shown to impart antibiofilm activity against Staphylococcus aureus, the first SPLUNC1-depenent 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.

Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

“…These data suggest that susceptibility to SPLUNC1 is strain dependent. SPLUNC1 has previously been shown to bind to bacterial lipopolysaccharide (LPS) (22,32). The LPS composition varies between the different Bcc strains, which may alter its susceptibility to SPLUNC1.…”

“…SPLUNC1 has previously been shown to exhibit antibiofilm activity against Gram-negative bacteria (22,30,32). To determine whether SPLUNC1 exerts antibiofilm activity against B. cenocepacia, increasing concentrations of SPLUNC1 were coincubated with 10 6 CFU/ml J2315 for 24 h. Biofilm biomass was then measured by crystal violet staining.…”

“…Since SPLUNC1 was effective against J2315, we then sought to determine which domains of SPLUNC1 were responsible for its antimicrobial activity. SPLUNC1's N-terminal S18 region is responsible for regulating ENaC (25), and we recently showed that deletion of the ␣4 helix resulted in attenuated antimicrobial activity (32). However, the following additional peptides and mutants of SPLUNC1 were generated to test whether the antimicrobial and antibiofilm activities were localized to a specific region of SPLUNC1: (i) the S18 peptide (residues G22 to A39), an N-terminal region containing only the ENaC inhibitory domain; (ii) the ⌬44 mutant (residues T45 to V256), which lacks the SPLUNC1 N terminus, including the S18 region; (iii) the ⌬␣4 mutant, which lacks the ␣4 helix (residues I76 to I105 and includes nonnative Gly-Ser-Gly-Ser linker to L75 to I106); (iv) a peptide corresponding to the ␣4 helix (residues K77 to L101); (v) the ⌬␣6 mutant, in which the ␣6 helix (residues I242 to V256) is absent; and (vi) the ␣6 helix peptide (residues I242 to V256) (Fig.…”

“…Furthermore, SPLUNC1 knockout mice are more susceptible to Klebsiella pneumoniae and P. aeruginosa infections (26,30). While SPLUNC1 has antimicrobial activity against P. aeruginosa, Haemophilus influenzae, and K. pneumoniae (22,26,30,31), its effects against Bcc strains have only recently been examined and are not fully understood (32).…”

“…Although NE is needed for killing of Gram-negative bacteria (39), increased levels of NE have been shown to cleave SPLUNC1 and have been proposed to impair airway epithelial defenses (25,31). We recently showed that SPLUNC1 affects Burkholderia cenocepacia J2315 (32). However, little is known about SPLUNC1's ability to affect different Bcc strains.…”