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

Abstract

The opportunistic bacteria of the Burkholderia cepacia complex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, including B. cenocepacia strain J2315 (50% inhibitory concentration [IC 50 ] ‫؍‬ 0.28 M), and reduced biofilm formation and attachment (IC 50 ‫؍‬ 0.11 M). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and ␣6 helix did not affect its function. However, deletion of the ␣4 helix attenuated antimicrobial activity, while the corresponding ␣4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease.

Crystal structure of the mouse innate immunity factor bacterial permeability-increasing family member A1

“…6c). This structural observation supports the hypothesis that the 4 motif is involved in the unique antibacterial activities of the BPIFA1 proteins, which have not been reported for latherin or Der f 7 (Walton et al, 2016;Ahmad et al, 2016;Emsley & Cowtan, 2004;Tan et al, 2012).…”

“…Bacterial permeability-increasing family member A1 (BPIFA1) is a member of the bacterial permeability-increasing (BPI) superfamily of proteins (Bingle et al, 2011), and has previously been known as short palate lung nasal epithelial clone 1 (SPLUNC1), secretory protein in upper respiratory tracts (SPURT), lung-specific X protein (LUNX) and nasopharyngeal carcinoma-related protein (NASG). BPIFA1 is one of the most abundantly secreted proteins in the upper airways of the lung, and demonstrates activities as a bacteriostatic agent, surfactant and lipopolysaccharidebinding factor (Liu et al, 2013;Di, 2011;Wei et al, 2014;Jiang et al, 2013;Chu et al, 2007;Walton et al, 2016;Ahmad et al, 2016). BPIFA1 employs these varied functions to maintain normal lung function by, for example, spreading fluid across the pulmonary epithelium and maintaining sterile airways (Tarran & Redinbo, 2014).…”

Role of CFTR in epithelial physiology

“…Indeed, it has been shown that the activity of molecules such as the protease inhibitor SPLUNC1, which regulates antimicrobial peptides (AMPs) and shows itself antimicrobial activity [97], depends on optimal ASL pH [36, 98], as does the activity of AMPs themselves [34]. On a more general level, when pathogens enter the airways, they are recognised by epithelial cells that activate inflammatory pathways to recruit immune cells, specifically neutrophils, into the airway lumen to eradicate the infection.…”

pH Dependent Antimicrobial Peptides and Proteins, Their Mechanisms of Action and Potential as Therapeutic Agents

“…More recent studies have shown that the Human Short Palate Lung Nasal Epithelial Clone 1 (SPLUNC1), which is a protein expressed in the upper airways of the lung, plays multiple roles in pulmonary innate immunity. These roles include: the direct inhibition of bacterial growth, the prevention of microbial biofilm formation and the regulation of other AMPs and antimicrobial proteins, such as LL-37, HBD-2 and lysozyme [173,174,175,176]. However, in CF, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to reduced HCO 3 − secretion and produces an abnormally acidic pH in ASL [177,178], which studies on humans and animal models have suggested can negatively affect the efficacy of ASL antimicrobial molecules and predispose CF airways to microbial infection (Figure 3) [165,179].…”