Background
Cystic fibrosis (CF) is a genetic, multi-organ disease that results from loss in function of the CF transmembrane conductance regulator (CFTR)-anion channel. Subsequently, diminished anion secretion results in dehydration and loss of pathogen clearance, causing an increase in chronic bacterial infections and inflammation in the lower airways, and eventual reduction in lung function [
1,
2]. Indeed, over 80% of the CF population ultimately succumbs to respiratory failure [
3]. Respiratory infections start early in life, with
Staphylococcus aureus and
Haemophilus influenza predominantly colonizing the lungs of CF children, and
Pseudomonas aeruginosa colonization occurring in older children and teenagers [
4]. The
Burkholderia cepacia complex (Bcc), which consists of 18 genetically distinct Gram-negative species and is relatively harmless in normal individuals, but especially virulent in CF patients [
5‐
8]. The epidemic
B. cenocepacia lineage ET-12 (strain J2315), is predominantly found in European and Canadian CF populations [
6]. Rather than colonizing the airways, it leads to cellular invasion and septicemia called Cepacia syndrome that is associated with a rapid decline in health and significantly increased mortality [
9,
10]. Moreover, J2315 are multidrug resistant, making treatment difficult [
1,
11,
12] and CF patients with J2315 infections are usually not considered for lung transplants as survival rates after transplantation are low [
13].
The short palate, lung, and nasal epithelial clone (SPLUNC1; gene name BPIFA1; also referred to as PLUNC, SPURT, NASG, and LUNX) is found in human saliva, sputum, nasal fluid, and bronchoalveolar lavage (BAL) at concentrations of up to 10 μM [
14,
15]. SPLUNC1 shares structural homology to the bactericidal/permeability-increasing (BPI) proteins and increases in concentration during Th1 inflammation. SPLUNC1 has surfactant-like and antimicrobial-activity against Gram-negative bacteria, such as
P. aeruginosa, Klebsiella pneumonaie, and J2315 [
15‐
21]. SPLUNC1 knockout mice infected with
P. aeruginosa are unable to clear bacterial infections [
17]. Additionally, SPLUNC1 maintains airway hydration through negative regulation of the epithelial sodium channel (ENaC). Knockdown of SPLUNC1 in human bronchial epithelial cultures (HBECs) leads to airway surface liquid (ASL) dehydration and in chinchilla models decreases mucociliary clearance [
22,
23]. We previously solved the crystal structure of SPLUNC1 and found that it possesses salt bridges that enables it to interact with ENaC in a pH-dependent fashion [
19]. These charged surfaces also maintain antimicrobial activity in a pH-dependent manner [
24]. Importantly, SPLUNC1 fails to bind to the apical surface of CF HBECs at acidic pH (≤ pH 7) and its failure to bind to ENaC at the acidic ASL pH contributes to Na
+ hyperabsorption and airway surface liquid dehydration in CF airways [
19]. As such, incapability of the CF lung’s innate defense system leads to chronic colonization with opportunistic bacteria [
25].
In normal airways, SPLUNC1 binds to the β-subunit of ENaC. While the αγENaC become internalized, βENaC remains at the apical membrane of HBECs and bound to SPLUNC1 to form a SPLUNC1-βENaC complex [
26]. SPLUNC1 has anti-J2315 effects in cell-free systems [
20] but its relevance in vivo, and to CF pathogenesis remains unclear. Here, we further explored the physiological role of the cell surface SPLUNC1-βENaC complex and we tested the hypothesis that a failure of SPLUNC1-ENaC interactions had adverse consequences on innate defense in the lung against J2315 infections.
Discussion
While relatively harmless to normal people, Bcc are an extremely virulent group of pathogens in immunocompromised patients, including those with CF. Infection with these pathogens often results in Cepacia syndrome, which is characterized by necrotizing pneumonia and septicemia followed by death [
8,
10]. Unlike other Gram-negative bacteria (e.g.
P. aeruguinosa) that tend to chronically colonize CF mucus, Bcc are invasive and rapidly enter the epithelia [
8]. Why CF patients are so susceptible to this organism is not known. However, the airways’ innate immune system provides the first line of defense against invading pathogens, and utilizes phagocytic cells, cytokines, mucins and AMPs to prevent bacterial colonization [
33,
34]. Whilst AMPs such as LL-37 and human beta defensins have been extensively studied [
12,
35], less is known about SPLUNC1’s antimicrobial abilities. However, here we demonstrated that SPLUNC1 played a hitherto underestimated role in preventing J2315 infection. Mice lacking SPLUNC1 (SPLUNC1
−/−) were significantly more susceptible to J2315 with increased bacterial growth than their wild-type littermate controls (Fig.
1), which is consistent with previous reports of increased susceptibility to
P. aeruginosa and
K. pneumonaie [
16,
17]. Additionally, stable knockdown of SPLUNC1 expression in normal HBECs similarly resulted in increased J2315 burden, while addition of rSPLUNC1 to the ASL reduced J2315 levels, suggesting that in vivo effects of SPLUNC1 on J2315 are reprised in vitro (Fig.
2). As part of the innate immunity, SPLUNC1 also increases mucocilliary clearance that can reduce bacterial burden in the airways [
15,
17,
21]. Indeed, mice that overexpress SPLUNC1 have increased protection against
Mycoplasma pneumonia and
P. aeruginosa [
36,
37]. Given the extent of J2315 burden in SPLUNC1
−/− mice and SPLUNC1 knockdown HBECs, we propose that SPLUNC1, therefore likely plays a pivotal role in preventing J2315 infection in mammalian airways.
Consistent with recent studies on the effect of SPLUNC1 on J2315 [
20,
21,
24], our data indicated that SPLUNC1 limited J2315 burden in the extracellular milieu independently of ENaC expression levels. However, SPLUNC1 in the absence of βENaC failed to limit J2315 invasion (Fig.
3). Thus, the interaction between βENaC and SPLUNC1 at the plasma membrane appeared necessary to reduce J2315 invasion. SPLUNC1 is a multifunctional protein, which has different domains that perform different functions. Its S18 domain interacts with ENaC [
38], the α6 domain interacts with the Orai1 Ca
2+ channel, while the α4 domain contains its antimicrobial activity [
21,
24,
39]. We have previously shown that αβγENaC dissociates once SPLUNC1 binds to βENaC, resulting in αγENaC being internalized while βENaC remains at the plasma membrane bound to SPLUNC1 [
26]. J2315 interacts with epithelial cells’ glycolipid receptors via its cable pili [
40] resulting in intracellular invasion through membrane-bound vacuoles or rearrangement of cytoskeleton [
41,
42]. However, SPLUNC1 is abundantly found in the airways and can bind to the lipopolysaccharide (LPS) of Gram-negative bacterial membranes [
18] to inhibit bacterial adhesion, and endocytosis into epithelial cells. Indeed, inhibition of J2315 attachment to epithelial cells by dextran resulted in reduced virulence in the lungs [
43]. We therefore speculate that αγENaC served as a chaperone to bring βENaC to the plasma membrane, where it is presented to SPLUNC1, which simultaneously serves as an antimicrobial shield to prevent bacterial internalization.
Despite being effective in normal HBECs, SPLUNC1 failed to affect J2315 burden in the airway surface liquid of CF HBECs and was also ineffective at reducing cellular invasion into CF cultures (Fig.
4). SPLUNC1 contains salt bridges that are pH-sensitive, which renders SPLUNC1 nonfunctional in acidic conditions such as that seen in CF HBECs. Non-functional SPLUNC1 is unable to bind to ENaC [
1] resulting in less SPLUNC1 at the apical membrane of CF HBECs. In normal conditions, SPLUNC1 binds to βENaC and remains at the plasma membrane [
2]. Additionally, SPLUNC1 fails to reduce bacterial burden in the acidic pH of the CF airways [
24]. Thus, we hypothesize that the abnormal pH in CF airway surface liquid leads to a double hit in that it (i) directly reduces SPLUNC1’s antimicrobial activity and (ii) prevents SPLUNC1 from binding to ENaC at the apical plasma membrane. These failures of SPLUNC1’s antimicrobial actions, combined with a failure to clear mucus, reduce CF airway epithelia’s ability to prevent J2315 invasions. Similar characteristics have been seen with other antimicrobial proteins: Attacin and collectin will bind to the LPS and remain in the outer membrane of Gram-negative bacteria to reduce bacterial burden [
44,
45]. Other peptides, such as CdsN peptide, have been shown to disrupt protein interaction of bacteria’s type III secretion system preventing bacterial invasion [
46]. Here SPLUNC1 is able to both i) remain in the bacteria’s outer membrane [
18], while ii) prevent invasion.
Acknowledgements
We thank Dr. Scott H. Randell (the UNC CF Center Tissue Procurement and Cell Culture Core) for providing cells, Dr. Colin Bingle (University of Sheffield) for providing the SPLUNC1 construct, Dr. Michael Miley and Richard Feng (UNC) for purifying rSPLUNC1 and rSPLUNC1 mutants, Dr. John LiPuma (University of Michigan) for sending the J2315 strain, Dr. Miguel Valvano (Queen’s University, Belfast) for sending the J2315-GFP strain, and Dr. Peter Y. Di (University of Pittsburg) for the SPLUNC1-/- mice.
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