PLUNC is a novel airway surfactant protein with anti-biofilm activity

Abstract

Background: The PLUNC ("Palate, lung, nasal epithelium clone") protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family--the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP)--are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways.

Methodology/principal findings: Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model.

Conclusions/significance: Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen.

Figure 1. Multiple sequence alignment of PLUNC and latherin.

Alignment depicts the amino acid sequence of human PLUNC (NP_057667), aligned with horse latherin (NP_001075328). The mature form of PLUNC was predicted using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/) to identify the likely location of the signal sequence . Hydrophobic residues are shown in black, hydrophilic residues are green, basic residues are blue, and acidic residues are red. Blue boxes indicate conserved residues. Alignment was constructed using the CLC Bio Workbench 4.1.1 (CLC Bio A/S, Aarhus, Denmark).

Figure 2. Expression and purification of recombinant human PLUNC.

(A) Recombinant PLUNC protein was expressed in E. coli as described in the Methods. After centrifugation, the crude bacterial lysate (Lane 1) was passed over amylose resin and bound fusion protein was eluted from the resin using maltose. The recovered elution fraction (Lane 2) indicated a predominant product of approximately 69 kDa on a reducing SDS-PAGE gel, consistent with the predicted size of the MBP-PLUNC-6xHis fusion protein. Fusion protein was further purified by gel filtration, followed by a 16 hour cleavage with Factor Xa protease to remove the MBP tag. Factor Xa treatment gave rise to two cleavage products, MBP at 43 kDa and PLUNC-6xHis at approximately 26 kDa (Lane 3). After passing the cleavage products over nickel resin, MBP was observed in the flowthrough fraction (Lane 4), while the elution fractions contained PLUNC-6xHis and any remaining uncleaved His-tagged fusion protein (Lane 5). A final gel filtration step was used to separate PLUNC-6xHis from the uncleaved protein (Lane 6). Cleaved PLUNC-6xHis, in its final form, is indicated by the arrow. MW  =  molecular weight ladder (BenchMark Unstained Protein Standard; BioRad) (B) Representative immunoblot, in which increasing concentrations of purified recombinant PLUNC-6xHis were electrophoresed and probed using a monoclonal antibody directed against the human PLUNC protein, revealing a single immunoreactive band at the expected molecular weight in the range of 20–25 kDa. Immunoblotting also demonstrated the presence of native PLUNC protein in apical secretions from primary cultures of well-differentiated human bronchial epithelia (BE) and nasal epithelia (NE). See Methods for details.

Figure 3. Circular dichroism spectra of recombinant human PLUNC.

Circular dichroism spectra of recombinant human PLUNC. Molar ellipticities in the far-UV range (197–260 nm) are plotted for PLUNC at 0.54 mg/mL (circles), 1.08 mg/mL (triangles), and 2.16 mg/mL (boxes). Analysis of this data using the K2d server predicts that the secondary structure of PLUNC is 24-33% alpha-helical, ∼15% beta-sheet and 51–61% random coil.

Figure 4. Contact angle measurements suggest that PLUNC possesses surface activity.

(A) Advancing contact angles were measured by the sessile drop technique for various solutions dispensed onto siliconized glass, a hydrophobic surface. Bars depict the mean values for contact angles (in degrees) measured one minute after drops were dispensed onto the solid surface. Error bars represent the standard error about the mean (n = 6). Contact angles (θ) of less than 90° (dotted line in panel A) indicate wetting of the surface by the drop, whereas contact angles greater than 90° (to the right of the vertical line) indicate that a sample is “non-wetting”. The solid vertical line separates the solutions that have greater wetting ability than buffer alone (bars pointing left) from the ones that have lesser wetting ability (bars pointing right). Asterisks denote measurements that are significantly different from buffer, as determined by Student's two-tailed t-test (P-value <0.01). On a hydrophobic surface, PLUNC solutions transitioned from “non-wetting” to “wetting” at concentrations greater than 10 µg/mL. Inset: spreading behavior over time was compared for Tris buffer and a PLUNC-containing solution dispensed onto a hydrophobic surface. On each coverslip, the drop on the left is buffer, while drops of PLUNC (140 µg/mL) are shown on the right. Drop spreading was photographed after 5 minutes, 25 minutes, and 45 minutes, revealing that the presence of PLUNC conferred an increased tendency for an aqueous solution to spread on a hydrophobic surface. In panel (B), drops of test solutions were formed on unmodified glass, a hydrophilic surface, and advancing contact angles were measured as described above. On a hydrophilic surface, PLUNC enhanced wetting when at lower concentrations (1–2 µg/mL), while higher PLUNC concentrations (5–150 µg/mL) appeared to reduce wetting ability. Infasurf, a commercial lung surfactant, displayed significant wetting ability on both hydrophilic and hydrophobic surfaces.

Figure 5. Reduction of surface tension at an air-liquid interface by PLUNC protein.

The pulsating bubble surfactometer was used to measure dynamic surface tension in solutions containing increasing concentrations of recombinant PLUNC, as described in Methods. For each sample, the minimum surface achieved at 5 minutes of pulsation is shown as a gray bar. The range, representing the difference between the minimum and maximum surface tensions at the 5 minute time point, is depicted in white. Error bars, where present, represent the standard error about the mean (n = 3 experiments).

Figure 6. PLUNC interaction with albumin in the pulsating bubble surfactometer.

The pulsating bubble surfactometer was used to measure dynamic surface tension in solutions containing mixtures of PLUNC and bovine serum albumin (BSA). Plotted values represent surface tension minima over time for Tris buffer (••••), BSA at 10 mg/mL (----), PLUNC at 10 µg/mL (― ―) or PLUNC (10 µg/mL) with BSA (10 mg/mL) (—). In the presence of BSA, it takes longer for PLUNC to reach its saturation value for minimum surface tension.

Figure 7. PLUNC inhibits biofilms growing at the air-surface interface.

(A) Crystal violet staining of P. aeruginosa PA14 biofilms shows that PLUNC (100 µg/mL) reduced the biofilm biomass accumulating on the sides of culture tubes after 72 hours of growth. (B) The amount of crystal violet retained by biofilms was quantitated using optical density measurements. Growth of PLUNC-treated biofilms (n = 3) was significantly inhibited compared to non-treated and buffer-treated biofilms. Error bars show standard deviation. * P<0.025. (C) Biofilms formed at the air-liquid interface of unshaken cultures after 48 hours of growth. (D) Magnification of areas outlined in panel C shows that in media containing 100 µg/mL PLUNC, biomass spontaneously separates from the air-liquid interface biofilm and sinks toward the bottom of the culture tube.