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. 2016 Sep 23;60(10):6003-12.
doi: 10.1128/AAC.00975-16. Print 2016 Oct.

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

Saira Ahmad  1 , Jean Tyrrell  1 , William G Walton  2 , Ashutosh Tripathy  3 , Matthew R Redinbo  2 , Robert Tarran  4
Affiliations

V体育安卓版 - Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

Saira Ahmad et al. Antimicrob Agents Chemother. .

"VSports注册入口" 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 VSports手机版. 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 [IC50] = 0. 28 μM), and reduced biofilm formation and attachment (IC50 = 0. 11 μM). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and α6 helix did not affect its function. However, deletion of the α4 helix attenuated antimicrobial activity, while the corresponding α4 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. .

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Figures

FIG 1
FIG 1
SPLUNC1 has antimicrobial activity against B. cepacia complex clinical isolates. (A) SPLUNC1 was coincubated with 106 CFU/ml B. cenocepacia J2315 (■), P. aeruginosa PAO1 (●), or S. aureus CDL (▲) for 24 h, and growth was measured. The number of CFU per milliliter was determined, and inhibition was calculated as follows: % inhibition = [(CFU/ml from vehicle − CFU/ml from SPLUNC1 present)/(CFU/ml from vehicle)] × 100. (B) SPLUNC1 (□), tobramycin (○), and polymyxin B (△) were incubated with 106 CFU/ml J2315 for 24 h, and growth was measured. (C to E) SPLUNC1 (0.4 μM) was incubated for 24 h with 106 CFU/ml of B. cenocepacia (C), B. cepacia (D), and Burkholderia multivorans (E) isolates, and growth was measured. (F) SPLUNC1 (0.4 μM) was incubated for 24 h with 106 CFU/ml of B. cenocepacia K56-2 and its ΔwbxE and ΔhldE LPS mutants, and growth was measured. Open bars, vehicle; closed bars, 0.4 μM SPLUNC1. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (n = 4 for all panels).
FIG 2
FIG 2
SPLUNC1 is bacteriostatic, not bactericidal. B. cenocepacia J2315 was incubated with or without 0.4 μM SPLUNC1 for 2 h (starting at −2 h). Bacteria were then washed at 0 h with 0.1% Triton X-100 to remove SPLUNC1, grown for an additional 9 h, and measured by determining the OD600 every 3 h. Open bars, vehicle; closed bars, 0.4 μM SPLUNC1. *, P < 0.05 compared to vehicle at −2 h; **, P < 0.01 compared to vehicle at −2 h; ++, P < 0.01 compared to 0.4 μM SPLUNC1 at −2 h (n = 3).
FIG 3
FIG 3
SPLUNC1 has antibiofilm activity against Bcc strains. (A) Dose-response curve for SPLUNC1 coincubated with 106 CFU/ml B. cenocepacia J2315 for 24 h. (B) Preformed (24 h) J2315 biofilms were incubated with increasing concentrations of SPLUNC1 for 1 h (●) or 24 h (▲). (C) Attachment assay for J2315 coincubated with 0.4 μM SPLUNC1 for up to 3 h. (D to F) SPLUNC1 (0.4 μM) was coincubated for 24 h with B. cenocepacia (D), B. cepacia (E), and B. multivorans (F) Bcc clinical isolates. (G) SPLUNC1 (0.4 μM) was incubated for 24 h with 106 CFU/ml of B. cenocepacia K56-2 and its ΔwbxE and ΔhldE LPS mutants. Bcc strains were stained with 1% crystal violet and measured by determining the OD590, and inhibition was calculated as follows: % inhibition = [(CFU/ml from vehicle − CFU/ml from SPLUNC1 present)/(CFU/ml from vehicle)] × 100. Open bars, vehicle; closed bars, 0.4 μM SPLUNC1. *, P < 0.05; **, P < 0.01 (n = 4 for all panels).
FIG 4
FIG 4
The α4 helix is required for SPLUNC1's antimicrobial activity against B. cenocepacia J2315. (A) Three-dimensional rendering of SPLUNC1 structure with the intrinsically disordered S18 region appended (labeled in red). Also indicated are Δ44 SPLUNC1, which lacks the S18 region (blue arrow); the α4 region (labeled in purple), which is absent in the Δα4 mutant; and the α6 region (labeled in green), which is absent in the Δα6 mutant. Increasing concentrations of SPLUNC1 (black closed circles), the Δ44 (blue closed squares), Δα4 (purple closed squares), and Δα6 (green closed triangles) SPLUNC1 mutants, and the α4 (purple open squares), α6 (green open triangles), and S18 (red closed triangles) peptides were coincubated with 106 CFU/ml J2315 for 24 h and measured for antimicrobial activity by CFU counts and calculation of % growth inhibition as previously described (B) and for antibiofilm activity by 1% crystal violet staining, OD590 reading, and calculation of % biofilm biomass inhibition as previously described (C). **, P < 0.001 for S18 peptide compared to SPLUNC1; ***, P < 0.0001 for S18 peptide compared to SPLUNC1; ++, P < 0.001 for Δα4 mutant compared to SPLUNC1; +++, P < 0.0001 for Δα4 mutant compared to SPLUNC1; #, P < 0.01 for Δα6 mutant compared to SPLUNC1; ××, P < 0.05 for α4 peptide compared to SPLUNC1; ×××, P < 0.001 for α4 peptide compared to SPLUNC1; °, P < 0.05 for α6 peptide compared to SPLUNC1; °°°, P < 0.001 for α6 peptide compared to SPLUNC1 (n = 3 for all panels).
FIG 5
FIG 5
SPLUNC1 does not dissociate and retains secondary structure after cleavage with NE. (A) Time course showing cleavage of 40 μM SPLUNC1 by 1 μM NE by SDS-PAGE with Coomassie blue staining. (B) Inhibition of 1 μM NE activity without (■) or with (□) 1 μM sivelestat and 10 μM substrate (Suc-Ala-Ala-Ala-MCA protein) (error bars are obscured by the symbols). A.U., arbitrary units. (C) Static light scattering of SPLUNC1 before and at timed intervals after exposure to NE and sivelestat. (D) Circular dichroism analysis of SPLUNC1 before and at timed intervals after exposure to NE and sivelestat. *, P < 0.01; **, P < 0.001 (n = 3 for all panels).
FIG 6
FIG 6
Cleaved wild-type SPLUNC1 exerts larger effects on B. cenocepacia J2315 growth and biofilm formation than those seen with whole SPLUNC1. Aliquots of SPLUNC1 were exposed to NE for timed intervals, and NE activity was then halted with sivelestat. NE-cleaved SPLUNC1 was then incubated for 24 h with 106 CFU/ml J2315. (A) CFU counts to show antimicrobial activity after incubation with whole versus cleaved SPLUNC1. (B) Inhibition of biofilm formation as measured by crystal violet staining followed by OD590 readings. NE (1 μM) plus sivelestat (1 μM) alone had neither antimicrobial activity (C) nor antibiofilm activity (D). White bars, vehicle; black bars, 0.4 μM SPLUNC1; gray bars, 0.4 μM NE-cleaved SPLUNC1; hatched bars, 1 μM NE plus 1 μM sivelestat (control). *, P < 0.05; **, P < 0.01 (n = 5 for all panels).
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