Dataset Information

Modulation of proinflammatory activity by the engineered cationic antimicrobial peptide WLBU-2.

ABSTRACT:

Background

Host-derived (LL-37) and synthetic (WLBU-2) cationic antimicrobial peptides (CAPs) are known for their membrane-active bactericidal properties. LL-37 is an important mediator for immunomodulation, while the mechanism of action of WLBU-2 remains unclear.

Objective

To determine if WLBU-2 induces an early proinflammatory response that facilitates bacterial clearance in cystic fibrosis (CF).

Methods

C57BL6 mice were given intranasal or intraperitoneal 1×10 (6) cfu/mL Pseudomonas aeruginosa (PA) and observed for 2h, followed by instillation of LL-37 or WLBU-2 (2-4mg/kg) with subsequent tissue collection at 24h for determination of bacterial colony counts and quantitative RT-PCR measurement of cytokine transcripts. CF airway epithelial cells (IB3-1, ?F508/W1282X) were cultured in appropriate media with supplements. WLBU-2 (25?M) was added to the media with RT-PCR measurement of TNF-? and IL-1? transcripts after 20, 30, and 60min. Flow cytometry was used to determine if WLBU-2 assists in cellular uptake of Alexa 488-labeled LPS.

Results

In murine lung exposed to intranasal or intraperitoneal WLBU-2, there was a reduction in the number of surviving PA colonies compared to controls. Murine lung exposed to intraperitoneal WLBU-2 showed fewer PA colonies compared to LL-37. After 24h WLBU-2 exposure, PA-induced IL-1? transcripts from lungs showed a twofold decrease (p<0.05), while TNF-? levels were unchanged. LL-37 did not significantly change transcript levels. In IB3-1 cells, WLBU-2 exposure resulted in increased TNF-? and IL-1? transcripts that decreased by 60min. WLBU-2 treatment of IB3-1 cells displayed increased LPS uptake, suggesting a potential role for CAPs in inducing protective proinflammatory responses. Taken together, the cytokine response, LPS uptake, and established antimicrobial activity of WLBU-2 demonstrate its ability to modulate proinflammatory signaling as a protective mechanism to clear infection.

Conclusions

The immunomodulatory properties of WLBU-2 reveal a potential mechanism of its broad-spectrum antibacterial activity and warrant further preclinical evaluation to study bacterial clearance and rescue of chronic inflammation.

SUBMITTER: Paranjape SM

PROVIDER: S-EPMC3894802 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Modulation of proinflammatory activity by the engineered cationic antimicrobial peptide WLBU-2.

Paranjape Shruti M SM Lauer Thomas W TW Montelaro Ronald C RC Mietzner Timothy A TA Vij Neeraj N

F1000Research 20130208

<h4>Background</h4>Host-derived (LL-37) and synthetic (WLBU-2) cationic antimicrobial peptides (CAPs) are known for their membrane-active bactericidal properties. LL-37 is an important mediator for immunomodulation, while the mechanism of action of WLBU-2 remains unclear.<h4>Objective</h4>To determine if WLBU-2 induces an early proinflammatory response that facilitates bacterial clearance in cystic fibrosis (CF).<h4>Methods</h4>C57BL6 mice were given intranasal or intraperitoneal 1×10 (6) cfu/mL ...[more]

PMID: 24555033

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Project description:Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

Dataset Information

Modulation of proinflammatory activity by the engineered cationic antimicrobial peptide WLBU-2.

Background

Objective

Methods

Results

Conclusions

Publications

Modulation of proinflammatory activity by the engineered cationic antimicrobial peptide WLBU-2.

Similar Datasets

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