Table 1.
Prediction of the potential antimicrobial domain in SPA4 peptide sequence based on physicochemical properties and algorithm models.
Fig 1.
SPA4 peptide does not bind to live P. aeruginosa.
Live non-GFP P. aeruginosa bacteria were incubated with 10, 50, 75, and 100 μM of FITC-SPA4 peptide, or 75 μM of Oregon Green (OG)-polymyxin B (positive control). No shift was observed in flow cytometric histograms of P. aeruginosa in the FL1 channel when incubated with FITC-SPA4 peptide. In contrast, a significant shift was observed in flow cytometric histograms of bacteria in the FL1 channel when incubated with OG-polymyxin B, which binds to bacteria (A). Confocal microscopic images of live non-GFP bacteria incubated with FITC-SPA4 peptide (75 μM) and OG-polymyxin B (75 μM). The confocal images were obtained using brightfield and FL1 channels. Absence of fluorescence indicates no binding of FITC-SPA4 peptide to the bacteria. Green fluorescence indicates binding of OG-polymyxin B to the bacteria (B).
Fig 2.
SPA4 peptide does not affect the growth of P. aeruginosa.
Growth curves (OD600 versus time in hours) of P. aeruginosa cultured over 17 h in the presence of increasing concentrations of SPA4 peptide or vehicle. Kanamycin (100 μg/ml) was used as a positive control for growth inhibition. Colony counts (CFU/ml) obtained at 17 h are included within the figure.
Fig 3.
SPA4 peptide induces phagocytosis of live GFP-P. aeruginosa.
Flow cytometric FSC versus SSC dot plots of JAWS II dendritic cells (gated in region P) and bacteria (gated in region R). The FSC versus FL1 dot plots show free GFP-P. aeruginosa in the R1 region (negative control) and cells only (negative control) in P2 region. A shift is noted when the cells take up the GFP-P. aeruginosa. The flow cytometric dot plot charts demonstrate the cells with phagocytosed bacteria in P1 area. Numbers indicate percent of cells with phagocytosed GFP-P. aeruginosa (in P1) or without any bacteria (in P2). Results shown here are from one representative experiment (A). The bar chart shows mean (+ SEM) of percent phagocytosis of GFP-P. aeruginosa as compared with one hundred percent basal phagocytosis from eight experiments, respectively. The p-values determined by t-test are shown within the figure (B). Representative confocal micrographs of cells alone or with phagocytosed GFP-P. aeruginosa. Images taken at brightfield and fluorescence channels were superimposed. Green fluorescence is of GFP-P. aeruginosa. Blue staining is of cell nuclei (C).
Fig 4.
SPA4 peptide treatment induces localization of bacteria inside acidic phagolysosomes of JAWS II dendritic cells and alveolar macrophages, but suppresses the TNF-α response.
The flow chart depicts the timing of adding the bacteria and SPA4 peptide, and of making assessments. Percent localization of pHrodo-labeled P. aeruginosa in acidic phagolysosomes of JAWS II dendritic cells (A) and alveolar macrophages (D) after treatment with SPA4 peptide. Confocal images show localization of red fluorescent pHrodo-labeled P. aeruginosa inside the acidic phagolysosomes of JAWS II dendritic cells (B) and alveolar macrophages (E). Secreted levels of TNF-α cytokine in cell-free supernatants of JAWS II dendritic cells (C) and alveolar macrophages (F) exposed to pHrodo-labeled P. aeruginosa. The cell nucleus stained with Hoechst 33342 dye is shown in blue. LAMP1 staining (in green) confirms the localization of pHrodo-labeled bacteria inside the LAMP1-expressing phagolysosome (G). Percent localization of pHrodo-labeled P. aeruginosa in phagolysosomes (H) and TNF-α in cell-free supernatants (I) of cells treated with heat-inactivated SPA4 peptide. The p-values determined by t-test are shown within figures.
Fig 5.
Effect of SPA4 peptide on localization of bacteria inside the acidic phagolysosomes and TNF-α levels in cell-free supernatants of JAWS II dendritic cells overexpressing TLR4.
The cells were co-transfected with NF-κB-luciferase reporter plasmid construct and plasmid construct encoding wild-type TLR4 or vector control (pDisplay-vector). Dot immunoblotting results demonstrated overexpression of TLR4 in cells transfected with wild-type TLR4. Total cell lysate proteins (5 and 10 μg) were immunoblotted with TLR4-specific antibody (A, i). Transfected cells were challenged with LPS (100 ng/ml) and arbitrary luminescence values for NF-κB-luciferase activity were normalized with total cellular protein (A, iii). Cells transfected with plasmid construct encoding wild-type TLR4 or vector control (pDisplay-vector) were exposed to pHrodo-labeled P. aeruginosa, and treated with SPA4 peptide (B, C). Red fluorescence due to internalized bacteria was quantified by fluorometry, and percent localization of bacteria was calculated relative to control; bars represent the mean + SEM of results from three separate experiments (B). TNF-α levels were measured in cell-free supernatants using ELISA and were normalized with total cellular protein (C). TNF-α results are from one representative experiment performed in triplicate. The p-values were determined by t-test (A, iii) or one-way ANOVA (Tukey’s post-hoc test) (B, C).
Fig 6.
SPA4 peptide treatment improves host defense in a mouse model of P. aeruginosa lung infection.
Flow chart depicts the schedule and dose of challenge with live P. aeruginosa and treatment with SPA4 peptide or heat-inactivated SPA4 peptide or vehicle via intratracheal route (A). Mice were sacrificed at 5 h of bacterial challenge, and their lungs were harvested. Before euthanasia, the symptoms of sickness were scored in mice. Average indices for the symptoms of sickness are shown in (B). The bar chart (Mean + SEM) shows recovered CFU of P. aeruginosa per g lung (C). Levels of cytokines (TNF-α, IL-1β, IL-6, IL-10, and IL-12p70) and chemokines (MCP-1, MIP-2, and KC) (pg per g lung) were measured in lung tissue homogenates by ELISA (D). Data are from 12 mice per group included in two separate experiments performed on different occasions. The p-values were determined by one-way ANOVA (Tukey’s post-hoc test).
Fig 7.
Representative micrographs of H & E-stained mouse lung tissues (with 20X and 40X objectives) harvested from P. aeruginosa-challenged and SPA4 peptide- or vehicle-treated mice (A). The lungs appear to be markedly hypercellular in vehicle-treated, P. aeruginosa-infected mice due to an acute inflammatory cell response consisting mostly of neutrophilic leukocytes (arrows) (A, i). Marked influx of the neutrophilic leukocytes (arrows) was multifocal to diffuse, with most cells present within the smaller vessels. Fewer cells appeared to be present within the interstitium, including the alveolar sacs (A, ii). In the SPA4 peptide-treated mice, the lungs appeared to be only mildly cellular (arrows) (A, iii). The mild influx of cells (arrows), consisting mostly of neutrophilic leukocytes, appears to have a predominantly intravascular location involving the smaller vessels. A few cells also appear to be located within the interstitium (A, iv). Lung edema was determined by lung wet/dry weight ratios (B). The lactate levels (pmol per g lung) were measured in lung tissue homogenates of P. aeruginosa-infected, SPA4 peptide- or vehicle-treated, mice (C). Data are from 12 mice per group included in two separate experiments performed on different occasions. The p-values were determined by one-way ANOVA (Tukey’s post-hoc test).
Fig 8.
Expression of total and phosphorylated (phospho) p42/p44 MAPK, SAPK/JNK, p38 MAPK, and NF-κB p65 in lung tissues.
Forty μg of lung tissue homogenate protein from P. aeruginosa-infected and SPA4 peptide- or vehicle-treated mice were separated on SDS-PAGE gel and immunoblotted for phospho NF-κB-p65 (phospho p65), NF-κB-p65 (p65), phospho p38 MAPK (phospho p38), p38 MAPK (p38), phospho p44/p42 MAPK (phospho p44 and phospho p42), total p42/p44 MAPK (total p42/p44), phospho SAPK/JNK (phospho p54 and phospho p46), and SAPK/JNK. The immunoblots were stripped and re-probed with β actin for protein loading. The immunoblots shown are from lung tissue homogenates of three mice in each group, representative of immunoblots performed in separate samples from 12 mice per group in two experiments (A). The densitometric units of immunoreactive bands for NF-κB and MAPK proteins in (A) were normalized with those of β actin. Average densitometric ratios are shown as mean + SEM (B). Ratios of phospho p65/p65, phospho p38/p38, phospho p54/total SAPK/JNK, and phospho p46/total SAPK/JNK are also shown. The p-values were determined by t-test.