Article Figures & Data
Figures
- FIG. 1.
Effects of temperature on peptide-induced killing of C. albicans (DIS) and C. neoformans (CN2). Fungal cells (2 × 103 in 20 μl of 10 mM sodium phosphate buffer) were incubated with 20 μl of MUC7 20-mer or Hsn-5 peptide (at concentrations ranging from 1.56 to 100 μM in 10 mM sodium phosphate buffer) for 1.5 h at 4 and 37°C. Cell viability was determined by plating on SAB plates. Results represent the mean and standard deviation of duplicate or triplicate experiments.
- FIG. 2.
Effects of CCCP and sodium azide on peptide-induced killing of C. albicans (DIS) and C. neoformans (CN2). Fungal cells were incubated with 20 mM sodium azide or 300 μM CCCP for 2 h at 37°C. Cells without any pretreatment were used as controls. A 20-μl aliquot (2 × 103 cells) of treated fungal cells was then incubated with 20 μl of MUC7 20-mer or Hsn-5 peptide (1.56 to 100 μM in 10 mM sodium phosphate buffer) for an additional 1.5 h at 37°C. Cell viability was determined by plating on SAB plates. Results represent the mean and standard deviation of duplicate or triplicate experiments.
- FIG. 3.
Effects of cations on peptide-induced killing of C. albicans (DIS) and C. neoformans (CN2). Fungal cells (2 × 103 in 20 μl of 10 mM sodium phosphate buffer) were incubated with 20 μl of MUC7 20-mer or Hsn-5 peptide (final concentrations, 25 μM) in 10 mM sodium phosphate buffer in the presence of either Mg2+ or Ca2+ at concentrations ranging from 1 to 50 mM for 1.5 h at 37°C. Cell viability was determined by plating on SAB plates. Results represent the mean and standard deviation of duplicate or triplicate experiments. At a 25 μM concentration of peptides, without the presence of cations, 100% peptide-induced killing was obtained.
- FIG. 4.
Secondary structure predictions and helical wheel projections for MUC7 15-mer, MUC7 20-mer, and Hsn-5. For the secondary structure prediction (shown at the top), a PSI Pred graphical viewer was used. Conf, confidence of prediction; Pred, predicted secondary structure; AA, target sequence. The cylinder denotes helix structure (H), and the line denotes coil structure (C). The helical wheel projections of the predicted helical region of each peptide were made by using Genetics Computer Group sequence analysis software. Hydrophobic residues are displayed in circles.
- FIG. 5.
Fluorescence light microscopy and confocal laser microscopy of internalization of FITC-MUC7 20-mer by C. albicans (DIS) and C. neoformans (CN2). (A to F) Fluorescence light microscopy. (G and H) Confocal laser microscopy. (A) 37°C. (B) 4°C. (C) Cells pretreated with 20 mM sodium azide. (D) Cells pretreated with 300 μM CCCP. (E) Cells in the presence of 50 mM Mg2+. (F) Cells in the presence of 50 mM Ca2+. Cells (107) were treated with 50 μM FITC- MUC7 20-mer in 100 μl of sodium phosphate buffer (10 mM, pH 7.4) for 45 to 90 min at 37°C (except for cells in panel B, which were treated at 4°C). Fluorescence light micrographs were made on a Nikon Optiphot microscope with a fluorescent light source. (G and H) Confocal fluorescence microscopy of cells labeled with FITC-MUC7 20-mer and a mitochondrion-specific marker, MitoTracker Red CMXRos (G), or (in a control experiment) with MitoTracker Red CMXRos and MitoTracker Green FM (H). Cells were incubated for 15 min at 37°C with 500 nM MitoTracker Red CMXRos in 10 mM phosphate buffer (pH 7.4), washed twice, and subsequently incubated for 20 min at 37°C with either 50 μM FITC-MUC7 20-mer in 10 mM phosphate buffer (pH 7.4) (G) or 500 nM MitoTracker Green FM (H). Confocal fluorescence microscopy was performed with a Bio-Rad MRC-1024 confocal microscope system. Bars, 1 μM.
Tables
- TABLE 1.
Amino acid sequences and charges of peptides under study
Peptide Amino acid sequence Charge + − Net MUC7 51-mer EGRERDHELRHRRHHHQSPKSHFELPHYPGLLAHQKPFIRKSYKCLHKRCR 13 5 +8 MUC7 15-mer RERDHELRHRRHHHQ 5 3 +2 MUC7 20-mer LAHQKPFIRKSYKCLHKRCR 7 0 +7 Hsn-5 DSHAKRHHGYKRKFHEKHHSHRGY 7 2 +5 Ins-A GIVEQCCASVCSLYQLENYCN 0 2 −2 - TABLE 2.
ED50s of MUC7 20-mer and Hsn-5a
Organism ED50 (95% confidence limit), μM MUC7 20-mer Hsn-5 Fungi C. albicans (DIS) 5.85 (4.17-8.67) 6.68 (6.05-7.37) C. glabrata 5.02 (3.75-8.22) 38.7 (30.8-50.7) C. krusei 5.16 (4.17-6.40) 6.47 (5.79-7.55) C. neoformans (CN2) 4.05 (3.16-5.81) 3.71 (1.92-5.60) S. cerevisiae 5.23 (4.01-6.76) 74.0 (58.0-101.) C. albicans (azole resistant) 2.40 (1.73-3.08) 6.40 (5.59-7.32) C. glabrata (fluconazole resistant) 12.3 (9.06-17.1) 85.3 (78.4-94.0) C. neoformans (amphotericin B resistant) 4.29 (3.59-4.26) 3.72 (2.90-4.87) Bacteria A. actinomycetemcomitansb 4.37 (3.74-5.04) >100 E. coli 1.61 (1.09-2.14) Not tested P. aeruginosa 4.41 (3.65-5.19) Not tested S. mutansc 1.39 (1.00-1.90) 92.0 (85.1-99.3) S. gordonii 2.43 (0.60-6.58) Not tested P. gingivalis <1.00 Not tested - TABLE 3.
Time-dependent killing activity of MUC7 20-mer and Hsn-5
Time (min) % Loss of viability of the following cells in the presence of the indicated agenta: C. albicans (DIS) C. neoformans (CN2) 20-Mer Hsn-5 20-Mer Hsn-5 5 31 ± 8 0 ± 0 97 ± 1 9 ± 2 15 42 ± 5 1 ± 3 97 ± 2 43 ± 3 30 52 ± 1 11 ± 4 96 ± 2 59 ± 5 45 64 ± 3 34 ± 2 98 ± 1 64 ± 1 90 74 ± 6 42 ± 3 100 ± 0 71 ± 7 ↵ a Peptide concentration, 6.25 μM. Data are reported as mean and standard deviation. P values for 20-mer versus Hsn-5 were <0.05.
