A peptide derived from the highly conserved protein GAPDH is involved in tissue protection by different antifungal strategies and epithelial immunomodulation

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an important role not only in glycolysis but also in nonmetabolic processes, including transcription activation and apoptosis. We report the isolation of a human GAPDH (hGAPDH) (2-32) fragment peptide from human placental tissue exhibiting antimicrobial activity. The peptide was internalized by cells of the pathogenic yeast Candida albicans and initiated a rapid apoptotic mechanism, leading to killing of the fungus. Killing was dose-dependent, with 10 μg ml (3.1 μM) and 100 μg ml hGAPDH (2-32) depolarizing 45% and 90% of the fungal cells in a population, respectively. Experimental C. albicans infection induced epithelial hGAPDH (2-32) expression. Addition of the peptide significantly reduced the tissue damage as compared with untreated experimental infection. Secreted aspartic proteinase (Sap) activity of C. albicans was inhibited by the fragment at higher concentrations, with a median effective dose of 160 mg l(-1) (50 μM) for Sap1p and 200 mg l(-1) (63 μM) for Sap2p, whereas Sap3 was not inhibited at all. Interestingly, hGAPDH (2-32) induced significant epithelial IL-8 and GM-CSF secretion and stimulated Toll-like receptor 4 expression at low concentrations independently of the presence of C. albicans, without any toxic mucosal effects. In the future, the combination of different antifungal strategies, e.g., a conventional fungicidal with immunomodulatory effects and the inhibition of fungal virulence factors, might be a promising treatment option.

Figure 1. Purification of the antimicrobial peptide hGAPDH (2-32) from human placental tissue

Each purification step was monitored by radial diffusion assay for detection of antimicrobial activity. The bars show the diameters of inhibition zones indicating the antimicrobial activity against E. coli. Fractions 19 and 20, corresponding to the maximum growth inhibition, were selected for further purification (a). Fractions 19 and 20 were pooled and fractionated by RP chromatography (b). Final purification of the antimicrobial peptide was performed, separating fraction 23 (b) using a strong cation-exchange column (c). MALDI-MS analysis of the purified peptide revealed a molecular mass of 3188 Da (d). Sequence analysis led to the identification of an N-terminal fragment of GAPDH. Amino acid sequence is shown in the single letter code (e).

Figure 1. Purification of the antimicrobial peptide hGAPDH (2-32) from human placental tissue

Each purification step was monitored by radial diffusion assay for detection of antimicrobial activity. The bars show the diameters of inhibition zones indicating the antimicrobial activity against E. coli. Fractions 19 and 20, corresponding to the maximum growth inhibition, were selected for further purification (a). Fractions 19 and 20 were pooled and fractionated by RP chromatography (b). Final purification of the antimicrobial peptide was performed, separating fraction 23 (b) using a strong cation-exchange column (c). MALDI-MS analysis of the purified peptide revealed a molecular mass of 3188 Da (d). Sequence analysis led to the identification of an N-terminal fragment of GAPDH. Amino acid sequence is shown in the single letter code (e).

Figure 1. Purification of the antimicrobial peptide hGAPDH (2-32) from human placental tissue

Each purification step was monitored by radial diffusion assay for detection of antimicrobial activity. The bars show the diameters of inhibition zones indicating the antimicrobial activity against E. coli. Fractions 19 and 20, corresponding to the maximum growth inhibition, were selected for further purification (a). Fractions 19 and 20 were pooled and fractionated by RP chromatography (b). Final purification of the antimicrobial peptide was performed, separating fraction 23 (b) using a strong cation-exchange column (c). MALDI-MS analysis of the purified peptide revealed a molecular mass of 3188 Da (d). Sequence analysis led to the identification of an N-terminal fragment of GAPDH. Amino acid sequence is shown in the single letter code (e).

Figure 1. Purification of the antimicrobial peptide hGAPDH (2-32) from human placental tissue

Each purification step was monitored by radial diffusion assay for detection of antimicrobial activity. The bars show the diameters of inhibition zones indicating the antimicrobial activity against E. coli. Fractions 19 and 20, corresponding to the maximum growth inhibition, were selected for further purification (a). Fractions 19 and 20 were pooled and fractionated by RP chromatography (b). Final purification of the antimicrobial peptide was performed, separating fraction 23 (b) using a strong cation-exchange column (c). MALDI-MS analysis of the purified peptide revealed a molecular mass of 3188 Da (d). Sequence analysis led to the identification of an N-terminal fragment of GAPDH. Amino acid sequence is shown in the single letter code (e).

Figure 1. Purification of the antimicrobial peptide hGAPDH (2-32) from human placental tissue

Each purification step was monitored by radial diffusion assay for detection of antimicrobial activity. The bars show the diameters of inhibition zones indicating the antimicrobial activity against E. coli. Fractions 19 and 20, corresponding to the maximum growth inhibition, were selected for further purification (a). Fractions 19 and 20 were pooled and fractionated by RP chromatography (b). Final purification of the antimicrobial peptide was performed, separating fraction 23 (b) using a strong cation-exchange column (c). MALDI-MS analysis of the purified peptide revealed a molecular mass of 3188 Da (d). Sequence analysis led to the identification of an N-terminal fragment of GAPDH. Amino acid sequence is shown in the single letter code (e).

Figure 2. Antimicrobial killing assays and electron microscopy

Flow cytometric antimicrobial killing assay of E. coli and C. albicans incubated with 10 μg/ml hGAPDH (2-32), LL-37 or hBD-3 (a). Dose dependent effect of hGAPDH (2-32). Suspensions of C. albicans were incubated with hGAPDH (2-32) for 90 min. The antimicrobial activity is shown as percentage of depolarized microorganisms (b). The data are means of one representative experiment in triplicate. Electron microscopy of C. albicans SC5314 cells grown without (c) and with (d) 125 μg/ml hGAPDH (2-32) for 24 h. Cells grown without hGAPDH (2-32) with a regular morphology (c). C. albicans grown under the influence of hGAPDH (2-32) shows enlargement of cytoplasmic vacuoles and disorganization of the internal organelles (d). Bar = 500 nm.

Figure 2. Antimicrobial killing assays and electron microscopy

Flow cytometric antimicrobial killing assay of E. coli and C. albicans incubated with 10 μg/ml hGAPDH (2-32), LL-37 or hBD-3 (a). Dose dependent effect of hGAPDH (2-32). Suspensions of C. albicans were incubated with hGAPDH (2-32) for 90 min. The antimicrobial activity is shown as percentage of depolarized microorganisms (b). The data are means of one representative experiment in triplicate. Electron microscopy of C. albicans SC5314 cells grown without (c) and with (d) 125 μg/ml hGAPDH (2-32) for 24 h. Cells grown without hGAPDH (2-32) with a regular morphology (c). C. albicans grown under the influence of hGAPDH (2-32) shows enlargement of cytoplasmic vacuoles and disorganization of the internal organelles (d). Bar = 500 nm.

Figure 2. Antimicrobial killing assays and electron microscopy

Flow cytometric antimicrobial killing assay of E. coli and C. albicans incubated with 10 μg/ml hGAPDH (2-32), LL-37 or hBD-3 (a). Dose dependent effect of hGAPDH (2-32). Suspensions of C. albicans were incubated with hGAPDH (2-32) for 90 min. The antimicrobial activity is shown as percentage of depolarized microorganisms (b). The data are means of one representative experiment in triplicate. Electron microscopy of C. albicans SC5314 cells grown without (c) and with (d) 125 μg/ml hGAPDH (2-32) for 24 h. Cells grown without hGAPDH (2-32) with a regular morphology (c). C. albicans grown under the influence of hGAPDH (2-32) shows enlargement of cytoplasmic vacuoles and disorganization of the internal organelles (d). Bar = 500 nm.

Figure 2. Antimicrobial killing assays and electron microscopy

Flow cytometric antimicrobial killing assay of E. coli and C. albicans incubated with 10 μg/ml hGAPDH (2-32), LL-37 or hBD-3 (a). Dose dependent effect of hGAPDH (2-32). Suspensions of C. albicans were incubated with hGAPDH (2-32) for 90 min. The antimicrobial activity is shown as percentage of depolarized microorganisms (b). The data are means of one representative experiment in triplicate. Electron microscopy of C. albicans SC5314 cells grown without (c) and with (d) 125 μg/ml hGAPDH (2-32) for 24 h. Cells grown without hGAPDH (2-32) with a regular morphology (c). C. albicans grown under the influence of hGAPDH (2-32) shows enlargement of cytoplasmic vacuoles and disorganization of the internal organelles (d). Bar = 500 nm.

Figure 3. Human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) (2-32) inhibits activity of secreted aspartic proteinases (Saps), a virulence attributes of C. albicans

Specific inhibition of Sap1-3 showed an ED₅₀ of 160 μg/ml (50 μM) for Sap1p and 200 μg/ml (63 μM) for Sap2p while Sap3p was not inhibited. Error bars indicate range of duplicates. Each duplicate consists of three background-normalized measures.

Figure 4. Experimental C. albicans infection

Release of LDH by monolayer epithelial cells 12 h after infection (or not) with C. albicans in the presence and absence of 5 μg/ml LL-37 or hGAPDH (2-32). Multiplicity of infection (MOI) 0.1. Epithelial cells were preincubated with the peptide for 1 h (a). Statistical significance was determined using the 2-tailed paired Student’s t test (n = 6). Light micrographs of reconstituted human oral epithelium (RHE) 18 h after infection with C. albicans SC5314 in the absence and presence of hGAPDH (2-32). Invasion by C. albicans of all epithelial layers with extensive edema and vacuolization (b) in the absence of hGAPDH (2-32). Strongly reduced virulence phenotype resulting in a protective effect in the presence of hGAPDH (2-32). Decreased number of C. albicans cells (c). Confocal laser microscopy of oral RHE after 12 h in the presence and absence of C. albicans and 5 μg/ml hGAPDH (2-32) (cell nuclei, green; hGAPDH (2-32), red; C. albicans, blue). No evidence for hGAPDH (2-32) in the uninfected and untreated oral RHE (d). Presence of hGAPDH (2-32) on the superficial layers of the uninfected RHE after external addition (e). Increased expression in the C. albicans infected but untreated oral RHE (f). Strong affinity of the peptide to the C. albicans cells ( f, g) after external addition of hGAPDH (2-32). Higher-magnification image demonstrating direct contact of the peptide with C. albicans cells (short arrow) and internalization by the fungal cells (long arrow). Bar = 30 μm.

Figure 4. Experimental C. albicans infection

Release of LDH by monolayer epithelial cells 12 h after infection (or not) with C. albicans in the presence and absence of 5 μg/ml LL-37 or hGAPDH (2-32). Multiplicity of infection (MOI) 0.1. Epithelial cells were preincubated with the peptide for 1 h (a). Statistical significance was determined using the 2-tailed paired Student’s t test (n = 6). Light micrographs of reconstituted human oral epithelium (RHE) 18 h after infection with C. albicans SC5314 in the absence and presence of hGAPDH (2-32). Invasion by C. albicans of all epithelial layers with extensive edema and vacuolization (b) in the absence of hGAPDH (2-32). Strongly reduced virulence phenotype resulting in a protective effect in the presence of hGAPDH (2-32). Decreased number of C. albicans cells (c). Confocal laser microscopy of oral RHE after 12 h in the presence and absence of C. albicans and 5 μg/ml hGAPDH (2-32) (cell nuclei, green; hGAPDH (2-32), red; C. albicans, blue). No evidence for hGAPDH (2-32) in the uninfected and untreated oral RHE (d). Presence of hGAPDH (2-32) on the superficial layers of the uninfected RHE after external addition (e). Increased expression in the C. albicans infected but untreated oral RHE (f). Strong affinity of the peptide to the C. albicans cells ( f, g) after external addition of hGAPDH (2-32). Higher-magnification image demonstrating direct contact of the peptide with C. albicans cells (short arrow) and internalization by the fungal cells (long arrow). Bar = 30 μm.

Figure 4. Experimental C. albicans infection

Release of LDH by monolayer epithelial cells 12 h after infection (or not) with C. albicans in the presence and absence of 5 μg/ml LL-37 or hGAPDH (2-32). Multiplicity of infection (MOI) 0.1. Epithelial cells were preincubated with the peptide for 1 h (a). Statistical significance was determined using the 2-tailed paired Student’s t test (n = 6). Light micrographs of reconstituted human oral epithelium (RHE) 18 h after infection with C. albicans SC5314 in the absence and presence of hGAPDH (2-32). Invasion by C. albicans of all epithelial layers with extensive edema and vacuolization (b) in the absence of hGAPDH (2-32). Strongly reduced virulence phenotype resulting in a protective effect in the presence of hGAPDH (2-32). Decreased number of C. albicans cells (c). Confocal laser microscopy of oral RHE after 12 h in the presence and absence of C. albicans and 5 μg/ml hGAPDH (2-32) (cell nuclei, green; hGAPDH (2-32), red; C. albicans, blue). No evidence for hGAPDH (2-32) in the uninfected and untreated oral RHE (d). Presence of hGAPDH (2-32) on the superficial layers of the uninfected RHE after external addition (e). Increased expression in the C. albicans infected but untreated oral RHE (f). Strong affinity of the peptide to the C. albicans cells ( f, g) after external addition of hGAPDH (2-32). Higher-magnification image demonstrating direct contact of the peptide with C. albicans cells (short arrow) and internalization by the fungal cells (long arrow). Bar = 30 μm.

Figure 4. Experimental C. albicans infection

Release of LDH by monolayer epithelial cells 12 h after infection (or not) with C. albicans in the presence and absence of 5 μg/ml LL-37 or hGAPDH (2-32). Multiplicity of infection (MOI) 0.1. Epithelial cells were preincubated with the peptide for 1 h (a). Statistical significance was determined using the 2-tailed paired Student’s t test (n = 6). Light micrographs of reconstituted human oral epithelium (RHE) 18 h after infection with C. albicans SC5314 in the absence and presence of hGAPDH (2-32). Invasion by C. albicans of all epithelial layers with extensive edema and vacuolization (b) in the absence of hGAPDH (2-32). Strongly reduced virulence phenotype resulting in a protective effect in the presence of hGAPDH (2-32). Decreased number of C. albicans cells (c). Confocal laser microscopy of oral RHE after 12 h in the presence and absence of C. albicans and 5 μg/ml hGAPDH (2-32) (cell nuclei, green; hGAPDH (2-32), red; C. albicans, blue). No evidence for hGAPDH (2-32) in the uninfected and untreated oral RHE (d). Presence of hGAPDH (2-32) on the superficial layers of the uninfected RHE after external addition (e). Increased expression in the C. albicans infected but untreated oral RHE (f). Strong affinity of the peptide to the C. albicans cells ( f, g) after external addition of hGAPDH (2-32). Higher-magnification image demonstrating direct contact of the peptide with C. albicans cells (short arrow) and internalization by the fungal cells (long arrow). Bar = 30 μm.

Figure 4. Experimental C. albicans infection

Release of LDH by monolayer epithelial cells 12 h after infection (or not) with C. albicans in the presence and absence of 5 μg/ml LL-37 or hGAPDH (2-32). Multiplicity of infection (MOI) 0.1. Epithelial cells were preincubated with the peptide for 1 h (a). Statistical significance was determined using the 2-tailed paired Student’s t test (n = 6). Light micrographs of reconstituted human oral epithelium (RHE) 18 h after infection with C. albicans SC5314 in the absence and presence of hGAPDH (2-32). Invasion by C. albicans of all epithelial layers with extensive edema and vacuolization (b) in the absence of hGAPDH (2-32). Strongly reduced virulence phenotype resulting in a protective effect in the presence of hGAPDH (2-32). Decreased number of C. albicans cells (c). Confocal laser microscopy of oral RHE after 12 h in the presence and absence of C. albicans and 5 μg/ml hGAPDH (2-32) (cell nuclei, green; hGAPDH (2-32), red; C. albicans, blue). No evidence for hGAPDH (2-32) in the uninfected and untreated oral RHE (d). Presence of hGAPDH (2-32) on the superficial layers of the uninfected RHE after external addition (e). Increased expression in the C. albicans infected but untreated oral RHE (f). Strong affinity of the peptide to the C. albicans cells ( f, g) after external addition of hGAPDH (2-32). Higher-magnification image demonstrating direct contact of the peptide with C. albicans cells (short arrow) and internalization by the fungal cells (long arrow). Bar = 30 μm.

Figure 4. Experimental C. albicans infection

Release of LDH by monolayer epithelial cells 12 h after infection (or not) with C. albicans in the presence and absence of 5 μg/ml LL-37 or hGAPDH (2-32). Multiplicity of infection (MOI) 0.1. Epithelial cells were preincubated with the peptide for 1 h (a). Statistical significance was determined using the 2-tailed paired Student’s t test (n = 6). Light micrographs of reconstituted human oral epithelium (RHE) 18 h after infection with C. albicans SC5314 in the absence and presence of hGAPDH (2-32). Invasion by C. albicans of all epithelial layers with extensive edema and vacuolization (b) in the absence of hGAPDH (2-32). Strongly reduced virulence phenotype resulting in a protective effect in the presence of hGAPDH (2-32). Decreased number of C. albicans cells (c). Confocal laser microscopy of oral RHE after 12 h in the presence and absence of C. albicans and 5 μg/ml hGAPDH (2-32) (cell nuclei, green; hGAPDH (2-32), red; C. albicans, blue). No evidence for hGAPDH (2-32) in the uninfected and untreated oral RHE (d). Presence of hGAPDH (2-32) on the superficial layers of the uninfected RHE after external addition (e). Increased expression in the C. albicans infected but untreated oral RHE (f). Strong affinity of the peptide to the C. albicans cells ( f, g) after external addition of hGAPDH (2-32). Higher-magnification image demonstrating direct contact of the peptide with C. albicans cells (short arrow) and internalization by the fungal cells (long arrow). Bar = 30 μm.

Figure 4. Experimental C. albicans infection

Release of LDH by monolayer epithelial cells 12 h after infection (or not) with C. albicans in the presence and absence of 5 μg/ml LL-37 or hGAPDH (2-32). Multiplicity of infection (MOI) 0.1. Epithelial cells were preincubated with the peptide for 1 h (a). Statistical significance was determined using the 2-tailed paired Student’s t test (n = 6). Light micrographs of reconstituted human oral epithelium (RHE) 18 h after infection with C. albicans SC5314 in the absence and presence of hGAPDH (2-32). Invasion by C. albicans of all epithelial layers with extensive edema and vacuolization (b) in the absence of hGAPDH (2-32). Strongly reduced virulence phenotype resulting in a protective effect in the presence of hGAPDH (2-32). Decreased number of C. albicans cells (c). Confocal laser microscopy of oral RHE after 12 h in the presence and absence of C. albicans and 5 μg/ml hGAPDH (2-32) (cell nuclei, green; hGAPDH (2-32), red; C. albicans, blue). No evidence for hGAPDH (2-32) in the uninfected and untreated oral RHE (d). Presence of hGAPDH (2-32) on the superficial layers of the uninfected RHE after external addition (e). Increased expression in the C. albicans infected but untreated oral RHE (f). Strong affinity of the peptide to the C. albicans cells ( f, g) after external addition of hGAPDH (2-32). Higher-magnification image demonstrating direct contact of the peptide with C. albicans cells (short arrow) and internalization by the fungal cells (long arrow). Bar = 30 μm.

Figure 5. Apoptotic marker induced by 5 μg/ml hGAPDH (2-32) on C. albicans cells

Representative micrographs showing cells stained with FITC-annexin V (green) and propidium iodide (PI, red) to detect apoptosis (phosphatidylserine externalization) and necrosis, respectively. The cells were untreated (a, b) or previously treated (c, d) with hGAPDH (2-32) for 1 h. Subpanels b and d are phase-contrast micrographs. Subpanels a and b show annexin and PI staining. (e) Percentage of 300 fungal cells that are classified as apoptotic [annexin (+) PI(−); green bars] and necrotic [annexin(+/−) PI(+); red bars] after treatment with hGAPDH (2-32) and H₂O₂. Bar=10 μm.

Figure 5. Apoptotic marker induced by 5 μg/ml hGAPDH (2-32) on C. albicans cells

Representative micrographs showing cells stained with FITC-annexin V (green) and propidium iodide (PI, red) to detect apoptosis (phosphatidylserine externalization) and necrosis, respectively. The cells were untreated (a, b) or previously treated (c, d) with hGAPDH (2-32) for 1 h. Subpanels b and d are phase-contrast micrographs. Subpanels a and b show annexin and PI staining. (e) Percentage of 300 fungal cells that are classified as apoptotic [annexin (+) PI(−); green bars] and necrotic [annexin(+/−) PI(+); red bars] after treatment with hGAPDH (2-32) and H₂O₂. Bar=10 μm.

Figure 5. Apoptotic marker induced by 5 μg/ml hGAPDH (2-32) on C. albicans cells

Representative micrographs showing cells stained with FITC-annexin V (green) and propidium iodide (PI, red) to detect apoptosis (phosphatidylserine externalization) and necrosis, respectively. The cells were untreated (a, b) or previously treated (c, d) with hGAPDH (2-32) for 1 h. Subpanels b and d are phase-contrast micrographs. Subpanels a and b show annexin and PI staining. (e) Percentage of 300 fungal cells that are classified as apoptotic [annexin (+) PI(−); green bars] and necrotic [annexin(+/−) PI(+); red bars] after treatment with hGAPDH (2-32) and H₂O₂. Bar=10 μm.

Figure 5. Apoptotic marker induced by 5 μg/ml hGAPDH (2-32) on C. albicans cells

Representative micrographs showing cells stained with FITC-annexin V (green) and propidium iodide (PI, red) to detect apoptosis (phosphatidylserine externalization) and necrosis, respectively. The cells were untreated (a, b) or previously treated (c, d) with hGAPDH (2-32) for 1 h. Subpanels b and d are phase-contrast micrographs. Subpanels a and b show annexin and PI staining. (e) Percentage of 300 fungal cells that are classified as apoptotic [annexin (+) PI(−); green bars] and necrotic [annexin(+/−) PI(+); red bars] after treatment with hGAPDH (2-32) and H₂O₂. Bar=10 μm.

Figure 5. Apoptotic marker induced by 5 μg/ml hGAPDH (2-32) on C. albicans cells

Representative micrographs showing cells stained with FITC-annexin V (green) and propidium iodide (PI, red) to detect apoptosis (phosphatidylserine externalization) and necrosis, respectively. The cells were untreated (a, b) or previously treated (c, d) with hGAPDH (2-32) for 1 h. Subpanels b and d are phase-contrast micrographs. Subpanels a and b show annexin and PI staining. (e) Percentage of 300 fungal cells that are classified as apoptotic [annexin (+) PI(−); green bars] and necrotic [annexin(+/−) PI(+); red bars] after treatment with hGAPDH (2-32) and H₂O₂. Bar=10 μm.

Figure. 6. Expression of cytokines and TLR4 by hGAPFH (2-32)

IL-8, GM-CSF secretion (a) and TLR expression (b) of epithelial cells 12 h after infection (or not) with C. albicans SC5314 in the presence and absence of 5 μg/ml hGAPDH (2-32) (n = 6). Data are expressed as means±SD from duplicate assays of three different experiments. Statistical significance was determined using the 2-tailed paired Student’s t test. A p value of 0.01 or less was considered significant (n = 6).

Figure. 6. Expression of cytokines and TLR4 by hGAPFH (2-32)

IL-8, GM-CSF secretion (a) and TLR expression (b) of epithelial cells 12 h after infection (or not) with C. albicans SC5314 in the presence and absence of 5 μg/ml hGAPDH (2-32) (n = 6). Data are expressed as means±SD from duplicate assays of three different experiments. Statistical significance was determined using the 2-tailed paired Student’s t test. A p value of 0.01 or less was considered significant (n = 6).

Figure. 6. Expression of cytokines and TLR4 by hGAPFH (2-32)

IL-8, GM-CSF secretion (a) and TLR expression (b) of epithelial cells 12 h after infection (or not) with C. albicans SC5314 in the presence and absence of 5 μg/ml hGAPDH (2-32) (n = 6). Data are expressed as means±SD from duplicate assays of three different experiments. Statistical significance was determined using the 2-tailed paired Student’s t test. A p value of 0.01 or less was considered significant (n = 6).