Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide

Abstract

The unexpected resistance of psoriasis lesions to fungal infections suggests local production of an antifungal factor. We purified Trichophyton rubrum-inhibiting activity from lesional psoriasis scale extracts and identified the Cys-reduced form of S100A7/psoriasin (redS100A7) as a principal antifungal factor. redS100A7 inhibits various filamentous fungi, including the mold Aspergillus fumigatus, but not Candida albicans. Antifungal activity was inhibited by Zn(2+), suggesting that redS100A7 interferes with fungal zinc homeostasis. Because S100A7-mutants lacking a single cysteine are no longer antifungals, we hypothesized that redS100A7 is acting as a Zn(2+)-chelator. Immunogold electron microscopy studies revealed that it penetrates fungal cells, implicating possible intracellular actions. In support with our hypothesis, the cell-penetrating Zn(2+)-chelator TPEN was found to function as a broad-spectrum antifungal. Ultrastructural analyses of redS100A7-treated T. rubrum revealed marked signs of apoptosis, suggesting that its mode of action is induction of programmed cell death. TUNEL, SYTOX-green analyses, and caspase-inhibition studies supported this for both T. rubrum and A. fumigatus. Whereas redS100A7 can be generated from oxidized S100A7 by action of thioredoxin or glutathione, elevated redS100A7 levels in fungal skin infection indicate induction of both S100A7 and its reducing agent in vivo. To investigate whether redS100A7 and TPEN are antifungals in vivo, we used a guinea pig tinea pedes model for fungal skin infections and a lethal mouse Aspergillus infection model for lung infection and found antifungal activity in both in vivo animal systems. Thus, selective fungal cell-penetrating Zn(2+)-chelators could be useful as an urgently needed novel antifungal therapeutic, which induces programmed cell death in numerous fungi.

Keywords: S100A7; antifungal; epithelial defense; innate immunity; psoriasin.

Fig. 1.

Identification of reduced S100A7 (redS100A7) as an antifungal protein. (A) Psoriatic scale extracts contain T. rubrum antifungal activity (n = 3 *P < 0.001). (B) SDS/PAGE and Western blot (WB)-analyses of the antifungal protein purified from the psoriasis scales. (C) redS100A7 is a potent antifungal protein compared with oxS100A7, reduced and alkylated S100A7 (red/alkS100A7), and S-sulfito-S100A7 (sulfS100A7). *P < 0.01, **P < 0.007 and ***P < 0.001. (D) redS100A7 decreases cell viability in T. rubrum dose-dependently. Viable fungi were detected by the MTT tetrazolium salt colorimetric assay. n = 3 independent experiments. Mean ± SD is shown. *P < 0.008. (E) Partial ascorbic acid (AA) sequences of redS100A7 and the three mutants. (F) Whereas antifungal activity is absent in all three Cys-Ala-mutants (*P < 0.002), antibacterial activity is still present. A minimum of three independent experiments was performed and the average ± SD is plotted.

Fig. S1.

Purification of the antifungal protein from psoriatic scales and properties. (A) Psoriatic scale extracts (molecular weight range: 3–50 kDa) contain T. rubrum antifungal activity (n = 3 independent experiments, *P < 0.0004). Psoriatic scale extracts (molecular weight range: 3–50 kDa) were first separated by heparin-affinity chromatography (B), followed by C₈ RP-HPLC of heparin-binding proteins (C). The fraction containing the highest antifungal activity was further purified by cation-exchange chromatography (D). As a final purification step, C₁₈ RP-HPLC was used (E). Extracts and HPLC fractions were tested in a microbroth dilution assay. (F) The molecular mass of the antifungal protein was analyzed by MALDI-MS using two internal calibration standards (IS). (G) The HPLC-purified protein fraction was incubated with a neutralizing monoclonal S100A7 antibody in phosphate buffer saline for 1 h. The mixture was tested against T. rubrum in a microbroth dilution assay. The S100A7 antibody (ab45091, Abcam) blocked antifungal activity of the purified protein preparation (n = 3 independent experiments, **P < 0.002). (H) ESI-MS analysis of Zn²⁺-bound redS100A7. Zinc-treated redS100A7 was analyzed by ESI-MS in ammonium formate buffer at pH 5.5. We found a peak at 22,958 Da, which indicates a dimer of red100A7 containing zinc and buffer ions and likely H₂O. (I) Antifungal activity of redS100A7 is pH-dependent. Note that redS100A7 works optimally between pH 4 and 7 and is sensitive to alkaline pH. n = 3 independent experiments, *P < 0.0001. Mean ± SD is shown. Statistical analysis was performed with two-tailed Student’s t test. Antifungal assays were performed in duplicate and repeated at least three times.

Fig. 2.

redS100A7 sequesters Zn²⁺ ions in fungal cells. (A) redS100A7 antifungal activity is inhibited by addition of Zn²⁺ (*P < 0.05, **P < 0.001 vs. 0 μM Zn²⁺). (B) CD spectroscopy of redS100A7 at pH 5.4 in the presence or absence of Zn²⁺. Note changes in α-helical content characterized by a deep minimum between 209 and 222 nm. (C) In redS100A7-treated T. rubrum, intracellular liable Zn²⁺ are stained with a specific fluorescent probe, Zinquin. (Magnification: 300×.) (D) Antifungal activity (T. rubrum) of different Zn²⁺-chelators. TPEN is a cell-permeable chelator, and DTPA and EDTA are cell-impermeable chelators (*P < 0.002, **P < 0.001). A minimum of three independent experiments was performed and the average ± SD is plotted.

Fig. S2.

Calcium (Ca²⁺)-dependent antifungal activity of redS100A7. (A) T. rubrum conidia were incubated with redS100A7 in the presence or absence of a 10 mM Ca²⁺ supplement. Fungal growth was measured at the optical density of 595 nm. Sterile saline was used as control. Addition of Ca²⁺ increased the efficacy of redS100A7 antifungal activity (n = 3 independent experiments, *P < 0.002 vs. Ca ²⁺-free control). (B) Ca²⁺ alone showed no effect on the fungal growth up to 50 mM. CaCl₂ was used as Ca^2+-source. Mean ± SD is shown. Statistical analysis was performed with two-tailed Student’s t test. (C) 5 mM of AA blocked the antifungal activity of redS100A7 (**P < 0.001). (D) Fungal Cu/Zn-SOD enzyme activity was measured in the presence or absence of redS100A7. (ns, not significant). (E) redS100A7-induced antifungal activity was not inhibited by ergosterol, in contrast to the control antimycotic amphotericin B (Amp-B) at 500 μM ergosterol. (***P < 0.0007). Mean ± SD is shown. Statistical analysis was performed with two-tailed Student’s t test. Data are representative of three independent experiments.

Fig. 3.

Action mechanism of redS100A7. (A) Broad-spectrum antifungal activity of redS100A7 against various fungi. (B) Broad-spectrum antifungal activity of TPEN. (C) Intracellular ROS (determined with the fluorescent probe carboxy-H2DCFA) were dose-dependently raised in redA100A7-treated conidia. The ROS-inhibitor AA prevented the ROS-generation (4 h, *P < 0.0003, n = 3 independent experiments). (D) TUNEL assay (to observe DNA fragmentation) and SYTOX-green staining (to observe disintegrated plasma membrane) of redS100A7-treated (4 μM) T. rubrum. (Scale bars, 20 μm.) (E) TUNEL assay and SYTOX-green staining of redS100A7-treated (4 μM) A. fumigatus. (Scale bars, 20 μm.) (F) TEM images of redS100A7 (4 μM)-treated T. rubrum. Arrows indicate electron-dense depositions and arrowhead represents dilated nucleolemmal cisterns. (Scale bars, 0.2 μm.) (G and H) SEM analyses of redS100A7-treated (4 μM) A. fumigatus (G) and controls (H). Note destroyed fungal spores (conidia), premature conidia and conidia-producing phialides (circle). (Scale bars, 2 μm.) CM, cell membrane; CW, cell wall; M, mitochondria; N, nucleus; R, ribosomes; V, vacuole. (I) A cell-permeant pan caspase inhibitor blocks the antifungal activity of redS100A7 in a dose-dependent manner. (n = 3 independent experiments, *P < 0.02.)

Fig. 4.

Target localization of redS100A7. (A) redS100A7-immunofluorescence visualization (arrows) on T. rubrum by confocal laser microscopy. Note redS100A7 accumulation on conidia (arrows). (Scale bars, 50 μm.) (B) Confocal laser microscopy of 2 h (Left) or 4 h (Center)-treated A. fumigatus. Arrows indicate the location of redS100A7 on conidia at 2 h and hyphae at 4 h. (Scale bars, 50 μm.) (C) Immunogold labeling electron microscopy (Upper and Lower). Arrows point toward gold particles that represent redS100A7 immune complexes. Arrowhead indicates gold particles accumulating within the conidia. (Scale bars, 0.2 μm.) C, conidium; CM, cell membrane; CW, cell wall; H, Hyphae; L, lipid droplet; M, mitochondria; N, nucleus; R, ribosomes; V, vacuole. Independent experiments have been repeated at least three times and showed similar results.

Fig. S3.

SEM of A. fumigatus. A. fumigatus conidia were cultured on a Sabouraud dextrose agar for 7 d. The agar was cut into 25 mm² and incubated with redS100A7 (A) or TPEN (B) for 24 h. Sunken filaments, fungal cell debris, and damaged conidia were observed in both, redS100A7- and TPEN-treated fungi.

Fig. S4.

redS100A7 accumulates primarily at conidia. (A) Confocal laser microscopic immunofluorescence analyses with S100A7 antibodies reveal after 4-h incubation preferential accumulation of redS100A7 (arrows) at conidia surfaces of T. rubrum and A. fumigatus. (Scale bars, 20 μm.) (B) Immunogold TEM images of redS100A7-treated T. rubrum. The possible T. rubrum-entry site of redS100A7 after 4 h treatment. (Scale bar, 0.5 μm.) (C) A boxed area in B is enlarged in C. Gold particles (which represent redS100A7) inside the cells, close to an area missing the cell wall (arrow). (Scale bar, 0.5 μm.) (D and E) Gold particles (arrowheads) were seen along the outer cell wall. Intact cell wall and intracelluar organelles were only detected in a time period when gold particles were outside the fungal cell. (Scale bar, 0.5 μm.) (F) The Cys46Ala-S100A7 mutant (mutant) is able to penetrate the fungal cell membrane. T. rubrum, grown on an agar plate, was treated with the antimycotically inactive Cys46Ala-S100A7 for 24 h. After pretreatment of T. rubrum with the mutant psoriasin, fungi were treated with a polyclonal S100A7 antibody (which recognized the mutant S100A7, mutant1, upon immunodot analysis) and then analyzed by confocal laser-scanning microscopy for immunofluorescence. Note the focal accumulation of the psoriasin mutant (arrows) close to conidia. (Scale bars, 50 μm.) CM, cell wall; CW. cell membrane; M, mitochondria; N, nucleus; Nu, nucleolus; V: vacuole. (G) Quantitation of redS100A7 gold particles in the conidia and hyphae. (n = 3, *P < 0.001).

Fig. S5.

Disulphide reduction of S100A7 protein in vivo. (A) S100A7 levels in healthy human skin (24.2 ± 5.3 U, n = 4) and dermatophyte-infected skin (64.2 ± 5.6 U, n = 5, P < 0.0001 versus healthy; Student’s t test), determined by RP-HPLC. (B) Redox status of S100A7 in healthy and infected skin. The ratios of oxS100A7/ redS100A7 in healthy (n = 4) and dermatophyte-infected skin scales (n = 5) were 100/85.1 ± 5.7 and 100/154.0 ± 6.1, respectively (redS100A7: P < 0.0001 vs. healthy; Student’s t test). redS100A7α, mono–CAM-S100A7; redS100A7β, bi–CAM-S100A7. (C) The TRX-R system reduces the disulphide-bridge of oxS100A7. The reduction was analyzed by RP-HPLC. A minimum of three independent experiments was performed and the average ± SD is plotted.

Fig. S6.

The GSH system reduces the oxS100A7. This experiment was performed by using HPLC. Results are representative of three independent experiments.

Fig. S7.

Reduction of S100A7 derivatives. (A) S-sulfito-S100A7 is produced by T. rubrum. oxS100A7 was incubated in T. rubrum culture medium for 24 h. The medium was collected and applied to a C₈ RP-HPLC column. The fraction containing S100A7 derivatives revealed only S-sulfito-S100A7 (11,445.60) as seen by MALDI-MS. (B) S-sulfito-S100A7 is converted toward redS1007 by the TRX-R system. The TRX-R system was able to reduce S-sulfito-S100A7, which shows a partial disproportionation toward oxS100A7 at experimental conditions, back to redS100A7 in a dose-dependent manner. (C) Oxidized GSH (GSSG) inhibits oxS100A7 antifungal activity (*P < 0.02; Student’s t test). Results are representative of three independent experiments.

Fig. 5.

In vivo antifungal activity of redS100A7 and TPEN. (A and B) After applied with vehicle, redS100A7 or TPEN, the plantar of guinea pigs was infected with T. mentagrophytes. Fungal invasion was determined histologically (A) and by scoring (B) (see SI Materials and Methods). (n = 4, *P < 0.005, **P < 0.0001). (C) Immunocompromised mice were infected with A. fumigatus and treated with redS100A7, TPEN, or the vehicle. The Kaplan–Meier survival curve comparing untreated control mice (black) with redS100A7-treated (red) and TPEN-treated mice (green) (n = 7 for each group, *P < 0.036 and **P < 0.003 vs. controls; log-rank test) is shown. (D) Histopathology and SEM analyses of mouse lungs infected with A. fumigatus. Arrowheads indicate A. fumigatus. Grocott’s Methenamine Silver (GMS), H&E. A, alveolus; Ad, alveolar duct; B, bronchiole; R, red blood cell; S, septum. (Magnification: D, GMS and H&E, 200×; SEM, 1,700×.)

Fig. S8.

Guinea pig model of tinea pedis. The foot of guinea pig was lightly abraded. Vehicle, redS100A7, and TPEN were applied to the skin and air-dried. The skin was then infected with 0.15 mL of 1 × 10⁸ conidia per milliliter of T. mentagrophytes and covered with a film. The infected sites were then covered with bandages. After 3 d of infection, the bandages were removed. The skin tissue was collected from the feet. The fungal tissues in the skin were observed microscopically after staining with PAS. (Magnification: 100×.)

Fig. S9.

(A) At the end of 7-d observation period, redS100A7- and TPEN-treated mice were cured from A. fumigatus infection, compared with the vehicle-treated mice at 3-d after infection. Histopathology of mouse lung after A. fumigatus infection and treatment with redS100A7 or TPEN. (B, Top) Untreated, redS100A7-treated and TPEN-treated mice after intranasal challenge with 2 × 10⁷ A. fumigatus conidia for 2 consecutive days (n = 7 mice for each group). Mouse lungs were stained with Grocott's Methenamine Silver (GMS), PAS, and H&E stain. In untreated control lungs, numerous A. fumigatus conidia and hyphae (arrows) were observed. Neutrophils and monocytes were the dominating leukocytes in infected tissue sites. In redS100A7- and TPEN-treated lungs, less fungi were observed (mainly conidia). After a 7-d observation period, redS100A7- and TPEN-treated lungs were cured from A. fumigatus infection. Monocytes and lymphocytes were the dominating leukocytes in the treated lungs.

Fig. S10.

redS100A7 inhibits the growth of hydrophobin-mutant A. fumigatus. A. fumigatus ΔKU80 (parent strain) (final 1 × 10⁵/mL) and hydrophobin-mutant A. fumigatus ΔKU80 ΔrodA (final 1 × 10⁵/mL) were cultured in the 96-well plate. Serial dilutions of redS100A7 were added to the assay medium. Fungal growth was measured at the absorbance of 595 nm. n = 3 independent experiments. Mean ± SD is shown. Both A. fumigatus ΔKU80 (parent strain) and A. fumigatus ΔKU80 ΔrodA were kindly provided by J. P. Latgé, the Aspergillus Unit at the Pasteur Institute in Paris, France.