All experimental protocols were approved by the Ethics Committee of the Institute for Laboratory Animal Research of the China-Japan Union Hospital of Jilin University (Jilin, Changchun) and were implemented in accordance with the guidelines for laboratory animal management of Jilin University. Female BALB/c mice (8–10-week-old; body weight: 16–20 g) were purchased from Yisi Laboratory Animal Technology Co., Ltd. (Changchun, Jilin, China). To reduce pain in the experimental animals, all operations were performed under adequate anesthesia with phenobarbital.

Sporothrix used in the experiment was isolated from a patient with disseminated sporotrichosis. The separated strains underwent strict transformation culture and molecular characterization and were identified as Sporothrix globosa. Sporothrix globosa was first cultured on Sabouraud’s medium at 25°C. Seven days later, the cultured fungi were transferred to brain heart infusion to culture at 37°C for 7 days with a shaker speed of 150 rpm. The culture fluid was centrifuged, resuspended in PBS, and diluted to 1 × 10⁸ cells/mL.

We previously reported ToAP2D, a 15-amino acid peptide (Antifungal Activity of ToAP2D 2021), which showed higher antifungal activity against S. globosa. Antifungal peptide ToAP2D was synthesized by ChinaPeptides Co., Ltd. (Suzhou, China), and its purity was determined to be higher than 95% using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry.

Fuse-55, the phage vector, was reproduced in the E. coli TG1 strains. The wild phages used in this experiment were maintained in our laboratory. Construction of the recombinant phage vector fuse-55 and recombinant phage are described in the corresponding literature (Malik et al., 1996; Yang et al., 2005). Briefly, the synthesized complementary oligonucleotides encoding peptide GK were connected to BglI-digested fuse-55. E. coli TG1 transfected with the recombinant phage vector inserted into the peptide was cultured in Luria-Bertani liquid medium containing ampicillin. The culture supernatant containing phage particles was collected and precipitated twice in polyethyleneglycol-6000 (PEG-6000; final concentration: 5%; 0.5M NaCl) for 12 h. The phage particles were then resuspended in 2 ml of phosphate-buffered saline (PBS). The concentration of phage particles was determined using a spectrophotometer (OD at 270 nm was 1.0, corresponding to 0.26 μg/μL).

Antibodies to AMPs displayed on bacteriophages were purchased from China Peptides Co., Ltd. (Suzhou, China). The protein was extracted from the recombinant phage, denatured, electrophoresed, and transferred onto the PVDG membrane, which was then placed in Tris-tricine buffer saline (TBST). Subsequently, the protein membrane was blocked with 3% skim milk powder at 37°C for 60 min, followed by membrane washing with TBST four times for 3 min each. Next, the nitrocellulose membrane was incubated with 1:2000 diluted serum in TBST and 5% skim milk at 37°C for 1 h. After adding oxidase-coupled goat anti-mouse IgG (Vector Laboratories Inc., United States), the mixture was incubated at 37°C for 1 h. Finally, the membrane was put into the chemiluminescence-based image analysis system for development for 3 min.

A total of 50 μL of bacterial solution (concentration: 1 × 10⁸ cells/mL) was coated onto broth solid medium, and autoclaved filter paper (0.5 cm in diameter) was pasted onto the surface of the solid medium. Then, 6 μL of hybrid phage expressing peptide GDKLIPGVMKLFRKK (phage-GK (PG)) (concentration: 4, 2, and 1 mg/ml by AMP concentration) and the control sample were dropped onto the filter paper and left in an incubator at 37°C for inverted culture for 5–7 days; the zone of inhibition was then observed.

The strains were divided into experimental and control groups; the former used a laser, while the latter was not illuminated. The two groups were cultured for 4 h in a 24-well plate on a cell culture slide. The supernatant was discarded, and each well was rinsed in PBS once and then fixed with 2.5% glutaraldehyde (0.2 M PBS, pH 6.0) for 1 h at 4°C. After fixation, the disks were rinsed three times with PBS and dehydrated in a graded series of ethanol (30, 50, 70, 80, and 90% for 7 min each and 100% for 10 min). The disks were critical point dried prior to being coated with a gold sputter and observed using SEM.

The strains were centrifuged at 5,000 × g for 5 min. Next, the cells were collected and resuspended in 800 μL of cell staining buffer. Hoechst 33342 (5 µL) staining solution was added to the solution and mixed well by turning it upside down. Then, 5 μL of propidium iodide (PI) was added to the solution and mixed as described above. The solution was then placed in an ice bath for 30 min. It was then rinsed once with PBS, smeared, and observed under a laser scanning confocal microscope (LSCM).

Sporothrix globosa treated with the recombinant phage (1 × 10⁸ CFU/ml) was washed and resuspended in PBS (pH 7.4). Then, JC-1 was added to the mixture at a final concentration of 5 μM, followed by incubation at 37°C for 20 min. Fluorescence absorption was detected using a fluorescence spectrophotometer at an excitation and emission wavelength of 490 and 530 nm, respectively.

DCFH-DA was diluted in yeast extract-peptone-dextrose (YPD) liquid medium at 1:1,000 to a final concentration of 10 μM. The Sporothrix solution was obtained after overnight culture, and the cells were collected and suspended in diluted DCFH-DA. The cells, at a concentration of 1 × 10⁸ cells/mL, were left for incubation at 37 °C for 20 min. The cells were washed with YPD liquid medium three times. Cells in the experimental and control groups were stimulated directly with reactive oxygen species (ROS)-positive controls and solvents. After 20 min, fluorescence absorption was measured using a fluorescence tester at an excitation and emission wavelength of 490 and 530 nm, respectively. ROS accumulation was calculated using the formula: (F_test-F_blank)/(F_control-F_blank), where F_test is the sample fluorescence value after laser treatment, F_control is the sample fluorescence value in the PBS treatment group, and F_blank is the fluorescence value in wells without cells.

After laser treatment, the Sporothrix solution from the experiment and control groups was centrifuged at 2000 × g for 3 min, and the cells were resuspended. Then, the cells were stained with 10 μM of CaspACE FITC-VAD-FMK for 20 min at 37°C, followed by rinsing with PBS twice. The cells were then smeared and observed under LSCM. The fluorescence absorption was tested using a fluorescence tester at an excitation and emission wavelength of 494 and 530 nm, respectively.

The mouse model of disseminated Sporothrix globosa was administered an intravenous injection of 0.2 ml (1 × 10⁸ cells/mL) of Sporothrix globosa suspension. Mice were randomly divided into four groups, each containing six mice. One day after infection, the following drugs were injected into the tail vein of each mouse every 3 days (6 injections in total): (1) 25 μg of PG in 100 μL PBS (PG group), (2) 25 μg of wild-type (WT) phage particles in 100 μL PBS (mock group), (3) ITR (5 mg/kg body weight, ITR group), or (4) PBS alone (PBS group; negative control). The mice were sacrificed on days 8 and 20 of infection.

On days 8 and 20, the mouse sera were isolated and frozen at −80°C before use. Mouse serum IFN-γ and IL-17 levels were measured using Mlbio (Shanghai, China) ELISA kits, according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a 96-well plate reader.

Mouse lung tissues were collected from each group and fixed in 10% buffered formalin for 24 h. After dehydrating and embedding in paraffin, the tissues were sectioned at approximately 5 µm using a paraffin slicer. Subsequently, the sections were stained with H&E to analyze the degree of inflammatory cell infiltration in the lung tissues, and the ImageJ software was used to quantify the percentage area of inflammatory cells on day 20. Neutrophils have very characteristic nuclei with three to five lobes connected by thin strands; and the size of neutrophils is about 10–15 µm. Lymphocytes are small cells, 8 to 10 microns in diameter and have large round nuclei.

Mice were randomly divided into four groups of 10 mice each. Sporothrix globosa suspension (0.2 ml; 5 × 10⁸ cells/mL) was injected into the tail vein of mice. On day 1 after infection, the mice were intraperitoneally injected with the above drugs. We observed a survival time of 2 weeks after treatment.

The mice were randomly divided into two groups of seven mice each. The experimental group was injected with 25 μg of PG in 100 μL of PBS into the tail vein, and the control group was injected with 100 μL of PBS. Blood was drawn from the tail vein on day 3, and aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (Cr) levels were measured.

Statistical analyses were performed using SPSS v24 (SPSS, Chicago, IL, United States). Values are expressed as the mean ± standard error of the mean for normally distributed data. One-way analysis of variance (ANOVA) and the Kruskal–Wallis H or Mann–Whitney U tests were performed to evaluate between-group differences. We examined the differences in survival time between different groups using the Kaplan-Meier test. The criterion for statistical significance was set at p < 0.05.

The expression of PG was tested using western blotting. According to the reaction of AMP antibodies with fusion proteins in the hybrid phage, AMP-GK was displayed on the surface of the recombinant phage (Figure 1).

The inhibitory effect of PG on Sporothrix globosa was analyzed using the bacteriostatic circle test. The results showed that the recombinant phage had no obvious antifungal effects at a concentration of 1 mg/ml. When the concentration was increased to 2 and 4 mg/ml, the recombinant phage had an inhibitory effect on Sporothrix globosa. The inhibitory effect was obvious (11.15 ± 1.38 mm) when the concentration of recombinant phage was 4 mg/ml. This suggests that PG (antifungal peptide at a concentration of 4 mg/ml) has an effective antifungal activity against S. globosa (Table 1). Therefore, we used PG (antifungal peptide at a concentration of 4 mg/ml) for our experiments.

Morphological changes on the surface of Sporothrix cells after recombinant phage treatment were observed using SEM. After the control treatment, S. globosa cells showed normal morphology, smooth and complete surface, regular shape, and clear boundaries. However, after recombinant phage treatment, Sporothrix became irregular in shape, with shrinkage and rupture of the cell membrane, cross-linked strains, and leakage of cell contents (Figure 2).

Hoechst 33342 can penetrate the cell membrane and bind to the chromosomes in apoptotic cells. Apoptotic cells showed significantly enhanced fluorescence compared to normal cells after staining (Yin et al., 2014). However, PI cannot penetrate the cell membrane. Normal or apoptotic cells with complete membranes were not stained. After double staining, under a fluorescence microscope, apoptotic cells showed faint red and strong blue fluorescence; necrotic cells showed strong red and strong blue fluorescence. In the recombinant phage treatment group, strong blue and red fluorescence (Hoechst+/PI+) were observed, indicating both apoptosis and necrosis in S. globosa. In contrast, in the control group, only faint blue and red fluorescence (Hoechst+/PI-) were observed, indicating a normal state of S. globosa (Figure 3).

Mitochondrial dysfunction initiates cell apoptosis (Eisenberg et al., 2007). At a higher mitochondrial membrane potential (MMP), JC-1 accumulates in the mitochondrial matrix and forms J-aggregates, producing red fluorescence. At lower MMPs, JC-1 cannot gather in the mitochondrial matrix but is a monomer that produces green fluorescence. The relative proportions of red and green fluorescence were used to measure mitochondrial depolarization. Through the transition of JC-1 from red to green fluorescence, it is easy to detect a decline in the cell membrane potential. Such a JC-1 transition can serve as an index for testing early cell apoptosis. According to the immunofluorescence results, compared with the control group, the recombinant phage treatment group showed a transition from red to green fluorescence. The fluorescence ratio of S. globosa cells in the control group was almost 25.84 ± 1.34, confirming no obvious depolarization. However, the fluorescence ratio of S. globosa cells in the recombinant phage group was reduced to 4.24 ± 0.26 (mean ± SEM) (p < 0.001), indicating no significant depolarization in S. globosa after the treatment (Figures 4A, B). In short, recombinant phages cause a decline in the MMP in S. globosa, indicating cell apoptosis.

Mitochondrial depolarization, accompanied by an endogenous increase in ROS, is a characteristic of cell apoptosis (Nury et al., 2021). After recombinant phage treatment, ROS accumulation in cells was significantly improved, without a significant difference from the positive control. No change was observed in ROS accumulation in the control group. This indicates that recombinant phage-induced cell apoptosis may be achieved through the ROS-mediated apoptosis pathway (Figure 5).

The fungal apoptotic pathway consists of various regulatory factors and effectors (Sharon et al., 2009). In this study, we focused on the caspase apoptotic pathway. Based on the experimental results, a small amount of fluorescence was detected in the blank control. However, in the recombinant phage treatment group, most cells showed bright green fluorescence, indicating that the recombinant phage activated the caspase-dependent cell apoptosis pathway in Sporothrix cells (Figure 6).

To assess the development of cell-mediated immunity, the levels of cytokines IFN-γ and IL-17 were assessed using ELISA on days 8 and 20. As shown in Figure 7A and C, the serum IFN-γ and IL-17 levels were significantly higher in the PG group (p < 0.001) than in the PBS group on day 8. Serum IL-17 levels were also significantly higher in the PG group than those in the WT and ITR groups (p < 0.01). On day 20 (Figures 7B, D), no significant changes in IFN-γ and IL-17 levels were observed in any of the study groups. These results showed that recombinant phage displaying ToAP2D peptide induced IFN-γ and IL-17 expression. IFN-γ and IL-17 are the main cytokines secreted by Th1 and Th17 cells, respectively. We speculated that phage displaying ToAP2D peptide activate Th1 and Th17 responses, promoting antifungal immunity (Figure 7).

The organs showed no obvious lesions as observed under naked eye. However, after H&E staining, on day 8, mice in the PBS (55.37 ± 13.20%) (Figures 8A, E) and wild phage groups (50.34 ± 7.29%) (Figures 8B, E) showed invasion of a large number of lymphocytes and neutrophils in the lungs, indicating significant inflammation. In contrast, in the recombinant phage (24.28 ± 5.69%) (Figures 8D, E) and ITR groups (19.95 ± 4.93%) (Figures 8C, E), lymphocytes and neutrophils of only medium levels were observed, indicating mild inflammation.

The detection time for mouse survival was 14 days (Figure 9). The experimental results showed that mice in the experimental group injected with recombinant phage and ITR showed the highest 14-day survival, reaching 80 and 70%, respectively, with no significant difference. Mice injected with PBS showed significantly reduced 14-day survival (only 20%), and survival of the WT group was 30%. Therefore, the survival rate of the recombinant phage-immunized mice was significantly higher than that of the PBS and WT groups.

Mice injected with recombinant phage showed clinical biochemical indexes, including AST, ALT, urea, and creatinine, within normal limits, with no significant difference from the control group (Figure 10).