Antifungal Peptide SP1 Damages Polysaccharide Capsule of Cryptococcus neoformans and Enhances Phagocytosis of Macrophages

C. neoformans is an opportunistic pathogen that causes invasive infections with a high mortality rate. Currently, the clinical drugs available for the treatment of cryptococcosis are limited to amphotericin B, azoles, and flucytosine. ABSTRACT Cryptococcus neoformans is a fungal pathogen which causes nearly half a million deaths worldwide each year. Under host-relevant conditions, it produces a characteristic polysaccharide capsule. The polysaccharide capsule is one of the main virulence factors of C. neoformans, which involves antiphagocytosis and immune responses of the host to cause a lack of an immune. Meanwhile, the polysaccharide capsule is a promising drug target because of the absence of analogs in the host. Here, we demonstrate that antifungal peptide SP1, which is derived from the N terminus of Saccharomyces cerevisiae GAPDH (glyceraldehyde-3-phosphate dehydrogenase), disrupts the polysaccharide capsule of C. neoformans H99. The mechanism is possibly due to the interaction of SP1 with glucuronoxylomannan (GXM). Disruption of the polysaccharide capsule enhances the adhesion and phagocytosis of C. neoformans H99 by macrophages and reduces the replication of C. neoformans H99 within macrophages. Additionally, SP1 exhibits antifungal activity against cryptococcal biofilms associated with the capsular polysaccharides. These findings suggest the potential of SP1 as a drug candidate for the treatment of cryptococcosis. IMPORTANCE C. neoformans is an opportunistic pathogen that causes invasive infections with a high mortality rate. Currently, the clinical drugs available for the treatment of cryptococcosis are limited to amphotericin B, azoles, and flucytosine. Amphotericin is nephrotoxic, and the widespread use of azoles and 5-flucytosine has led to a rapid development of drug resistance in C. neoformans. There is an urgent need to develop new and effective anticryptococcal drugs. Targeting virulence factors is a novel strategy for developing antifungal drugs. The antifungal peptide SP1 is capable of disrupting the polysaccharide capsule, which is a principal virulence factor of C. neoformans. Studying the mechanism by which SP1 damages the polysaccharide capsule and investigating the potential benefits of SP1 in removing C. neoformans from the host provides baseline data to develop a therapeutic strategy against refractory cryptococcal infections. This strategy would involve both inhibiting virulence factors and directly killing C. neoformans cells.

with Escherichia coli or Saccharomyces cerevisiae when treated with the same concentration or even a higher concentration (256 mM) of SP1 ( Fig. 1C and D).
Given that the virulence of C. neoformans H99 decreased with the increase of the degree of flocculation (34), we investigated the possible mechanism of flocculation caused by SP1. For C. neoformans H99, treatment with 8 mM SP1 for 30 min killed only 11% of the cells (see Fig. S1 in the supplemental material) with obvious flocculation (Fig. 2B), although we did not observe any cell debris after treatment. Thus, it is unlikely that the flocculation was due to the aggregation of cellular substances released from the cells killed by SP1. Previous studies have shown that the lung surfactant protein SP-D can bind to the carbohydrates of the cell wall and induce the aggregation of acapsular C. neoformans in the presence of calcium ions (35,36). We performed EDTA and carbohydrate competition experiments to verify whether SP1 triggered C. neoformans H99 flocculation by the same mechanism. As shown in Fig. 2A and B, the presence of EDTA, mannose, galactose, maltose, or glucose did not inhibit the SP1-induced flocculation of C. neoformans H99 cells (P . 0.05). This observation suggested that SP1 caused flocculation by a different mechanism from that of SP-D.
The transient flocculation that occurs during the cultivation of C. neoformans is easily dispersed by vortexing (34). However, the aggregation of H99 cells caused by SP1 was not reversed by vigorous vortexing (Fig. S2). We observed that the acapsular cap59D mutants naturally flocculated in the logarithmic phase of culturing and that the degree of aggregation was increased after 8 mM SP1 was added to the culture of the cap59D mutant (Fig. 2D). Similarly, the cell aggregation of the cap59D mutant without SP1 or in the presence of SP1 was resistant to vortexing ( Fig. 2D and Fig. S4). Combining these observations of the cap59D mutants and the fact that the dense network structure of polysaccharide capsule was severely damaged after treatment of SP1 (32), we postulated that SP1 destroyed the capsule to expose proteins or polysaccharide, and the interactions of these exposed substances might induce the flocculation of C. neoformans H99. When the flocs were treated with 3 mg/mL proteinase K, the cell aggregation caused by SP1 was dispersed (Fig. 2C). In contrast, proteinase K digestion did not affect the spontaneous cell aggregation of the cap59D mutant (Fig. 2E). These observations implied that the SP1-induced flocculation of C. neoformans H99 might be due to the interactions mediated by SP1 or the exposed proteins. The mechanism is also different from the mechanism of spontaneous flocculation in cap59D mutants that might be mediated by polysaccharide. SP1 interacts with the glucuronoxylomannan of polysaccharide capsule. As SP1 damaged the polysaccharide capsule in a very short time, we hypothesized that SP1 would directly bind and break the polysaccharide capsule rather than regulating the expression of genes involved in capsule synthesis. Furthermore, our previous study showed that the killing activity of SP1 on C. neoformans H99 was due to an apoptosislike cell death rather than direct damage to the capsule. SP1 does not form pores on the cell membrane of C. neoformans H99 but interacts with membrane ergosterol and enters the vacuole to induce apoptosis-like cell death (32). Theoretically, the capsule can prevent SP1 from reaching the cell membrane or inside of C. neoformans H99, and the MIC of SP1 against C. neoformans H99 would change if damage to the capsule caused by SP1 was altered. If SP1 hardly damaged the capsule of a C. neoformans H99 Effects of carbohydrates and EDTA on the flocculation of C. neoformans H99 induced by SP1. After adding 5 mM EDTA, 20 mM mannose, 20 mM maltose, 20 mM galactose, or 20 mM glucose to C. neoformans H99 cultures, the cell cultures were treated with 8 mM SP1 for 30 min. Flocculation was observed through a microscope. Cells without any treatment were set as the negative control, and cells treated only with SP1 were the positive control. (B) Flocculation in 100 randomly selected visual fields was analyzed under the indicated experimental conditions. Student's t test was used to evaluate the significance between data sets, and a P value of ,0.05 (*) indicates a significant difference between the two data sets. (C) The treatment of proteinase K dispersed SP1-induced cell aggregation of C. neoformans H99. After flocculation was induced with SP1, 3 mg/mL proteinase K was added. Images were taken after 30 min of treatment. (D) Flocculation of acapsular mutant cap59D with or without 8 mM SP1. (E) Cell aggregation that formed spontaneously in the cap59D mutant was not dispersed by the treatment of protease K. mutant, the intact capsule would prevent the access of SP1 to the cell membrane. Under this condition, we expected that the MIC would increase. Therefore, we examined the MIC of SP1 against a series of capsular defect mutants to determine the possible interacting targets of SP1 in the capsule ( Table 1). The capsule of C. neoformans is composed primarily of two types of polysaccharides, GXM and glucuronoxylomannogalactan (GXMGal) (37,38). Both GXM and GXMGal contain xylose, glucuronic acid, and O-acetyl modifications, and O-acetyl modification mainly occurs on GXM (39). As shown in Table 1, the MIC of SP1 against wild-type C. neoformans H99 cells was 8 mM, which was same as that of SP1 against GXMGal-deficient strains [(uge1D [CNAG_00697] and uge1D [CNAG_03096])] and 2-fold higher than that of the xylose-deficient mutant uxs1D. The MIC of SP1 against O-acetyl-deficient mutant cas1D was increased by 7-fold compared with that of the wild-type cells (Table 1). These results implied that oxyacetylation modification would be related to SP1 interaction with capsule.
Furthermore, using the polysaccharide capsule monoclonal antibody 18B7 (MAb 18B7) against GXM oxyacetylation, we performed an immunofluorescence competition experiment to examine whether SP1 interacted with GXM. As shown in Fig. 3A, fluorescent images of control-peptide-treated cells displayed an annular pattern, whereas SP1treated cells showed a punctate pattern when incubated with fluorescent MAb 18B7. These results indicated that the O-acetyl modification on GXM would be a potential binding site for SP1. There is another possibility that SP1 breaks the capsule to form a punctate structure. To obtain independent evidence for the binding of SP1 to GXM, we used a microscale thermophoresis (MST) assay to detect the binding ability of SP1-fluorescein isothiocyanate (FITC) with GXM or deoxyacetylated GXM. As shown in Fig. 3, there is an evident binding between SP1 and GXM with a dissociation constant (K d ) of 4.13 6 0.857. The K d value for the interaction between SP1 and deoxyacetylated GXM was 107 6 15.8. Collectively, these observations suggested that SP1 interacted with GXM and that the oxyacetylation modification of GXM played an important role during the interaction.
To determine how the oxyacetylation modification affects the interaction between SP1 and GXM, we analyzed the molecular docking of SP1 and GXM using AutoDock Vina and PDBePISA (https://www.ebi.ac.uk/msd-srv/prot_int/pistart.html). The structure of SP1 was predicted using AlphaFold2 (Fig. 4A), and the structure of GXM decasaccharide with or without oxyacetylation was predicted using the GLYCAM carbohydrate builder (Fig. 4B). These analyses showed that the amino acids involved in the interaction between SP1 and GXM were polar amino acids such as arginine (Arg), isoleucine (Ile), asparagine (Asn), aspartic acid (Asp), and glutamic acid (Glu). If GXM decasaccharide was deoxyacetylated, Asp became the only amino acid that was involved in the interaction (Fig. S3). The results suggested that the GXM decasaccharide formed hydrogen bonds with the Asn. However, when the GXM decasaccharide was deoxyacetylated, no hydrogen bond was formed in the interaction between SP1 and GXM decasaccharide (Fig. S3). Thus, the predicted binding affinity decreased from 22.3 to 21.7 kcal/mol (Fig. 4C). SP1 treatment inhibits the GXM release and upregulates expression of genes related to polysaccharide capsule synthesis. To investigate how SP1 destroys the capsules through interaction with GXM, we first used India ink negative staining to examine the effect of SP1 on the size of the capsule. The SP1-treated cells had significantly smaller   neoformans H99 culture incubated with SP1 was not significantly different from that of the supernatant without incubation with SP1 ( Fig. S5), indicating that SP1 does not interfere with the measurement of GXM content in the ELISA. Therefore, SP1 prevents the release of GXM rather than causing GXM to be shed from the capsule during the damage to capsule. Moreover, we noticed that SP1 only significantly reduced the level of extracellular deoxyacetylated-GXM at a concentration of 64 mM (Fig. S6), which is 8-fold higher than that found in wild-type strains (8 mM). This observation supports the hypothesis that the oxyacetylation modification of GXM might be important for the binding of SP1. Additionally, given that C. neoformans can sense the content of the extracellular capsule to regulate the synthesis and release of capsule components (40), we evaluated the expression levels of capsular-related genes (CMT1, MAN1, GRASP1, and CAP10) after treating C. neoformans H99 cells with SP1. SP1-treated cells demonstrated an ;1.5-fold upregulation of CMT1 and MAN1 expression compared with that in control-peptide-treated cells ( Fig. 5D; P , 0.05). Furthermore, CAP10, an essential capsular gene, displayed an ;2-fold increase in expression in SP1-treated fungi compared with that in control-peptide-treated cells ( Fig. 5D; P , 0.01). Collectively, our results indicate that SP1 interacted with GXM to cause serious damage to the capsular network structure, which decreased the extracellular GXM content and upregulated expression of capsule-related genes. SP1 exhibits antifungal activity against C. neoformans H99 biofilms. GXM is a key constituent of the exopolymeric matrix of cryptococcal biofilm (41). We evaluated the effect of SP1 on growing and mature biofilms of C. neoformans H99. SP1 was added at the beginning of biofilm formation or after the biofilm reached maturity. The biomass and metabolic activity of biofilms were determined using crystal violet staining and the XTT (tetrazolium salt) reduction method, respectively. As shown in Fig. 6A and B, the biomass was reduced by 86.4%, and metabolic activity was reduced by 96.9% with the addition of 2 Â MIC (16 mM) of SP1 (P , 0.01) before biofilm formation. This suggested that SP1 could prevent C. neoformans H99 from efficiently forming a biofilm. As previously reported, we observed that amphotericin B inhibited biofilm formation but that fluconazole did not (31). For the mature biofilms, SP1 treatment did not reduce the biomass, which is different from the effect following treatment with amphotericin B (Fig. 6C). However, SP1 reduced the metabolic activity of the mature biofilm in a concentration-dependent manner (Fig. 6D). At concentrations of 8 Â MIC (64 mM), SP1 decreased the metabolic activity of biofilm by 73.6%  6D; P , 0.01). Hence, our results suggest that SP1 shows strong activity against cryptococcal biofilms. SP1 promotes the phagocytosis of C. neoformans H99 by macrophages. The polysaccharide capsule of C. neoformans is known to inhibit macrophage phagocytosis of C. neoformans (42,43). Because SP1 damages the polysaccharide capsule, we examined whether the treatment of SP1 enhanced the phagocytosis of C. neoformans. The adhesion and phagocytosis of macrophages were assessed for SP1-pretreated C. neoformans H99 cells. The adhesion of macrophage J774A.1 to C. neoformans H99 cells in the SP1-treated group was significantly enhanced compared with that of the control group ( Fig. 7A and C; P , 0.05), and the adhesive efficiency was increased by 49.7% (Fig. 7C). After treating C. neoformans H99 cells with SP1, the number of cells phagocytosed by macrophage was nearly 2-fold that in the control group ( Fig. 7B and D).
As C. neoformans can proliferate within macrophages owing to the protection of the polysaccharide capsule (18), we examined the proliferation of SP1-pretreated C. neoformans H99 that were actively ingested in macrophages. The C. neoformans H99 cells with an intact capsule or damaged capsule induced by 8 mM SP1 for 30 min were phagocytosed by J774A.1 macrophages, and fluconazole was added to inhibit the growth of C. neoformans that escaped to outside the macrophages. After 12 h from phagocytosis, the C. neoformans H99 cells with intact capsules in the control group displayed an average intracellular replication rate of 6.795-fold. However, the intracellular replication rate of SP1-pretreated cells in macrophages was only 2.805-fold ( Fig. 7E; P , 0.05). These data implied that the destruction of polysaccharide capsule by SP1 facilitated the killing by macrophages and prevented the immune escape of C. neoformans H99.

DISCUSSION
The options for anticryptococcosis treatment are limited because only three major classes of drugs have been approved for clinical use. The situation is deteriorating because the extraordinary genomic plasticity and physiological adaptability of C. neoformans has enabled the development of extensive drug resistance (11). SP1 is a peptide that shows activity against Cryptococcus spp. and can disrupt the integrity and reduce the thickness of Cryptococcus polysaccharide capsule. In this study, we investigated the mechanism whereby SP1 damages the structure of capsule. SP1 interaction with GXM may cause the destruction of capsule. Owing to capsule damage, the treatment of SP1 produced flocculation, inhibited the formation of Cryptococcus biofilm, and enhanced the clearance of C. neoformans by macrophages. This study laid a foundation for the potential of SP1 to treat cryptococcosis.
The polysaccharide capsule is one of the primary virulence factors in C. neoformans and can enhance pathogenicity, modulate immune responses, and protect against oxidative stress (15,44). Animal model studies suggest that the capsular mutants display reduced virulence or even avirulence (29,(45)(46)(47)(48)(49). The ability of SP1 to damage the capsule is a considerable advantage for the potential application in clinics. Our results demonstrated that SP1 caused the flocculation of C. neoformans H99, which may be the outcome of capsule disruption. The flocculation decreased the virulence because the adhesion property of aggregated cells enhanced the phagocytosis and clearance by lung macrophages (34). We also found that SP1 treatment enhanced the adhesion and phagocytosis of C. neoformans H99 by macrophages and hampered the replication of C. neoformans H99 within macrophages. Moreover, the obstruction of GXM release by SP1 alleviated the inhibitory effect of GXM on host immune regulation. Accordingly, SP1 not only directly killed C. neoformans but also benefited the host immune system to clear C. neoformans during the infection. In addition, SP1 would be a good candidate for drug combinations against C. neoformans, as polysaccharide capsule notoriously blocks the absorption of antifungal drugs. Studies reported that the capsule protects C. neoformans against polyenes and glycolipid-hydrolyzed inhibitors (50, 51). SP1 severely disrupts the structure of the polysaccharide capsule, which would facilitate the entry of antifungal drugs into the cells to exert fungicidal effects. The combination of SP1 and other antifungal agents would be a feasible strategy to improve therapeutic efficacy, lower side effects, and minimize development of drug resistance.
Cryptococcal biofilm has been reported to reduce the sensitivity to antifungal agents and various antimicrobial molecules produced by the innate immune system (52). GXM plays a key role in the biofilm formation of C. neoformans (30). We evaluated the effects of SP1 on biofilm formation and maturation using fluconazole and amphotericin B as the negative and positive controls, respectively. Consistent with the previous report, fluconazole showed no effect on the formation and metabolic activities of cryptococcal biofilms (31). In contrast, amphotericin B prevented the formation of cryptococcal biofilms at .0.5 mg/mL (2 Â MIC) and reduced the metabolic activity of mature biofilms by at least 50% at .1 mg/mL (4 Â MIC). The formation of the cryptococcal biofilm was efficiently prevented at 53.3 mg/mL (2 Â MIC) SP1. Thus, SP1 prevents the release of GXM to inhibit the formation of biofilm in C. neoformans. SP1 reduced the metabolic activity of mature biofilms by at least 50% at 213.3 mg/mL (8 Â MIC). Although the killing concentration required for SP1 to act on biofilms was higher than that in planktonic cells (8 Â versus 1 Â MIC), this is much lower than the estimated concentration (100 to 1,000 Â MIC) of antifungal agents according to previous studies (53,54). Thus, SP1 could be used to diminish the formation of cryptococcal biofilm.
GXM and GXMGal are two main polysaccharides in the C. neoformans capsule, and GXM accounts for approximately 90% of the capsule mass (55). Our study determined that SP1 directly interacted with GXM, and that O-acetyl modification played an important role during the interaction. The O-acetyl modification does not change the helical structure of GXM decasaccharide but exposes more polar groups of the GXM decasaccharide (56). Our molecular docking assay implied that the polar amino acids are important for the formation of hydrogen bonds. In addition, the isoelectric point of SP1 is 11.83, and SP1 is positively charged under neutral pH. Studies have shown that capsular polysaccharides have a negative charge. Therefore, SP1 could also interact with the polysaccharide capsule through electrostatic interaction. Although it is unclear how SP1 disrupts the polysaccharide capsule after binding to GXM, it is reasonable to postulate that SP1 disrupted the structure of the polysaccharide capsule by cross-linking the polysaccharide with SP1. Moreover, SP1 caused the flocculation of C. neoformans, and the treatment of proteinase K dispersed the flocculation. Thus, SP1 may bind to GXM and further disrupt the structure of the capsule by the aggregation of SP1. Future studies will seek to clarify the detailed molecular mechanism of how SP1 disrupts the capsule.
In conclusion, we demonstrated that SP1 interacts with GXM in the polysaccharide capsule of C. neoformans H99. The O-acetyl modification of GXM dramatically reinforces the interaction between SP1 and GXM. This interaction possibly leads to the disruption of the polysaccharide capsule. SP1 treatment reduces the release of GXM to the supernatant of liquid culture of C. neoformans H99. The capsule damage caused by SP1 enhances the adhesion and phagocytosis of C. neoformans H99 and impedes the proliferation of C. neoformans H99 in macrophages. These characteristics of SP1 enhance the innate immune response against the infection of C. neoformans. Furthermore, SP1 inhibits the formation of cryptococcal biofilms. As SP1 specifically kills Cryptococcus, the present findings highlight the additional benefits of SP1 for fighting against refractory C. neoformans infection by impairing C. neoformans capsule. Future studies will focus on advancing the promising clinical application of SP1.

MATERIALS AND METHODS
Strains, cells, and growth conditions. C. neoformans strain H99 and H99 mutant strains (cap59D were kindly provided by Linqi Wang at the Institute of Microbiology, Chinese Academy of Sciences. C. neoformans strains were revived on YPD (yeast extract peptone dextrose) solid medium at 30°C and grown in RPMI 1640 medium supplemented with MOPS (morpholinepropanesulfonic acid; 0.165 M, pH 7.0) at 37°C unless otherwise specified. Escherichia coli DH5a was cultured in LB medium at 37°C. Saccharomyces cerevisiae BY4741 was cultured in YPD medium at 30°C. Mouse macrophage cell line J774A.1 was acquired from Cuihua Liu at the Institute of Microbiology, Chinese Academy of Sciences. J774A.1 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and cultured at 37°C with 5% CO 2 .
Flocculation experiment. C. neoformans H99, E. coli DH5a, and S. cerevisiae BY4741 in logarithmic phase (optical density at 600 nm [OD 600 ], 1) were treated with different concentrations of SP1 (0, 4, 8, 16, 32, 64, 128, 256 mM). The time required for the appearance of flocculation was recorded. Acapsular mutant cap59D in logarithmic phase (OD 600 , 1) was treated with 0 or 8 mM SP1 for 30 min. In all samples, cell-cell aggregation was checked by confocal microscopy (Olympus IX81, Japan) before and after SP1 was added. All experiments were repeated at least three times, with similar results.
Competitive inhibition experiment. A total of 5 mM EDTA (Sigma), 20 mM mannose (Hushi), 20 mM maltose (Hushi), 20 mM galactose (Hushi), and 20 mM glucose (Hushi) were separately added to the H99 cultures. Then 8 mM SP1 was added, and all samples were incubated at 37°C and 220 rpm for 30 min. Flocculation was determined microscopically. H99 without any treatment was the negative control, and H99 treated only with SP1 was the positive control. The probability of flocculation in 100 visual fields was counted. All experiments were repeated at least three times.
Proteinase K treatment experiment. After the H99 culture was treated with 8 mM SP1 for 30 min or not, it was washed three times with phosphate-buffered saline (PBS) and resuspended in an equal volume of fresh RPMI 1640 medium (supplemented with 0.165 M MOPS). Then cells were incubated with 3 mg/mL proteinase K (Amresco) at 37°C and 220 rpm for another 30 min. The results were evaluated by confocal microscopy (Olympus IX81, Japan). All experiments were repeated at least three times, with similar results.
MIC assay. The MIC was measured according to the previously described Clinical and Laboratory Standards Institute (CLSI) M27-A4 protocol (57). To determine the MIC of the C. neoformans strains (H99, Indirect immunofluorescence microscopy. According to the previous indirect immunofluorescence protocol, the polysaccharide capsule of H99 treated with 8 mM SP1 or control peptide was labeled with the capsular monoclonal antibody (MAb) 18B7 (Millipore) (58,59). In brief, the SP1-treated or control-peptide-treated H99 culture was washed three times with PBS and resuspended in 200 mL PBS containing 20 mg/mL MAb 18B7 at a concentration of 10 7 cells/mL. The samples were incubated at room temperature for 20 rpm for 1 h. After being washed in PBS, the samples were finally incubated with a goat anti-mouse IgG conjugate (H1L) Qdot 655 secondary antibody (Life Technologies) under the same conditions. All samples were washed again with PBS to remove unbound secondary antibodies and then examined by confocal microscopy.
The combination of SP1 and GXM was detected by MST. GXM was extracted and purified according to the previous experimental protocol with slight modifications (60,61). We cultured H99 using capsule induction medium (minimal medium: 20 mg/mL thiamine, 30 mM glucose, 26 mM glycine, 20 mM MgSO 4 Á 7H 2 O, and 58.8 mM KH 2 PO 4 ; pH 7; Sigma) and then extracted GXM by ethanol precipitation of polysaccharides and hexadecyltrimethylammonium bromide (CTAB) precipitation of GXM. The GXM solution was dialyzed (dialysis tube, molecular weight cutoff of 3,500) versus sterile distilled water and then lyophilized. Then GXM was weighed, aliquoted, and stored at 280°C for later use. After dissolving a part of the GXM, the GXM solution was adjusted to pH 11.25 with concentrated NH 4 OH and then incubated at 23°C for 24 h. The samples were dialyzed and lyophilized to obtain de-O-acetylated derivative of GXM.
The interactions between SP1 and GXM or de-O-acetylated GXM were determined by microscale thermophoresis (MST) assay (62). GXM (2 mg/mL), or de-O-acetylated GXM (2 mg/mL) was mixed with FITC-labeled SP1 after a 2-fold serial dilution. After the mixture was uniform, the solution was loaded into 16 capillaries in a gradient of concentration from high to low and placed in the MST Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany) to measure the interaction.
Indian ink staining and capsule thickness measurement. C. neoformans H99 was cultured to the logarithmic phase. Subsequently, 8 mM SP1 was added to the above-mentioned H99 cultures (OD 600 , 1) and then incubated at 37°C and 220 rpm for 30 min. After centrifugation, C. neoformans solution and Indian ink were mixed 1:1 and visualized with confocal microscopy (Olympus IX81, Japan). H99 not treated with SP1 was used as the control. The capsule size of 100 cells was measured using Image J software. Capsule size was defined as the width of the white part from the cell wall to the outer edge of the cell.
Measuring the change of GXM content in the culture supernatant of C. neoformans H99. H99 was cultured to the logarithmic phase (OD 600 , 1), and the supernatant and fungal cells were collected. The collected fungal cells were resuspended with the collected supernatant at a concentration of 2 Â 10 7 cell/ mL. In the meantime, the collected supernatant was used to prepare a stock solution containing 2 Â 128 mM SP1, and used to perform 2-fold gradient dilution of the stock solution. The cell suspension and different concentrations of SP1 (2 Â 8, 2 Â 16, 2 Â 32, 2 Â 64, and 2 Â 128 mM) solution were mixed 1:1. Immediately after mixture, the mixed solution was centrifuged at 5,000 Â g for 2 min, and 20 mL of the supernatant was taken to measure the content of GXM. The GXM content at this time was defined as the initial content in the supernatant before the treatment of H99 with SP1. The centrifuged mixtures were vortexed to mix them evenly and incubated at 37°C for 30 min. Subsequently, the mixtures were centrifuged, and 20 mL of the supernatant was taken to measure the content of GXM. The GXM content at this time was defined as the content in the supernatant after treatment of H99 with SP1. The content of GXM in all samples was measured using the C. neoformans capsular polysaccharide ELISA kit (Beijing Hua Bo Deyi Biotechnology Co., China) according to the instructions.
The formula for the GXM content change in the supernatant of H99 treated with different concentrations of SP1 is as follows: change of GXM content in supernatant (pg/mL) = GXM content in supernatant after SP1 treatment of H99 (pg/mL) -GXM content in supernatant before SP1 treatment of H99 (pg/mL).
Real-time PCR of capsule-related genes. H99 was treated with 8 mM SP1 or control peptide and then incubated at 37°C for 1 h. RNA was extracted and reverse transcribed into cDNA using the yeast RNA extraction kit (Omega) and the cDNA synthesis kit (Biotechrabbit), respectively according to the manufacturers' instructions. The synthesized cDNA was diluted 10-fold with diethyl pyrocarbonate (DEPC) water and stored at 220°C.
CAP10, MAN1, CMT1, and GRASP1 were selected for real-time PCR, and these genes are involved in polysaccharide capsular synthesis or transport (48,(63)(64)(65). GAPDH was used as the reference gene, and its quantitative PCR (qPCR) primers were as follows: 59-TGAGAAGGACCCTGCCAACA-39 (forward) and 59-ACTCCGGCTTGTAGGCATCAA-39 (reverse). The real-time PCR primers for CAP10, MAN1, CMT1, and GRASP1 were derived from the research of Lee et al. (66). qPCR of H99 cDNA was performed using Hieff qPCR SYBR green master mix (Yeasen) according to the manufacturer's instructions. Target gene expression was measured using expression relative to the GAPDH reference gene. The relative expression was calculated using the 2 -DDCT method. All experiments were performed in triplicate.
Antibiofilm activity. The biofilm formation of C. neoformans H99 and the antibiofilm activity of SP1 were evaluated according to the previously described method with slight modifications (67,68). Amphotericin B (AMB) and fluconazole (FLC) were used as biofilm inhibition and noninhibition controls, respectively, in all experiments. Here, H99 was cultured with RPMI 1640 to form biofilm. Drugs were added at 0 h and 72 h at the beginning of the culture of cryptococcal biofilm to assess the effect of the drug on biofilm formation and mature biofilm, respectively. At 0 h, SP1 at a concentration of 8 to 32 mM, AMB at a concentration of 0.25 to 2 mg/mL, and FLC at a concentration of 1 to 128 mg/mL were added and incubated at 37°C for 72 h. At 72 h, SP1 at a concentration of 8 to 128 mM, AMB at a concentration of 0.25 to 64 mg/mL, and FLC at a concentration of 32 to 256 mg/mL were added and incubated at 37°C for 48 h. The metabolic activity and total biomass of the biofilms were evaluated using the 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay and crystal violet staining, respectively (67). Here, the OD 492 (for the XTT assay) and OD 590 (for the crystal violet assay) were determined in a microplate reader (BioTek, USA). The percentage of total biomass (metabolic activity) of cryptococcal biofilm treated with SP1 (AMB/FLC) was compared to that of the untreated control. Each sample was evaluated in triplicate in three independent experiments.
Adherence and phagocytosis assays. The adhesion and phagocytosis assay of macrophages J774A.1 to H99 was performed according to the previously described method with slight modifications (34,69). Here, H99 was treated with SP1 for 30 min and then washed three times with PBS and resuspended in DMEM (with 10% FBS). We treated H99 with the control peptide in the same manner. Then, SP1-treated and controlpeptide-treated H99 (3 Â 10 6 cells/well) infected J774A.1 cells, respectively. After incubating C. neoformans with macrophages for 90 min and 180 min, the adhesion and phagocytosis of macrophages to C. neoformans were evaluated by determining the CFU and imaged by confocal microscopy (Olympus IX81, Japan). C. neoformans within macrophages was stained with calcofluor white (CFW; Sigma). CFW specifically stains chitin in the cell walls of several eukaryotic microorganisms, including C. neoformans (70).
Fluconazole protection assays. The experiment was carried out according to the previous experiment protocol (69,71). In short, after SP1-treated or control-peptide-treated H99 infected macrophages J774A.1 for 3 h, we washed these host cells with PBS 8 to 10 times and then added fresh DMEM medium containing 20 mg/mL fluconazole. Fluconazole was used to inhibit the replication of host extracellular H99 (69). After incubation for 12 h at 37°C, the H99 cells were harvested from the DMEM medium, which contained fungal cells that escaped from the J774A.1 macrophages. At the same time, the H99 cells within the J774A.1 macrophages were also collected. The numbers of H99 cells inside and outside J774A.1 were counted by CFU. The replication number of H99 cells in the host cells is the sum of the number of H99 inside and outside the cells.
Statistical analysis. Student's t test was used to compare the data for CFU (macrophage) and capsule sizes. One-way analysis of variance (ANOVA) was used to compare the data for GXM content, gene expression, and antibiofilm activity. A P value of ,0.05 (*) or ,0.01(**) was considered statistically significant.

SUPPLEMENTAL MATERIAL
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