Novel gene that regulates blood-brain barrier passage of cryptococcus neoformans and use thereof

Abstract

The present invention relates to a method for screening for an inhibitor against the blood-brain barrier passage of Cryptococcus neoformans, a method for screening for an antifungal agent, and an antifungal composition. The screening method of the present invention uses a novel gene that regulates the blood-brain barrier passage of Cryptococcus neoformans, and thus can effectively screen for an inhibitor against blood-brain barrier passage and an antifungal agent.

Description

Novel genes regulating blood-brain barrier passage in Cryptococcus neoformans and their uses

[0001] The present invention relates to a novel gene that regulates passage of the blood-brain barrier of Cryptococcus neoformans and its use, and more particularly, to a method for screening a blood-brain barrier inhibitor for Cryptococcus neoformans, a method for screening an antifungal agent, and an antifungal composition.

[0002]

[0003] Cryptococcus neoformans is a fruiting fungus commonly found in various terrestrial and aquatic environments. Under certain environmental conditions, this widespread fungus can produce infectious ascospores through either asexual or parthenogenetic reproduction. Inhalation of these spores by human hosts can reach the lower respiratory tract. In this ecosystem, Cryptococcus neoformans resists phagocytosis by alveolar macrophages using unique antiphagocytic factors, including a polysaccharide capsule and polyphenolic melanin pigments. Furthermore, even after phagocytosis, Cryptococcus neoformans inhibits phagosome maturation and proliferates within the macrophage, often escaping via insoluble exocytosis. Cryptococcus neoformans then spreads into the bloodstream, infecting the central nervous system and causing life-threatening meningitis.

[0004] Unlike other human pathogenic fungi, this fungus exhibits unique neuronal motility and has the ability to cross the blood-brain barrier (BBB). In our previous study, we identified several BBB-permeability factors using an in vitro BBB model using a transwell membrane grown on human brain capillary endothelial cells (HBMECs). These factors include inositol transporters (Itr1a and Itr3c), hyaluronic acid synthase (Cps1), a metalloproteinase (Mpr1), urease (Ure1), phospholipase (Plb1), Fnx1, and Rub1. Cryptococcus neoformans utilizes the high inositol levels in the brain to induce the expression of Cps1, which then produces hyaluronic acid on the cell surface. Cryptococcus hyaluronic acid is recognized by the CD44 glycoprotein on HBMECs, promoting lateral migration of Cryptococcus. However, recent systematic analysis of a mutant library of transcription factors and kinases from Cryptococcus neoformans has revealed a broad network of signaling components involved in BBB passage and survival within the brain parenchyma. Furthermore, using a newly developed three-dimensional human neurovascular unit (hNVU) chip, specific transcription factors and kinases involved in neural tracing and BBB penetration were identified. These findings suggest that the signaling network and regulatory mechanisms for BBB passage in Cryptococcus neoformans are far more complex than previously anticipated.

[0005] Based on previous studies, we focused on the homeobox transcription factor Hob1. Deletion of HOB1 results in severe defects in the adhesion and BBB penetration of Cryptococcus neoformans, significantly attenuating its pathogenicity, and induction of other BBB penetration factors such as ITR1a, ITR3c, MPR1, FZC3, and PDR802 is reduced in the hob1Δ mutant.

[0006] In contrast, Cryptococcus gatiai, a sibling species of C. neoformans, primarily infects the lungs but not the brain, and Hob1 does not contribute to BBB passage or pathogenicity. This highlights the role of Hob1 as a key regulator of BBB passage and pathogenicity in C. neoformans, a role not observed in C. gatiai. However, the downstream signaling network governed by Hob1 in C. neoformans and the direct mechanisms regulating BBB penetration remain poorly understood.

[0007] Through research into the mechanism by which Cryptococcus neoformans crosses the blood-brain barrier, the present inventors discovered Hob1, a homeobox transcription factor that regulates this process (Korean Patent Publication No. 10-2017-0077066). Subsequently, the present inventors explored the downstream network of Hob1 through transcriptome analysis. Among 100 Hob1-dependent proteins, we discovered Hbf1, a key blood-brain barrier penetration factor that is significantly induced by Hob1 under host-mimetic conditions. Furthermore, we confirmed that deletion of Hbf1 significantly impairs the blood-brain barrier penetration ability of Cryptococcus neoformans, thereby completing the present invention.

[0008]

[0009] The technical problem to be achieved by the present invention is to provide a method for screening an antifungal agent against Cryptococcus neoformans.

[0010] Another object of the present invention is to provide a method for screening for a blood-brain barrier penetration inhibitor of Cryptococcus neoformans.

[0011] In addition, the technical problem to be achieved by the present invention is to provide an antifungal composition against Cryptococcus neoformans.

[0012]

[0013] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0014]

[0015] In order to achieve the above technical task, one embodiment of the present invention provides a method for screening a blood-brain barrier (BBB) ​​penetration inhibitor of Cryptococcus neoformans, comprising the steps of: contacting a candidate substance with Cryptococcus neoformans; and measuring the expression level of mRNA or the protein of the HBF1 gene of Cryptococcus, or measuring the activity of the protein.

[0016] In an embodiment of the present invention, if the measured expression level or activity decreases compared to a control group that has not come into contact with the candidate substance, a step of selecting the candidate substance as a blood-brain barrier (BBB) ​​penetration inhibitor may be further included.

[0017] In order to achieve the above technical task, another embodiment of the present invention provides a method for screening an antifungal agent against Cryptococcus neoformans, comprising the steps of: contacting a candidate substance with Cryptococcus neoformans; and measuring the expression level of mRNA or the protein of the HBF1 gene of Cryptococcus, or measuring the activity of the protein.

[0018] In an embodiment of the present invention, if the measured expression level or activity decreases compared to a control group that has not come into contact with the candidate substance, a step of selecting the candidate substance as an antifungal agent may be further included.

[0019] In order to achieve the above technical task, another embodiment of the present invention provides an antifungal composition comprising an inhibitor screened according to the above method.

[0020] In order to achieve the above technical task, another embodiment of the present invention provides an antifungal composition for Cryptococcus neoformans, comprising a substance that inhibits the expression of the HBF1 gene of Cryptococcus neoformans, or the expression or activity of a protein expressed therefrom.

[0021] In an embodiment of the present invention, the material may be a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound, or a natural product.

[0022] In an embodiment of the present invention, the material may include an antisense, siRNA (small interfering RNA), or shRNA (short hairpin RNA) nucleic acid having a sequence complementary to the mRNA of the HBF1 gene.

[0023]

[0024] The present invention relates to a method for screening a blood-brain barrier penetration inhibitor for Cryptococcus neoformans, a method for screening an antifungal agent, and an antifungal composition. The screening method of the present invention utilizes a novel gene that regulates blood-brain barrier penetration of Cryptococcus neoformans, and thus can effectively screen for blood-brain barrier penetration inhibitors and antifungal agents.

[0025]

[0026] The effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0027]

[0028] Figure 1 shows the results of transcriptome analysis of Cryptococcus neoformans. Figure 1A shows differentially expressed genes, and the genes with increased and decreased expression among the transcriptome expression levels of the wild-type strain and the HOB1-deficient strain are shown in R using the Enhanced volcano package. Figures 1B and 1D show the results of Gene-ontology (GO-term) analysis of genes corresponding to the basal condition (B) and HMC condition (D) among the differentially expressed genes analyzed in Figure 1A. Figures 1C and 1E show the results of KEGG analysis of genes corresponding to the basal condition (C) and HMC condition (E) among the differentially expressed genes analyzed in Figure 1A.

[0029] Figure 2 shows images of Cryptococcus neoformans wild type strain, hob1Δ (YSB2308, 2309), and hob1Δ::HOB1 (YSB7467) strains cultured for 3 days in YPD medium containing 1 ㎍ / ml of latruculin A.

[0030] Figure 3 shows the results of transcriptome analysis analyzed through Venn diagram analysis. Figure 3A shows the results of analyzing up-regulated genes, and Figure 3B shows the results of analyzing down-regulated genes.

[0031] Figure 4 shows the structure of HBF1 and HBF101 proteins. Figure 4A shows a domain schematic diagram of HBF1 and HBF101 proteins and their sizes. Figure 4B shows the IPR018824 domain predicted by RoseTTAFold and the overall structure prediction diagram of HBF1 and HBF101. Figure 4C shows the results of ClusterW analysis of the sequence similarity of HBF1 and HBF101. Figure 4D shows the structures of HBF1 and HBF101 proteins predicted by RoseTTAFold.

[0032] Figure 5 shows the production of strains lacking or overexpressing the HBF1 gene. Figures 5A and 5B show the production strategy and schematic diagram of strains lacking or overexpressing the HBF1 gene, Figures 5C and 5D show the diagnostic PCR results of strains lacking or overexpressing the HBF1 gene, Figures 5E and 5F show the Southern blotting results of strains lacking or overexpressing the HBF1 gene, and Figure 5G shows the results of measuring the expression level of the produced strains lacking or overexpressing the HBF1 gene.

[0033] Figure 6 shows the results of an experiment on the involvement of the HBF1 gene in oxidative stress.

[0034] Figure 7 shows the results of an experiment on the involvement of the HBF1 gene in melanin formation.

[0035] Figure 8 shows the intracellular location of the HBF1 gene.

[0036] Figure 9 shows the measurement of the change in fluorescence intensity of the HBF1 gene.

[0037] Figure 10 shows the results of blood-brain barrier (BBB) ​​passage analysis of Cryptococcus neoformans in the Hbf1 mutant.

[0038] Figure 11 shows the production of yeast strains expressing HBF1 in Saccharomyces cerevisiae. Figures 11A, 11B, and 11C show the HBF1cDNA synthesis and insertion, vector generation strategy through two-step PCR, Figure 11D shows cloning using Gibson assembly and the results thereof, and Figures 11E and 11F show the diagnostic PCR results for HBF1 expression and empty vector insertion transformation in Saccharomyces cerevisiae.

[0039] Figure 12 shows the results of analyzing the expression of HBF1 in a yeast strain using qRT PCR.

[0040]

[0041] Hereinafter, the present disclosure will be described in detail through examples and experimental examples. The present disclosure can be modified in various ways and can have various embodiments. Therefore, specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present disclosure to specific embodiments, but should be understood to include all modifications, equivalents, and alternatives included in the spirit and technical scope of the present disclosure. In describing the present disclosure, if a detailed description of a related known technology is judged to obscure the gist of the present disclosure, the detailed description thereof will be omitted.

[0042]

[0043] Hereinafter, the present invention will be described in detail.

[0044]

[0045] The present invention relates to a method for screening for a blood-brain barrier penetration inhibitor of Cryptococcus neoformans.

[0046] The present invention comprises a step of contacting a candidate substance with Cryptococcus neoformans; and a step of measuring the expression level of mRNA or protein of the HBF1 gene of Cryptococcus, or measuring the activity of the protein.

[0047] The above Cryptococcus neoformans strain can be cultured using methods and conditions known in the art.

[0048] The step of contacting the candidate substance with the Cryptococcus neoformans may involve treating the candidate substance with Cryptococcus neoformans using known means and under known methods and conditions in the art. The treatment may be appropriately performed depending on the desired treatment amount and treatment method.

[0049] The above candidate substances may include, but are not limited to, nucleic acids, antibodies, aptamers, peptides, proteins, compounds, or natural products, for example.

[0050] Measurement of the mRNA expression level can be performed by a method known in the art, for example, by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay (RPA), Northern blotting, DNA chip (Microarray), or RNA sequencing analysis.

[0051] Measurement of the expression level of the above protein can be performed by a method known in the art, for example, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), or protein chip.

[0052] If the measured expression level or activity is reduced compared to a control group that has not been in contact with the candidate substance, a step of selecting the candidate substance as a blood-brain barrier penetration inhibitor may be further included.

[0053] The above HBF1 gene may be composed of a sequence identified by the unique number (CNAG_03759) described in Table 2.

[0054]

[0055] The present invention relates to a method for screening antifungal agents against Cryptococcus neoformans.

[0056] The present invention comprises a step of contacting a candidate substance with Cryptococcus neoformans; and a step of measuring the expression level of mRNA or protein of the HBF1 gene of Cryptococcus neoformans, or measuring the activity of the protein.

[0057] The cultivation, candidate substances, expression or activity measurement methods of Cryptococcus neoformans are as described above.

[0058] If the measured expression level or activity decreases compared to a control group that has not come into contact with the candidate substance, a step of selecting the candidate substance as an antifungal agent may be further included.

[0059] The above antifungal agent is a drug that prevents and treats fungal infections. Cryptococcus neoformans is a fungus that causes meningitis by passing through the blood-brain barrier, and thus, it can exhibit an antifungal effect by significantly inhibiting the expression or activity of HBF1, which is related to the ability to penetrate the blood-brain barrier.

[0060]

[0061] The present invention relates to an antifungal composition against Cryptococcus neoformans.

[0062] The present invention includes a substance that inhibits the expression of the HBF1 gene of Cryptococcus neoformans, or the expression or activity of a protein expressed therefrom.

[0063] The above material may be, but is not limited to, a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound or a natural product.

[0064] The above material may be, but is not limited to, an antisense, siRNA (small interfering RNA), or shRNA (short hairpin RNA) nucleic acid having a sequence complementary to the mRNA of the HBF1 gene.

[0065] The antifungal composition of the present invention can be utilized as an antifungal agent, a pharmaceutical composition, a skin cleansing composition, an antibacterial building material and household goods composition, or a cosmetic composition.

[0066] The above antifungal agent is a drug that prevents and treats fungal infections by inhibiting the proliferation and growth of fungi.

[0067] The above pharmaceutical composition may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, etc. in the form of a solid, semi-solid, or liquid by adding a commercially available inorganic or organic carrier.

[0068] Examples of preparations for oral administration include tablets, pills, granules, soft / hard capsules, powders, granules, powders, emulsions, syrups, pellets, and beverages. In addition, examples of preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, and suppositories.

[0069] In order to formulate the effective ingredient of the present invention, it can be easily formulated by following a conventional method, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspending agents, and other commonly used auxiliary agents can be appropriately added and used.

[0070] Additionally, the dosage of the above-mentioned active ingredient will vary depending on the age, sex, and weight of the subject being treated, the specific disease or pathological condition being treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determining the dosage based on these factors is within the skill of those skilled in the art, and typically, the dosage may range from 0.001 mg / kg / day to approximately 2,000 mg / kg / day, but this does not limit the scope of the present invention in any way.

[0071]

[0072] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0073]

[0074] Materials and Methods

[0075] 1. Transcriptome analysis (RNA sequencing)

[0076] Wild-type and hob1Δ mutants of Cryptococcus neoformans and Cryptococcus gattii were initially cultured overnight at 30°C, and then OD 600 The cells were transferred to fresh YPD medium until they reached a confluency of 0.8. The cells were washed three times with PBS buffer and then cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C and 5% CO2 for 3 h. After incubation, the cells were collected, rapidly frozen, and lyophilized. Total RNA was extracted using the Easy-BLUE kit (17061, iNtRON, South Korea) and further purified using the RNeasy Mini Kit (74106, Qiagen, Germany). This procedure was repeated in three independent cultures for each strain.

[0077] For RNA sequencing, 1 μg of total RNA from each sample was used to prepare cDNA libraries using the TruSeq mRNA Library Kit (Illumina, USA), and sequencing was performed on the Illumina platform. Post-sequencing processing included removing adapters and low-quality sequences using Cutadapt v2.4 and Python 3.7.4. Cleaned reads were aligned to the Cryptococcus neoformans H99 reference genome using Hisat2 v2.2.1, utilizing the Hisat and Bowtie2 algorithms. Annotation data were obtained from the NCBI FTP server. Hisat2 was run with the "-p 30" and "--dta -1" options and default values. Aligned reads were managed using Samtools v0.1.19, modifying the default parameters for conversion ("-Sb -@ 8") and alignment ("-@ 20 -m 2000000000"). Transcriptome assembly and abundance estimation were performed using Stringtie v1.3.6 with the “-p 12” setting, and transcript abundance was quantified by FPKM values. Data matrices were generated and analyzed using the R package “isoformswitchanalyzerR.” Quality control was performed using DEBrowser. Differential expression analysis was performed using DESeq2 v1.24, and results were visualized using the Enhanced Volcano package in R v4.1.0, with a threshold of twofold change and a P-value less than 0.05 applied for significance.

[0078]

[0079] 2. Construction of gene deletion mutants

[0080] Mutant strains of HBF1 were constructed within the genetic background of Cryptococcus neoformans serotype A strain H99S. For gene disruption, a cassette conferring norsotricin resistance (encoded by the norsotricin acetyltransferase gene, NAT) was constructed using conventional nested PCR or NAT split marker / double-junction (DJ) PCR methodologies. In the first PCR step to construct this cassette, the 5' and 3' flanking sequences of the HBF1 gene were amplified using the L1 / L2 and R1 / R2 primer sets, respectively, using H99S genomic DNA as a template. The full-length NAT marker was amplified using the M13Fe (M13 forward extended) and M13Re (M13 reverse extended) primer pairs from the pNAT-STM plasmid, which contains the NAT gene including a unique signature tag sequence. In the split marker / DJ-PCR approach, the 5' and 3' portions of the NAT marker were amplified individually using the M13Fe / SM2 and M13Re / SM1 primer combinations, respectively. After the initial amplification, a nested PCR method involved merging the initially amplified products using the L1 and R2 primer pairs as templates to synthesize the complete gene disruption cassette. In contrast, the split marker / DJ-PCR strategy amplified the 5' and 3' portions of the NAT split gene disruption cassette using the L1 / SM2 and R2 / SM1 primer combinations, respectively, using the initial PCR products as templates. For the biotransformation process, the H99S strain was cultured overnight at 30°C in yeast extract-peptone-dextrose (YPD) medium, collected by centrifugation, and resuspended in 5 mL of sterile distilled water. Approximately 200 μL of this cell suspension was evenly distributed onto YPD solid medium containing 1 M sorbitol and incubated at 30°C for 3 h. The PCR-generated disruption cassette was 0.600 μg.After adsorption onto 6 μm diameter gold microcarrier beads (Bio-Rad), the cells were delivered using the PDS-100 particle delivery system (Bio-Rad). After transformation, the cells were incubated at 30°C for 4 h to promote restoration of cell membrane integrity, then harvested and cultured on YPD solid medium (YPD+NAT) containing 100 μg / mL norsotricin to select stable transformants. These were then purified through multiple passages on YPD+NAT medium. For initial confirmation, NAT-positive strains were screened by diagnostic PCR. To ensure accurate gene deletion and exclude off-target integration, Southern blot analysis was performed on each mutant strain. Further validation of the mutant phenotypes to rule out unrelated mutational effects involved generating multiple independent deletion strains for each mutant.

[0081]

[0082] 3. Construction of HBF1 overexpression, complementation, and fluorescent protein-tagged strains

[0083] To generate a strain that persistently overexpresses HBF1 in Cryptococcus neoformans, the native promoter of the HBF1 gene was replaced with the histone H3 promoter. This was achieved by constructing a homologous recombination cassette that was amplified in multiple steps. In the initial PCR step, the 5'-flanking region and 5'-coding region of HBF1 were amplified using the L1 / OEL2 and OER1 / PO primer pairs, respectively. Simultaneously, the NAT-H3 promoter region was amplified using the primer pairs listed in Table 1.

[0084]

[0085] In the second PCR step, double joint (DJ) PCR is used to H3 : The 5' region of the HBF1 cassette was constructed. It was amplified using the 5'-flanking region of HBF1 and the NAT-H3 promoter region as templates with the primer pairs listed in Table 1. Similarly, PH3 :The 3' region of the HBF1 cassette was synthesized by DJ-PCR using a mixed template of the 3'-flanking region of HBF1 and the NAT-H3 promoter region, and amplified with another primer pair. The assembled P H3 The HBF1 cassette was introduced into the wild-type H99S strain via biotransformation. Stable transformants were selected on yeast extract-peptone-dextrose (YPD) medium supplemented with norsotricin (NAT), and correct insertion was confirmed by diagnostic PCR using the SO / B79 primer pair. Integration and proper orientation of the cassette were confirmed by Southern blot analysis using specific probes PCR-amplified with the L1 and PO primers.

[0086] To construct untagged and mRuby3-tagged strains, the open reading frame (ORF) of HBF1, including the promoter region excluding the stop codon, was amplified by PCR and cloned into the pNEO-mRuby3 vector using the Gibson Assembly Master Mix kit from New England BioLabs (USA). To avoid Dcm methylation, the pNEO-mRuby3 plasmid was transformed into dam- / dcm-competent Escherichia coli cells (provided by New England BioLabs, USA). After transformation, the plasmid was extracted. The pNEO_HBF1-mRuby3 plasmid was linearized by StuI digestion and then introduced into the hbf1Δ (YSB10384) strain by biotransformation.

[0087]

[0088] 4. Construction of HBF1-expressing strain of the nonpathogenic fungus Saccharomyces cerevisiae

[0089] To construct a strain that continuously overexpresses HBF1 in the nonpathogenic fungus Saccharomyces cerevisiae, the cDNA of the HBF1 gene was synthesized from the cDNA of the Cryptococcus neoformans H99 strain using a primer set. Since the cDNA of the HBF1 gene is very short at 89 amino acids, the promoter of the TEF1 gene, a highly expressed gene, was synthesized for smooth cloning. This was achieved by constructing a homologous recombination cassette amplified in several steps. In the initial PCR step, the 5'-flanking region and the 5'-coding region of HBF1 were amplified using the L1 / OEL2 and OER1 / PO primer pairs, respectively. Simultaneously, the TEF1 promoter region was also amplified using the corresponding primer pairs. In the second PCR step, double-joint (DJ) PCR was used to amplify P TEF : The 5' region of the HBF1 cassette was constructed. It was amplified with the corresponding primer pair using the 5'-flanking region of HBF1 and the TEF1 promoter region as templates. Similarly, P TEF :The 3' region of the HBF1 cassette was synthesized by DJ-PCR using a mixed template of the 3'-flanking region of HBF1 and the TEF1 promoter region and amplified with another primer pair.

[0090] Assembled P TEF : The HBF1 cassette was inserted into the pLEU_TEF plasmid. The PLEU_TEF plasmid and HBF1 cDNA treated with restriction enzymes BamHI and SacI were cloned using the Gibson assembly method. The nonpathogenic fungus Saccharomyces cerevisiae BY4742 strain was transformed, and the transformation was performed using the following procedure. The BY4742 strain, cultured in YPD at 30°C for more than 16 hours, was subcultured and OD 600After growing until the value reached 0.6, the cells were collected and diluted in 2.5 ml of sterile distilled water. PEG / LiOAc mixture containing 1 ml of 50% PEG, 180 μl of 1 M LiOAc, and 125 μl of SS-DNA was prepared, and 300 μl of the mixture was mixed with 1 μg of plasmid. 100 μl of the diluted strain cultured above was mixed and incubated at 42°C for 45 minutes. The strain was collected by centrifugation, the supernatant was discarded, and a portion of the dilution was spread on SD-Leu medium and incubated at 30°C for 3-4 days. The colony-forming groups selected on the SD-Leu medium were confirmed to be the correct strain by diagnostic PCR using a primer pair.

[0091]

[0092] 5. Quantitative RT-PCR to confirm HBF1 gene expression

[0093] Leucine empty vector transformed strain and P TEF _HBF1 was cultured overnight in EBM-2 medium, and OD6 00nm Re-cultured in EBM-2 medium until OD = 0.8 600nmAfter reaching =0.8, cells were collected and washed with PBS, and the remaining cells were sampled for 6, 12, and 24 h in EBM-2 medium while shaking at 120 rpm. The collected cells were immediately frozen and lyophilized. Total RNA was extracted according to the method described above. For cDNA synthesis, total RNA was adjusted to a concentration of 5 μg with DEPC-treated water, and oligo(dT)-pdN6 was added and heated at 65°C for 5 min. Then, RNAse inhibitor (M007, Enzynomics, South Korea), dNTP, and reverse H minus reverse transcriptase (EP0752, Thermo Scientific, USA) were added, and the mixture was incubated at 50°C for 60 min, and then heat inactivated at 85°C for 10 min. Gene expression levels were normalized using the expression values ​​of the actin gene (ACT1) and leucine gene (LEU2), and the fold change relative to the basal expression level in the leucine empty vector transformed strain was calculated.

[0094]

[0095] 6. Growth and chemical sensitivity analysis

[0096] To evaluate the growth kinetics and chemical susceptibility of the Cryptococcus neoformans HBF1 mutant strain, the strain, cultured overnight at 30°C, was serially diluted 10-fold (from 1:1 to 1:10,000). These dilutions were spot-applied onto YPD agar plates containing various concentrations of chemicals. Chemicals were used to induce specific types of stress (sorbitol for osmotic stress; NaCl and KCl for cation / salt stress; hydrogen peroxide (H2O2), tert-butyl hydroperoxide, menadione, diamide for oxidative stress; cadmium sulfate (CdSO4) for heavy metal stress; methyl methanesulfonate and hydroxyurea for genotoxic stress; sodium lauryl sulfate (SDS) for membrane destabilization; calcofluor white and Congo red for cell wall destabilization; tunicamycin (TM) and dithiothreol (DTT) for endoplasmic reticulum (ER) and reductive stress; and several antifungal agents (fludeoxynil, fluconazole, amphotericin B, flucytosine)) to assess antifungal susceptibility.

[0097] These media were cultured with or without glucose to test stress responses under various nutrient conditions. After treatment, the media were incubated at 30°C, and images were taken daily from day 2 to day 5 to monitor colony growth. Additionally, to determine the temperature tolerance of each mutant strain, YPD media containing serially diluted cells were spot-applied and cultured at four different temperatures: 25°C, 30°C, 37°C, and 39°C. Growth was documented through photographs taken from day 2 to day 4. This comprehensive experimental design allowed for a detailed evaluation of the mutant strains' tolerance to environmental stresses and antifungal agents, as well as their adaptability to various temperatures.

[0098]

[0099] 7. Analysis of in vitro virulence factor production

[0100] To measure capsule production efficiency, each mutant was cultured at 30°C, spotted onto Dulbecco's modified Eagle's (DME) agar, and cultured at 37°C for 2 days. The cells were then scraped, washed with distilled water, fixed with 10% formalin, and washed again with distilled water. To measure capsule production efficiency in other media, 5 μl of each culture was spotted onto Littman's agar and FBS agar (10% fetal bovine serum and 90% PBS), cultured at 37°C for 2 days, scraped, and resuspended in distilled water. The resuspended cells were stained with India ink (BactiDrop; Remel, San Diego, CA, USA) and observed using a differential interference contrast (DIC) microscope (BX51, Olympus, Tokyo, Japan). Capsule thickness was measured by subtracting the cell diameter (total diameter - cell body diameter) from the capsule diameter. For quantitative measurements of capsule thickness, 50 cells were measured for the H99S strain and each phosphatase mutant. To measure melanin production efficiency, the mutants, including the wild-type strain, were cultured in YPD medium at 30°C for 16 h, washed with PBS, and cultured in 3 μl of Niger seed medium. Cultured cells were photographed after 1–3 days of incubation at 37°C.

[0101]

[0102] 8. Visualization of the subcellular localization of mRuby3-tagged HBF1

[0103] The Hbf1-mRuby3-tagged strain was initially cultured overnight in YPD medium at 30°C. The culture was grown in YPD medium until OD 600The culture was re-cultured until it reached a confluency of 0.8. The culture was then divided into two 25 ml aliquots, centrifuged, and washed three times with phosphate-buffered saline (PBS). One aliquot was stored in a liquid nitrogen tank for analysis of the basal localization of Hbf1, and the other was resuspended in an equal volume of RPMI 1640 medium supplemented with 10% FBS. The samples were cultured in a CO2 incubator at 37°C with horizontal shaking at 120 rpm. Cells were harvested at 10, 30, 60, and 180 min, fixed with 4% paraformaldehyde solution containing 3.4% sucrose for 15 min at room temperature, and then washed with a solution containing 0.1 M KPO4 and 1.2 M sorbitol. For nuclear staining, cells were treated with 10 μg / mL Hoechst 33342 (Thermo Fisher, USA) for 30 min in the dark. Finally, stained cells were observed under differential interference contrast (DIC) and fluorescence microscopy (Nikon Eclipse, Japan).

[0104]

[0105] 9. In vitro BBB passage assay

[0106] BBB passage assays were performed using the method described in a previous study (Lee KT et al. Nat Commun, 2020). Human brain microvascular endothelial cells (HBMEC line hCMEC / D3, Merck, USA) were cultured on collagen-precoated plates (354236, Corning, USA) until 80% confluence was reached. 5 × 10 4 hCMEC / D3 cells were seeded on 8-μm porous collagen-coated membranes (353097, BD Falcon) and EndoGRO TM - MV Complete Media Kit (SCME004, Sigma-Aldrich, USA) was maintained. After 24 h, the medium was replaced with fresh EndoGR supplemented with 2.5% human serum (H4522, Sigma, USA).TM The medium was replaced with EndoGRO containing 1.25% human serum, and the cells were cultured for an additional 4 days. The day before yeast inoculation, the medium was replaced with EndoGRO containing 1.25% human serum. TM The medium was replaced with medium. Cell culture was performed at 5% CO2, 37°C. To assess the integrity of cell junctions, transendothelial electrical resistance (TEER) was measured using an Epithelial Volt per Ohm Meter (EVOM2, World Precision Instruments, USA). Wild-type and HBF1 mutant yeast cells cultured overnight were washed three times with PBS and seeded at 5 × 10 5 Cells were adjusted to 100 μL of PBS and added to hCMEC / D3-seeded membranes. After 24 h of incubation, yeast cells that had passed through the membrane were collected. The relative BBB passage rate of each mutant strain was determined compared to that of the wild type.

[0107]

[0108] <Result>

[0109] 1. Comparative transcriptome analysis of the hob1Δ mutants of Cryptococcus neoformans and Cryptococcus gatiai.

[0110] To analyze the downstream networks regulated by Hob1, we performed RNA-seq-based transcriptome analysis of wild-type (WT) and hob1Δ mutant strains (Fig. 1). Recognizing the distinct phenotypic changes observed after Hob1 deletion in Cryptococcus neoformans and Cryptococcus gatiai, we performed transcriptome analyses between these two species. Furthermore, we confirmed the critical role of Hob1 in blood-brain barrier (BBB) ​​passage and closely examined the transcriptome under host-mimicking conditions. For this purpose, we used tissue culture medium (RPMI) supplemented with 10% fetal bovine serum (FBS) and cultured at 37°C and 5% CO2.

[0111] In Cryptococcus neoformans, the hob1Δ mutant showed upregulation of 661 genes and downregulation of 817 genes (compared to the WT strain) under basal conditions (YPD medium, 30°C). The upregulated gene set mainly included capsule biosynthesis genes (e.g., CAS32, CAP4, CAP60) and genes related to cell cycle, division, and meiosis (e.g., IRR1, CDC2801, CDC7). In contrast, the downregulated genes mainly belonged to the mitogen-activated protein kinase (MAPK) signaling pathway (e.g., HSP12, PTP1, PTP2), exocytosis, and actin binding gene categories. In particular, ERG11, which is generally associated with the MAPK signaling pathway, was paradoxically upregulated. Further analysis using gene ontology terms and KEGG pathways highlighted dynamic changes in signaling pathways, showing induction of RNA degradation and meiotic pathways and decreases in metabolic pathways, starch and sucrose metabolism, and carbon metabolism. In contrast, transcriptomic changes in the hob1Δ mutant in Cryptococcus gatii were relatively minimal, identifying only three upregulated and five downregulated genes. These genes did not belong to specific GO terms or KEGG pathways, and their functions were largely undefined. The actin-binding gene category was indirectly affected by the significantly increased growth of the HOB1 deletion mutant when grown in drug media containing latrunculin A, a drug that interferes with actin binding (Fig. 2).

[0112] A striking contrast was then observed under HMC conditions. The hob1Δ mutant of Cryptococcus neoformans significantly regulated 453 genes (320 induced and 133 downregulated), suggesting a crucial role for Hob1 in adaptation to host-like conditions. This adaptation contrasts sharply with the hob1Δ mutant of Cryptococcus gatiai, where only seven genes were significantly regulated, highlighting the differences in the importance of Hob1 between the two species under both basal and HMC conditions. Interestingly, the genes induced in Cryptococcus neoformans under HMC closely matched those under basal conditions, but the number of downregulated genes was reduced. This change may reflect the stringent survival requirements imposed by HMC, namely, elevated temperature and changes in nutrient availability.

[0113] Comparative analysis between basal and HMC conditions revealed that 187 genes were commonly upregulated in Cryptococcus neoformans under both conditions, highlighting pathways involved in chromatin remodeling, carbohydrate metabolism, and cell division (Figure 3). This commonality suggests a core function regulated by Hob1, regardless of the external environment. Conversely, distinct conditions-dependent gene regulation was clearly evident, suggesting that distinct sets of genes activated under basal and HMC conditions represent tailored responses to environmental challenges. Variations in the number of downregulated genes further emphasize this conditional specificity, with a significantly reduced number of downregulated genes in the HMC condition compared to the basal condition. This pattern suggests a complex role for Hob1, particularly in regulating metabolic and biosynthetic processes under stress conditions typified by HMC.

[0114] In conclusion, our detailed comparative transcriptome analysis reveals the profound impact of Hob1 deletion in Cryptococcus neoformans on gene expression regulation, highlighting a wide range of biological processes affected by diverse environmental conditions. These contrasting responses between Cryptococcus neoformans and Cryptococcus gatiai highlight the central role of Hob1 in the pathogenicity and adaptive strategies of Cryptococcus neoformans within its host environment.

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[0116] 2. Identifying downstream targets of Hob1: A transcriptome-based approach

[0117] Previous studies have shown that the functional differences of Hob1 in Cryptococcus neoformans and Cryptococcus gatiai do not lie in its sequence. Instead, they suggest that downstream target proteins of Hob1 may function differently in these two species, suggesting the interplay of complex, species-specific regulatory mechanisms. To further explore this hypothesis, we performed a comprehensive transcriptome analysis of the Cryptococcus neoformans hob1Δ mutant under both basal and host-mimetic (HMC) conditions. This analysis was instrumental in identifying a set of genes presumed to be under direct or indirect regulation by Hob1, which are summarized in Table 2 (Potential downstream target predictions and candidate gene list based on Hob1 transcriptome analysis).

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[0119] Among the downstream targets, CNAG_03759 emerged as a gene of particular interest for several compelling reasons. First, Hbf1 was the gene whose expression was most consistently downregulated upon Hob1 deletion, both under basal conditions and in HMC experimental settings. This consistent regulatory pattern suggests a strong and specific interaction between Hbf1 and the Hob1-regulated pathway. Second, unlike other genes with well-documented functions, CNAG_03759 encodes a protein whose role is not yet understood, making it a novel target for further functional characterization.

[0120] The protein encoded by CNAG_03759 was characterized by its short length (only 89 amino acids) and the presence of a domain with a conidia-specific function (Fig. 4). This domain is also present in CNAG_01056, a structural isoform with a similar protein size and domain structure. However, in contrast to CNAG_03759, CNAG_01056 appeared to be unregulated by Hob1 due to its extremely low expression levels under the tested conditions. The differential expression between these isoforms provided intriguing insights into the selective regulatory role of Hob1.

[0121] Despite the absence of conidial formation common in other fungal species, the role of CNAG_03759 in C. neoformans virulence remained speculative. However, its strong regulatory association and unique protein structure led us to designate it as Hob1-dependent blood-brain barrier penetration factor 1 (HBF1), and its structural isoform, CNAG_01056, was designated HBF101. We hypothesize that HBF1 may play a crucial role in facilitating C. neoformans BBB passage, a key step in the development of meningitis. This opens new avenues for understanding the molecular mechanisms by which C. neoformans invades the central nervous system and suggests a potential target for therapeutic intervention.

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[0123] 3. Hbf1 is involved in the oxidative stress response of Cryptococcus neoformans.

[0124] To better understand the functional importance of Hbf1 in the growth and survival of Cryptococcus neoformans, hbf1Δ and P H3 : An HBF1 mutant strain was developed. In the hbf1Δ mutant strain, the HBF1 gene was completely deleted and replaced with an antifungal resistance marker, which is a common strategy for evaluating gene function by observing the phenotypic consequences of gene deletion. On the other hand, P H3 : In the HBF1 mutant strain, the original promoter of HBF1 was replaced with a highly active H3 promoter to induce overexpression of the HBF1 gene, and the effect of increased gene expression on fungal physiology was confirmed (Fig. 5).

[0125] In vitro phenotypic analyses using various oxidative and endoplasmic reticulum (ER) stress inducers, namely hydrogen peroxide (H2O2) and dithiothreol (DTT), revealed interesting resistance patterns (Fig. 6). H2O2, a potent oxidant, inflicts severe oxidative stress on cellular systems, while DTT disrupts the environment required for oxidative protein conjugation, inducing ER stress. The hbf1Δ strain exhibited slightly increased resistance to both agents. This suggests that the presence of Hbf1 increases the susceptibility to oxidative stress, and thus, its deletion may enhance the fungus' resistance to these stresses.

[0126] Additionally, we investigated the effect of Hbf1 overexpression on melanin production, a key protective mechanism of Cryptococcus neoformans. Melanin not only protects bacterial cells from UV and strong ionizing radiation, but also plays a crucial role in mitigating oxidative damage. Our results showed that melanin production was significantly reduced when Hbf1 was overexpressed (Fig. 7). Given that melanin plays a crucial role in the pathogenicity of Cryptococcus neoformans, this reduction may impair the fungus' ability to survive in the hostile environmental conditions encountered during host infection.

[0127] Unexpectedly, while Hbf1 deletion enhanced resistance to oxidative stress, its overexpression prevented effective utilization of melanin to protect against this damage. These contradictory results highlight the complex regulatory role of Hbf1 in oxidative stress management and suggest that Hbf1 may be essential not only for survival but also for pathogenicity in Cryptococcus neoformans under oxidative stress. These findings provide important insights into the biological function of Hbf1 and highlight its potential as a target for therapeutic strategies aimed at suppressing cryptococcal infection by modulating its expression or function.

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[0129] 4. Analysis of the subcellular localization of Hbf1

[0130] To investigate the subcellular localization of Hbf1 in Cryptococcus neoformans, we constructed an hbf1Δ::HBF1-mRuby3 reporter strain, in which the mRuby3 fluorescent tag was tagged to the C-terminus of the Hbf1 protein. This genetic modification was used to assess whether the fusion protein retained biological activity under stress conditions (Fig. 8). This strain demonstrated restoration of tolerance to oxidative and endoplasmic reticulum (ER) stress to wild-type levels, confirming that the Hbf1-mRuby fusion protein retained full functionality.

[0131] Subsequently, we focused on elucidating the subcellular localization of Hbf1 under standard conditions and host-like conditions (HMC), which strongly induce Hbf1 expression. Under standard, untreated conditions, Hbf1 was distributed throughout the cytoplasm with weak fluorescence intensity. However, under HMC conditions, Hbf1 was relocated to specific subcellular locations, indicating that it is localized to specific sites under host-like conditions. This targeted relocation suggests that Hbf1 may perform specialized functions related to stress responses or cell signaling pathways.

[0132] Further analysis using fluorescence microscopy confirmed an increase in the brightness of the fluorescence signal under HMC conditions compared to untreated cells (Figure 9). This increase in fluorescence intensity not only confirms the upregulation of Hbf1 expression but also indicates that Hbf1 is more actively involved in the fungal cell response to host-like conditions. Furthermore, the timing of the peak fluorescence increase within 30 min after HMC treatment suggests that Hbf1 is an early-response protein, likely regulated by a Hob1-dependent transcriptional mechanism. These temporal expression and localization patterns indicate that Hbf1 plays a crucial role in the immediate cellular response to environmental stress.

[0133] These observations highlight the complex regulation and critical functional role of Hbf1 within Cryptococcus neoformans, particularly in response to environmental challenges that mimic host conditions. This expanded analysis provides important insights into Hbf1's cellular behavior and fungal biology, contributing to our understanding of the molecular mechanisms by which Cryptococcus neoformans adapts and survives in a hostile host environment.

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[0135] 5. Analysis of the blood-brain barrier (BBB) ​​passage of Cryptococcus neoformans with Hbf1 mutants

[0136] In this study, we investigated whether Hbf1 is essential for BBB penetration, as transcriptome analysis identified Hbf1 as a critical downstream target. The in vitro BBB model, first developed by Vu et al., utilizes human brain microvascular endothelial cells (HBMECs) cultured on transwell membranes, mimicking the physical separation between the blood and brain compartments. This model has been previously validated using the mpr1Δ mutant, nonpathogenic Saccharomyces cerevisiae (strain S288C), and pathogenic Cryptococcus neoformans (strain H99). We employed a similar transwell-based system to assess whether Hbf1 can promote BBB penetration.

[0137] As a result, wild-type (WT) Cryptococcus neoformans successfully crossed the BBB within 24 h of culture. In contrast, the hbf1Δ mutant showed a significantly reduced ability to cross the BBB, highlighting the potential role of Hbf1 in this process. To confirm whether Hbf1 directly affects BBB permeability, we compared the Hbf1 complemented strain (hbf1Δ::HBF1) and the mutant strain overexpressing the Hbf1 gene (P H3:HBF1) was evaluated. The complemented strain restored BBB penetration ability to wild-type levels, while the overexpressing mutant strain did not exceed wild-type levels (Fig. 10). These results indicate that Hbf1 is essential for BBB passage, but overexpression does not proportionally increase BBB permeability. RNA-seq transcriptome analysis confirmed that HBF1 was upregulated 4.55-fold under HMC conditions, suggesting a functional impact under these conditions. This correlation highlights the important role of Hbf1 in human BBB passage.

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[0139] The present invention relates to a novel gene that regulates passage of the blood-brain barrier of Cryptococcus neoformans and its use, and more particularly, it can be used as a method for screening a blood-brain barrier inhibitor for Cryptococcus neoformans, a method for screening an antifungal agent, and an antifungal composition.

Claims

1. A step of contacting a candidate substance with Cryptococcus neoformans; and A method for screening a blood-brain barrier (BBB) ​​penetration inhibitor of Cryptococcus neoformans, comprising the step of measuring the expression level of the HBF1 gene of Cryptococcus mRNA or its protein, or measuring the activity of the protein.

2. A method for screening a blood-brain barrier penetration inhibitor of Cryptococcus neoformans, further comprising a step of selecting the candidate substance as a blood-brain barrier penetration inhibitor when the measured expression level or activity decreases compared to a control group that has not come into contact with the candidate substance in claim 1.

3. A step of contacting the candidate substance with Cryptococcus neoformans; and A method for screening an antifungal agent against Cryptococcus neoformans, comprising the step of measuring the expression level of the mRNA or protein of the HBF1 gene of the Cryptococcus, or measuring the activity of the protein.

4. A method for screening an antifungal agent against Cryptococcus neoformans, further comprising a step of selecting the candidate substance as an antifungal agent when the measured expression level or activity decreases compared to a control group that has not come into contact with the candidate substance in claim 3.

5. An antifungal composition comprising an inhibitor screened according to the method of claim 1.

6. An antifungal composition for Cryptococcus neoformans, comprising a substance that inhibits the expression of the HBF1 gene of Cryptococcus neoformans, or the expression or activity of a protein expressed therefrom.

7. An antifungal composition for Cryptococcus neoformans according to claim 6, wherein the substance is a nucleic acid, an antibody, an aptamer, a peptide, a protein, a compound or a natural product.

8. In claim 6, the material is an antifungal composition for Cryptococcus neoformans comprising an antisense, siRNA (small interfering RNA) or shRNA (short hairpin RNA) nucleic acid having a sequence complementary to the mRNA of the HBF1 gene.

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