Novel genes for regulating the passage through the blood-brain barrier of Cryptococcus neoformans and their use

Abstract

The present invention relates to a screening method for blood-brain barrier crossing inhibitors against Cryptococcus neoformans, a screening method for antifungal agents, and an antifungal composition. Since the screening method of the present invention utilizes a novel gene that regulates the blood-brain barrier crossing of Cryptococcus neoformans, it is possible to effectively screen for blood-brain barrier crossing inhibitors and antifungal agents.

Description

Technology Field

[0001] The present invention relates to a novel gene that regulates the passage of Cryptococcus neoformans across the blood-brain barrier and its uses, and specifically to a method for screening inhibitors of blood-brain barrier passage against Cryptococcus neoformans, a method for screening antifungal agents, and an antifungal composition. Background Technology

[0003] Cryptococcus neoformans is a fruiting fungus commonly found in various terrestrial and aquatic environments. Under specific environmental conditions, the widespread fungus can produce infectious ascospores through sexual or parthenogenesis. When a human host inhales these spores, they can reach the lower respiratory tract. In this ecosystem, Cryptococcus neoformans resists phagocytosis by alveolar macrophages by utilizing unique anti-phagocytic factors, such as polysaccharide capsules and polyphenol melanin pigments. Furthermore, even after phagocytosis, Cryptococcus neoformans inhibits the maturation of phagosomes, proliferates within macrophages, and frequently escapes via insoluble exocytosis. Subsequently, Cryptococcus neoformans spreads into the bloodstream, infects the central nervous system, and causes fatal meningitis.

[0004] Unlike other human pathogenic fungi, the above fungus possesses the ability to cross the blood-brain barrier (BBB) ​​by performing unique neural tracking movements. In previous research, the inventors identified several BBB crossing factors using an in vitro BBB model utilizing a Transwell membrane in which human brain capillary endothelial cells (HBMECs) were grown. These factors include inositol transporters (Itr1a and Itr3c), hyaluronic acid synthase (Cps1), metalloproteinase (Mpr1), urease (Ure1), phospholipase (Plb1), Fnx1, and Rub1. Cryptococcus neoformans utilizes high inositol levels in the brain to induce the expression of Cps1, which subsequently produces hyaluronic acid on the surface of Cryptococcus cells. The hyaluronic acid of Cryptococcus is recognized by the CD44 glycoprotein of HBMECs, which promotes the transverse movement of Cryptococcus. However, a recent systematic analysis of a library of transcription factor and kinase mutations of Cryptococcus neoformans has revealed an extensive network of signaling components involved in BBB crossing and survival within the brain parenchyma. Additionally, specific transcription factors and kinases involved in neural tracking movements and BBB penetration were identified using a newly developed 3D human neurovascular unit (hNVU) chip. These findings suggest that the signaling networks and regulatory mechanisms for BBB crossing by Cryptococcus neoformans are far more complex than previously anticipated.

[0005] Based on previous research, the inventors focused on Hob1, a homeobox transcription factor. HOB1 The deficiency causes serious defects in the attachment and BBB penetration of Cryptococcus neoformans, significantly weakening its pathogenicity, and hob1Δ In mutations ITR1a, ITR3c, MPR1, FZC3, PDR802 The induction of other BBB crossing factors, such as [mention specific factors], is reduced.

[0006] On the other hand, Cryptococcus gatiei, a sibling of Cryptococcus neoformans, primarily infects the lungs but not the brain, and Hob1 does not contribute to BBB penetration or pathogenicity. This highlights the role of Hob1 as the primary regulator of BBB penetration and pathogenicity in Cryptococcus neoformans, a role that is absent in Cryptococcus gatiei. However, the downstream signaling networks dominated by Hob1 and the direct mechanisms regulating BBB penetration in Cryptococcus neoformans remain largely unknown.

[0007] The inventors discovered Hob1, a homeobox transcription factor that regulates this process, through research on the mechanism by which Cryptococcus crosses the blood-brain barrier (Korean Patent Publication No. 10-2017-0077066). Subsequently, in the present invention, the downstream network of Hob1 was explored through transcriptome analysis. Among 100 proteins dependent on Hob1, Hbf1, an important blood-brain barrier crossing factor significantly induced by Hob1 under host-mimicking conditions, was discovered, and the present invention was completed by confirming that the deletion of Hbf1 significantly inhibits the blood-brain barrier penetration ability of Cryptococcus neoformans. Prior art literature

[0009] Korean Published Patent No. 10-2017-0077066 Korean Published Patent No. 10-2023-0098758 The problem to be solved

[0010] The technical problem that the present invention aims to solve is to provide a method for screening antifungal agents against Cryptococcus neoformans.

[0011] Another objective of the present invention is to provide a screening method for inhibitors of Cryptococcus neoformans crossing the blood-brain barrier.

[0012] In addition, the technical problem that the present invention aims to solve is to provide an antifungal composition against Cryptococcus neoformans.

[0014] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0016] To achieve the above technical problem, one embodiment of the present invention is Cryptococcus neoformans ( C. neoformans A step of contacting a candidate substance with ); and the Cryptococcus HBF1 The present invention provides a method for screening inhibitors of the blood-brain barrier (BBB) ​​of Cryptococcus neoformans, comprising the step of measuring the expression level of the mRNA of a gene or the protein thereof, or measuring the activity of the protein thereof.

[0017] In an embodiment of the present invention, if the measured expression level or activity is reduced compared to a control group that has not come into contact with the candidate substance, the step of selecting the candidate substance as a blood-brain barrier (BBB) ​​penetration inhibitor may be further included.

[0018] To achieve the above technical objective, another embodiment of the present invention comprises the steps of: contacting a candidate substance with Cryptococcus neoformans (C. neoformans); and the Cryptococcus HBF1 The present invention provides a method for screening antifungal agents for Cryptococcus neoformans, comprising the step of measuring the expression level of the mRNA of a gene or the protein thereof, or measuring the activity of the protein thereof.

[0019] In an embodiment of the present invention, if the measured expression level or activity is reduced compared to a control group that has not come into contact with the candidate substance, the step of selecting the candidate substance as an antifungal agent may be further included.

[0020] To achieve the above technical problem, another embodiment of the present invention provides an antifungal composition comprising an inhibitor screened according to the above method.

[0021] To achieve the above technical problem, another embodiment of the present invention is of Cryptococcus neoformans HBF1 The present invention provides an antifungal composition for Cryptococcus neoformans comprising a substance that inhibits the expression of a gene, or the expression or activity of a protein expressed therefrom.

[0022] In an embodiment of the present invention, the material may be a nucleic acid, antibody, aptamer, peptide, protein, compound, or natural product.

[0023] In an embodiment of the present invention, the material is HBF1 It may include antisense, siRNA (small interfering RNA), or shRNA (short hairpin RNA) nucleic acids having a sequence complementary to the mRNA of the gene. Effects of the invention

[0025] The present invention relates to a screening method for blood-brain barrier crossing inhibitors against Cryptococcus neoformans, a screening method for antifungal agents, and an antifungal composition. Since the screening method of the present invention utilizes a novel gene that regulates the blood-brain barrier crossing of Cryptococcus neoformans, it is possible to effectively screen for blood-brain barrier crossing inhibitors and antifungal agents.

[0027] The effects of the present invention are not limited to the effects described above, 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. Brief explanation of the drawing

[0029] Figure 1 shows the transcriptome analysis results of Cryptococcus neoformans, and Figure 1A shows the wild-type strain and by performing differential expression gene analysis. HOB1The genes with increased expression and genes with decreased expression among the transcriptome expression levels of the deficient strain are shown in R using the Enhanced volcano package, Figures 1B and 1D are the results of analyzing the gene-ontology (GO-term) of the genes corresponding to the basal condition (B) and HMC condition (D) among the differential expression genes analyzed in Figure 1A, and Figures 1C and 1E are the results of KEGG analysis using the genes corresponding to the basal condition (C) and HMC condition (E) among the differential expression genes analyzed in Figure 1A. Figure 2 is an image of Cryptococcus neoformans wild-type strains, hob1Δ(YSB2308, 2309), and hob1Δ::HOB1(YSB7467) cultured in YPD medium containing 1 μg / ml of Latruculin A for 3 days. Figure 3 shows the results of transcriptome analysis analyzed through Venn diagram analysis, where Figure 3A shows the results of analyzing upregulated genes and Figure 3B shows the results of analyzing downregulated genes. Fig. 4 is HBF1 , HBF101 Figure 4A shows the structure of a protein. HBF1 , HBF101 This shows a schematic diagram of the protein domain and its size, and Figure 4B shows the IPR018824 domain predicted by RoseTTAFold and HBF1 , HBF101 This is a predicted diagram of the overall structure, and Fig. 4C is HBF1 class HBF101 This is the result of the ClusterW analysis of the sequence similarity of, and Fig. 4D is HBF1 class HBF101 The structure of the protein was predicted using RoseTTAFold. Fig. 5 is HBF1 Figures 5A and 5B illustrate the production of gene deletion and overexpression strains. HBF1 This shows the production strategy and schematic diagram of gene deletion or overexpression strains, and Figures 5C and 5D are HBF1This shows the diagnostic PCR results of strains with gene deletion or overexpression, and Figures 5E and 5F are HBF1 This shows the Southern blotting results of gene deletion or overexpression strains, and Fig. 5G is fabricated HBF1 This shows the results of measuring the expression levels of gene deletion and overexpression strains. Fig. 6 is HBF1 This shows the experimental results regarding the involvement of genes in oxidative stress. Fig. 7 is HBF1 This shows the experimental results regarding the involvement of genes in melanin formation. Fig. 8 is HBF1 This indicates the intracellular location of the gene. Fig. 9 is HBF1 It measured the change in fluorescence intensity of the gene. Figure 10 shows the results of the analysis of the blood-brain barrier (BBB) ​​passage of Cryptococcus neoformans of the Hbf1 variant. Fig. 11 is HBF1 This shows the production of Saccharomyces cerevisiae yeast strains, and Figures 11A, 11B, and 11C are via Two-step PCR HBF1 This shows the strategies for cDNA synthesis, insert, and vector generation; Fig. 11D shows cloning using Gibson assembly and the results; and Figs. 11E and 11F show Saccharomyces cerevisiae. HBF1 This shows the diagnostic PCR results for expression and covector insertion transformation. Figure 12 shows a yeast strain HBF1 This shows the results of analyzing the expression of using qRT PCR. Specific details for implementing the invention

[0030] The present invention will be described in detail below.

[0032] The present invention relates to a screening method for inhibitors of the blood-brain barrier passage of Cryptococcus neoformans.

[0033] The present invention relates to Cryptococcus neoformans ( C. neoformans A step of contacting a candidate substance with ); and the Cryptococcus HBF1 The method includes the step of measuring the expression level of the mRNA of a gene or the protein thereof, or measuring the activity of the protein thereof.

[0034] The above Cryptococcus neoformans strain can be cultured using methods and conditions known in the art.

[0035] The step of contacting the candidate substance with the Cryptococcus neoformans may involve treating the Cryptococcus neoformans with the candidate substance using means known in the art, under methods and conditions known in the art. It may be appropriately treated according to the desired throughput and treatment method.

[0036] The above candidate substances may include, for example, nucleic acids, antibodies, aptamers, peptides, proteins, compounds, or natural products, but are not limited thereto.

[0037] The measurement of the mRNA expression level can be performed by methods known in the art, for example by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction, RNase protection assay (RPA), Northern blotting, DNA chip (Microarray), or RNA sequencing analysis.

[0038] The expression level of the above protein can be measured by methods known in the art, for example, by Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), or protein chip.

[0039] If the above-mentioned measured expression level or activity is reduced compared to a control group that has not come into contact with the candidate substance, the method may further include a step of selecting the candidate substance as a brain-blood barrier penetration inhibitor.

[0040] The above HBF1 The gene may consist of a sequence identified by the unique number (CNAG_03759) listed in Table 2.

[0042] The present invention relates to a method for screening antifungal agents against Cryptococcus neoformans.

[0043] The present invention relates to Cryptococcus neoformans ( C. neoformans A step of contacting a candidate substance with ); and the Cryptococcus neoformans HBF1 It includes the step of measuring the expression level of the mRNA of a gene or the protein, or measuring the activity of the protein.

[0044] The methods for the culture, candidate substances, expression, or activity measurement of Cryptococcus neoformans, etc., are as described above.

[0045] If the above-mentioned measured expression level or activity is reduced compared to a control group that has not come into contact with the candidate substance, the step of selecting the candidate substance as an antifungal agent may be further included.

[0046] The aforementioned antifungal agent is a drug that prevents and treats fungal infections; as Cryptococcus neoformans is a fungus that crosses the blood-brain barrier and causes meningitis, related to the ability to penetrate the blood-brain barrier HBF1 It can exhibit an antifungal effect by significantly inhibiting the expression or activity of.

[0048] The present invention relates to an antifungal composition against Cryptococcus neoformans.

[0049] The present invention relates to Cryptococcus neoformans HBF1 It includes a substance that inhibits the expression of a gene, or the expression or activity of a protein expressed therefrom.

[0050] The above-mentioned substances may be nucleic acids, antibodies, aptamers, peptides, proteins, compounds, or natural products, but are not limited thereto.

[0051] The above substance is HBF1 It may be an antisense, siRNA (small interfering RNA), or shRNA (short hairpin RNA) nucleic acid having a sequence complementary to the mRNA of the gene, but is not limited thereto.

[0052] 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 product composition, or a cosmetic composition.

[0053] The above antifungal agent is a drug that prevents and treats fungal infections by inhibiting the proliferation and growth of fungi.

[0054] The above pharmaceutical composition may be administered orally in solid, semi-solid, or liquid form by adding a commercially available inorganic or organic carrier, or administered parenterally, rectally, topically, transdermally, intramuscularly, intraperitoneally, subcutaneously, etc.

[0055] Examples of preparations for oral administration include tablets, pills, granules, soft and hard capsules, powders, fine granules, powders, emulsions, syrups, pellets, beverages, etc. Additionally, examples of preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, etc.

[0056] The active ingredient of the present invention can be easily formulated by following conventional methods, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspending agents, and other commonly used auxiliary agents may be appropriately added.

[0057] In addition, the dosage of the active ingredient will vary depending on the age, gender, and weight of the subject to treatment, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. The determination of the dosage based on these factors is within the level of a person skilled in the art, and generally, the dosage may range from 0.001 mg / kg / day to approximately 2000 mg / kg / day, but this does not limit the scope of the invention in any way.

[0059] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0061] Materials and Methods

[0062] 1. Transcriptome analysis (RNA sequencing)

[0063] Cryptococcus neoformans ( Cryptococcus neoformans ) and Cryptococcus gati ( Cryptococcus gattii ) wild type and hob1 The Δ mutant was initially cultured overnight at 30°C, and subsequently OD 600The cells were transferred to fresh YPD medium until a value of 0.8 was reached. After washing the cells three times with PBS buffer, they were cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C and 5% CO2 for 3 hours. After culture, 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 for three independent cultures for each strain.

[0064] For RNA sequencing, a cDNA library was prepared using the TruSeq mRNA Library Kit (Illumina, USA) with 1 μg of total RNA from each sample, 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. The purified reads were aligned to the Cryptococcus neoformans H99 reference genome using Hisat2 v2.2.1, utilizing the Hisat and Bowtie2 algorithms. Annotation data was retrieved from the NCBI FTP server. Hisat2 was executed using default values ​​with the "-p 30" and "--dta -1" options. Aligned reads were managed using Samtools v0.1.19 by modifying the default parameters for transformation ("-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 transcriptome 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; for significance, thresholds were applied for doubling changes and P-values ​​less than 0.05.

[0066] 2. Construction of gene deletion mutants

[0067] HBF1The mutant strain was constructed within the genetic background of Cryptococcus neoformans serotype A strain H99S. For genetic disturbance, a cassette conferring norosothricin resistance (the norosothricin acetyltransferase gene, encoded by NAT) was constructed using conventional nested PCR or NAT splitting marker / double junction (DJ) PCR methodologies. In the first PCR step to construct this cassette, HBF1The 5' and 3' flanking sequences of the gene were amplified using L1 / L2 and R1 / R2 primer sets, respectively, using H99S genomic DNA as a template. Full-length NAT markers were amplified from the pNAT-STM plasmid using M13Fe (M13 forward extended) and M13Re (M13 reverse extended) primer pairs, which contain the NAT gene including its unique signature tag sequence. In the split marker / DJ-PCR approach, the 5' and 3' portions of the NAT marker were amplified individually using M13Fe / SM2 and M13Re / SM1 primer combinations, respectively. Following the initial amplification, the nested PCR method involved synthesizing a complete gene perturbation cassette by merging the initially amplified products into a template using L1 and R2 primer pairs. On the other hand, the split marker / DJ-PCR strategy utilized the initial PCR product as a template to amplify the 5' and 3' portions of the NAT split gene perturbation cassette using L1 / SM2 and R2 / SM1 primer combinations, respectively. For the biological transformation process, H99S strains were 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 hours. The PCR-generated perturbation cassettes were adsorbed onto 600 μg of 0.6 μm diameter gold microcarrier beads (Bio-Rad) and then delivered to the cells using a PDS-100 particle delivery system (Bio-Rad). After transformation, cells were collected after being cultured at 30°C for 4 hours to promote the recovery of cell membrane integrity, and stable transformants were selected by culturing them in YPD solid medium (YPD+NAT) containing 100 μg / mL norsothricin. Subsequently, they were purified by passing them through YPD+NAT medium several times.For initial verification, NAT-positive strains were screened using diagnostic PCR. Southern blot analysis was performed on each mutant strain to ensure accurate gene deletions and exclude off-target integration. Further verification of mutant phenotypes to exclude irrelevant mutation effects involved generating multiple independent deletion strains for each mutation.

[0069] 3. HBF1 Construction of overexpression, complement, and fluorescent protein tagging strains

[0070] In Cryptococcus neoformans HBF1 To produce a sustained overexpression strain, HBF1 The original promoter of the gene is a histone H3 It was replaced with a promoter. This was achieved by constructing a homologous recombinant cassette amplified in multiple steps. In the initial PCR step, HBF1 The 5'-flanking and 5'-coding regions were amplified using L1 / OEL2 and OER1 / PO primer pairs, respectively. At the same time, the NAT-H3 promoter region was amplified using the primer pairs listed in Table 1.

[0071]

[0072] In the second PCR step, Double Joint (DJ) PCR was used to P H3 : HBF1 The 5' area of ​​the cassette was constructed. This HBF1 Amplification was performed using the primer pairs listed in Table 1, utilizing the 5'-flanking region and the NAT-H3 promoter region as templates. Likewise, P H3 : HBF1 The 3' area of ​​the cassette is HBF1 It was synthesized by DJ-PCR using a mixed template of the 3'-flanking region and the NAT-H3 promoter region and amplified with another primer pair. The assembled P H3 :HBF1The cassette was introduced into the wild-type H99S strain via biological transformation. Stable transformants were selected in yeast extract-peptone-dextrose (YPD) medium supplemented with norsothricin (NAT), and proper insertion was confirmed by diagnostic PCR using SO / B79 primer pairs. The integration and proper orientation of the cassette were confirmed by Southern blot analysis using specific probes PCR-amplified with L1 and PO primers.

[0073] To construct untagged and mRuby3-tagged strains, the open read frame (ORF) of HBF1, including the promoter region excluding the stop codon, was PCR-amplified 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 E. coli cells (provided by New England BioLabs, USA). The plasmid was extracted after transformation. The pNEO_HBF1-mRuby3 plasmid was cleaved with the StuI enzyme, linearized, and then subjected to biological transformation hbf1 It was introduced into the Δ(YSB10384) strain.

[0075] 4. of the non-pathogenic fungus Saccharomyces cerevisiae HBF1 Construction of expression strains

[0076] In the non-pathogenic fungus Saccharomyces cerevisiae HBF1 To produce a sustained overexpression strain, HBF1 The cDNA of the gene was synthesized from the cDNA of the Cryptococcus neoformans H99 strain using a primer set. HBF1 Since the gene's cDNA is extremely short at 89 amino acids, for smooth cloning, a high-expression gene TEF1 The gene promoter was synthesized. This was achieved by constructing a homologous recombinant cassette amplified in multiple steps. In the initial PCR step, HBF1The 5'-flanking and 5'-coding regions were amplified using L1 / OEL2 and OER1 / PO primer pairs, respectively. Simultaneously, TEF1 The promoter region was also amplified using the corresponding primer pair. In the second PCR step, P was amplified using double joint (DJ) PCR. TEF :HBF1 The 5' region of the cassette was constructed. This is the 5'-flanking region of HBF1 and TEF1 The promoter region was used as a template and amplified with the corresponding primer pair. Similarly, P TEF :HBF1 The 3' area of ​​the cassette is HBF1 The 3'-planking area of ​​and TEF1 It was synthesized by DJ-PCR using a mixed template of the promoter region and amplified with another primer pair. The assembled P TEF :HBF1 The cassette was inserted into the pLEU_TEF plasmid. Cloning was performed using the Gibson assembly method with the pLEU_TEF plasmid and HBF1 cDNA treated with restriction enzymes BamHI and SacI. The plasmids were transformed into the non-pathogenic fungal strain Saccharomyces cerevisiae BY4742, and the transformation was carried out according to the following procedure. The BY4742 strain, cultured at 30°C for at least 16 hours, was subcultured, and the OD 600After growing the cells until the value reached 0.6, they were collected and diluted in 2.5 ml of sterile distilled water. A PEG / LiOAc mixture containing 1 ml of 50% PEG, 180 µl of 1M LiOAc, and 125 µl of SS-DNA was prepared, and 300 µl of this mixture was combined with 1 µg of plasmid. 100 µl of the diluted strain cultured above was added, and the mixture was incubated at 42°C for 45 minutes. The strain was collected by centrifugation, the supernatant was discarded, and the solution was diluted with 100 µl of 0.9% NaCl solution. A portion of the diluted solution was plated onto SD-Leu medium and incubated at 30°C for 3–4 days. The correct strain was confirmed using primer pairs via diagnostic PCR on selected colony-forming groups from SD-Leu medium.

[0078] 5. HBF1 Quantitative RT-PCR for Confirmation of Gene Expression

[0079] Leucine covector transformed strain and P TEF _ HBF1 After incubating each in EBM-2 medium overnight, OD6 00nm Recultured 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 at 6, 12, and 24 hours in EBM-2 medium while shaking at 120 rpm. The collected cells were immediately frozen and freeze-dried. 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, oligo(dT)-pdN6 was added, and the mixture was heated at 65°C for 5 minutes. Then, an RNAse inhibitor (M007, Enzynomics, South Korea), dNTPs, and reverse H minus Reverse Transcriptase (EP0752, Thermo Scientific, USA) were added, incubated at 50°C for 60 minutes, and then inactivated by heating at 85°C for 10 minutes. Gene expression levels were measured for the actin gene ( ACT1 ) and leucine gene( LEU2 ) expression values ​​were normalized, and the fold change relative to the basal expression level in the leucine covector transformed strain was calculated.

[0081] 6. Growth and Chemosus Analysis

[0082] Cryptococcus neoformans HBF1To evaluate the growth kinetics and chemosensitivity of mutant strains, strains cultured overnight at 30°C were serially diluted tenfold (from 1:1 to 1:10,000). These dilutions were applied dropwise to YPD agar media containing chemicals of various concentrations. 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, and diamide for oxidative stress; cadmium sulfate (CdSO4) for heavy metal stress; methyl methanesulfonate and hydroxyurea for genotoxic stress; sodium lauryl sulfate (SDS) for membrane instability; chalcofluor white and Congo red for cell wall instability; tunicamycin™ and dithiothreol (DTT) for endoplasmic reticulum (ER) and reduction stress; and several antifungal agents (fludioxynil, fluconazole, amphotericin B, flucytosine))).

[0083] These media were cultured under conditions containing or excluding glucose to test stress responses under various trophic 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 confirm the temperature tolerance of each mutant strain, YPD media with serially diluted cells applied dropwise was cultured at four different temperatures: 25°C, 30°C, 37°C, and 39°C. Growth was recorded via photographs taken from day 2 to day 4. This comprehensive experimental design allowed for a detailed evaluation of the adaptability of the mutant strains to various temperatures, as well as their robustness to environmental stress and antifungal agents.

[0085] 7. Analysis of in vitro toxicity factor production

[0086] To measure capsule production efficiency, each mutant was cultured at 30°C, then spotted onto Dulbecco's modified Eagle's (DME) agar medium and incubated at 37°C for 2 days. Subsequently, the cells were scraped off, 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 medium and FBS agar medium (10% fetal bovine serum and 90% PBS), incubated at 37°C for 2 days, scraped off, 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 microscope (DIC) (BX51, Olympus, Tokyo, Japan). Capsule thickness was measured by subtracting the cell diameter (total diameter minus cell body diameter) from the capsule diameter. To quantitatively measure capsule thickness, 50 cells were measured for the H99S strain and each phosphatase mutant. Subsequently, to measure melanin production efficiency, mutants including the wild-type strain were cultured in YPD medium at 30°C for 16 hours, washed with PBS, and cultured in 3 μl aliquots of Niger seed medium. The cultured cells were incubated at 37°C and photographed for 1 to 3 days.

[0088] 8. Visualization of the intracellular localization of mRuby3-tagged HBF1

[0089] The Hbf1-mRuby3-tagged strain was initially cultured overnight in YPD medium at 30°C. The culture medium's OD in YPD medium 600Recultured until it reached 0.8. Subsequently, the culture medium was 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 to analyze the basal position of Hbf1, and the other was resuspended in an equal volume in RPMI 1640 medium supplemented with 10% FBS. The samples were cultured in a CO2 incubator at 37°C under horizontal vibration conditions of 120 rpm. Cells were harvested at 10, 30, 60, and 180 minutes, fixed in a 4% paraformaldehyde solution containing 3.4% sucrose for 15 minutes 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) in the dark for 30 minutes. Finally, the stained cells were observed using differential interference contrast (DIC) and fluorescence microscopy (Nikon Eclipse, Japan).

[0091] 9. In vitro BBB crossing analysis

[0092] BBB crossing analysis was 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-free coated plates (354236, Corning, USA) until 80% fusion was reached. Subsequently, 5 x 10 4 hCMEC / D3 cells were seeded onto an 8-μm porous collagen-coated membrane (353097, BD Falcon), and EndoGRO TM - It was maintained in the MV Complete Media Kit (SCME004, Sigma-Aldrich, USA). After 24 hours, the medium was changed to fresh EndoGR supplemented with 2.5% human serum (H4522, Sigma, USA). TMThe medium was replaced, and the cells were cultured for an additional 4 days. On the day before yeast inoculation, the medium was EndoGRO containing 1.25% human serum. TM The medium was replaced. Cell culture was conducted at 5% CO2 and 37°C. To assess the integrity of cell junctions, trans-endothelial 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 5 x 10⁶ 5 Cells were prepared in 100 μL of PBS and added to an hCMEC / D3 seeding membrane. After 24 hours of incubation, yeast cells that passed through the membrane were collected. The BBB passage rate of each mutant strain was determined as a relative BBB passage rate compared to the wild type.

[0094] <Result>

[0095] 1. Comparative Transcriptome Analysis of Hob1Δ Mutations in Cryptococcus neoformans and Cryptococcus gatiai

[0096] To analyze the downstream network regulated by Hob1, RNA sequencing (RNA-seq)-based transcriptome analysis was performed on wild-type (WT) and hob1Δ mutant strains (Fig. 1). Distinct phenotypic changes following Hob1 deletion in Cryptococcus neoformans and Cryptococcus gatiai were identified, and transcriptome analysis was performed between these two species. Additionally, the important role of Hob1 in crossing the blood-brain barrier (BBB) ​​was confirmed, and transcriptomes were closely investigated under host-mimicking conditions. For this purpose, tissue culture medium (RPMI) supplemented with 10% FBS was used, and culture was performed at 37°C and 5% CO2.

[0097] In Cryptococcus neoformans hob1ΔUnder baseline conditions (YPD medium, 30°C), 661 genes were upregulated and 817 genes were downregulated in the mutation (compared to the WT strain). The set of upregulated genes was mainly capsule biosynthetic genes ( CAS32, CAP4, CAP60 genes related to the cell cycle, division, and meiosis ( IRR1, CDC2801, CDC7 It included (etc.). On the other hand, the downregulated genes were mainly genes of the mitotic activation protein kinase pathway (MAPK signaling pathway) ( HSP12, PTP1, PTP2 It belonged to the categories of extracellular secretion and actin-binding genes (e.g.). In particular, it was generally associated with the MAPK signaling pathway. ERG11 ...was paradoxically upregulated. Subsequent analysis via gene ontology terms and the KEGG pathway highlighted dynamic changes in signaling pathways, demonstrating the induction of RNA degradation and meiotic pathways, as well as metabolic pathways, starch and sucrose metabolism, and carbon metabolism. On the other hand, in Cryptococcus gatiai hob1 Transcriptomic changes in the Δ mutations were relatively minor, and only three upregulated and five downregulated genes were identified. These genes do not belong to specific GO terms or the KEGG pathway, and their functions were largely undefined. The actin-binding gene category is HOB1 The effect could be indirectly confirmed through the result that the growth rate of the defective variant increased significantly when grown in a drug medium containing latrunculin A, a drug that interferes with actin binding (Fig. 2).

[0098] Subsequently, a distinct contrast was observed under HMC conditions. Cryptococcus neoformans hob1Δ Mutations significantly regulated 453 genes (320 induced, 133 reduced), suggesting a crucial role for Hob1 in adapting to host-like conditions. This adaptation is characteristic of Cryptococcus gatiai hob1ΔIn stark contrast to mutations, only seven genes were significantly regulated here, highlighting the difference 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 decreased. This change may reflect the strict survival requirements imposed by HMC, namely increased temperature and changes in nutrient availability.

[0099] In a comparative analysis between basal and HMC conditions, 187 genes in Cryptococcus neoformans were generally upregulated under both conditions, highlighting pathways related to chromatin remodeling, carbohydrate metabolism, and cell division (Fig. 3). This commonality indicates a core function regulated by Hob1 regardless of the external environment. Conversely, it was clearly revealed that gene regulation varies depending on the conditions; consequently, the different sets of genes activated under basal and HMC conditions can be interpreted as representing tailored responses to environmental challenges. Changes in the number of downregulated genes further emphasize this condition specificity, showing a pattern of significantly reduced downregulated genes under HMC conditions compared to basal conditions. This pattern suggests a complex role for Hob1 in regulating metabolic and biosynthetic processes, particularly under the stress conditions represented by HMC.

[0100] In conclusion, our detailed comparative transcriptome analysis reveals the significant impact of Hob1 deletion on gene expression regulation within Cryptococcus neoformans and highlights the broad range of biological processes affected under various environmental conditions. These contrasting responses between Cryptococcus neoformans and Cryptococcus gatiai emphasize the key role of Hob1 in the pathogenicity and adaptive strategies of Cryptococcus neoformans within the host environment.

[0102] 2. Identification of Hob1 Downstream Target Candidates: A Transcriptome Analysis-Based Approach

[0103] Previous research revealed that the reason for the difference in Hob1 function between Cryptococcus neoformans and Cryptococcus gatiai is not due to the nucleotide sequence of Hob1. Instead, it suggested that Hob1's downstream target proteins may function differently in these two species, implying the interaction of complex, species-specific regulatory mechanisms. To explore this hypothesis further, we [investigated] Cryptococcus neoformans under both basal and host-mimicking (HMC) conditions hob1Δ A comprehensive transcriptome analysis of the mutations was performed. This analysis played a significant role in identifying a group of genes presumed to be under the direct or indirect regulation of Hob1, which is summarized in Table 2 (Prediction of potential downstream targets and list of candidate genes through Hob1 transcriptome analysis).

[0104]

[0105] Among the downstream targets, CNAG_03759 has emerged as a gene of particular interest for several compelling reasons. First, Hbf1 was the gene with the most consistently reduced expression when Hob1 was deficient in both baseline conditions and HMC experimental setups. This consistent regulatory pattern implies a strong and specific interaction in which Hbf1 is present on the pathway regulated by Hob1. Second, unlike other well-documented genes, CNAG_03759 encodes a protein whose role is not yet understood, making it a new subject for further functional characterization.

[0106] The protein encoded by CNAG_03759 was characterized by its short length (only 89 amino acids) and a domain with conidial-specific functions (Fig. 4). This domain is also present in CNAG_01056, a structural isogeneic with similar protein size and domain structure. However, in contrast to CNAG_03759, CNAG_01056 appeared not to be regulated by Hob1 due to extremely low expression levels under the tested conditions. The differential expression between these isogeneics provided interesting insights into the selective regulatory role of Hob1.

[0107] Although Cryptococcus neoformans lacks the conidial processes common in other fungal species, the function of CNAG_03759 in the pathogenicity of Cryptococcus neoformans was previously merely speculative. However, the protein's strong regulatory associations and unique protein structure led us to designate it as the Hob1-dependent blood-brain barrier crossing factor (HBF1), and its structural isogeneic, CNAG_01056, HBF101 It was named as. The research team HBF1 It has been hypothesized that Cryptococcus neoformans may play a crucial role in facilitating the passage of this virus across the blood-brain barrier, which is a key stage in the development of meningitis. This opens a new avenue for understanding the molecular mechanisms by which Cryptococcus neoformans invades the central nervous system and suggests potential targets for therapeutic intervention.

[0109] 3. Hbf1's involvement in the oxidative stress response of Cryptococcus neoformans

[0110] To better understand the functional importance of Hbf1 in the growth and survival of Cryptococcus neoformans hbf1Δ and P H3 :HBF1 A mutant strain was developed. hbf1Δ In mutant strains HBF1The gene was completely removed and replaced with an antifungal resistance marker, which is a common strategy to evaluate gene function by observing the phenotypic consequences of gene deletion. On the other hand, P H3 :HBF1 In mutant strains HBF1 The original promoter of is highly active H3 Replace with a promoter HBF1 We induced gene overexpression and sought to confirm the effect of increased gene expression on fungal physiology (Fig. 5).

[0111] Interesting resistance patterns were observed through various in vitro phenotypic analyses using oxidative and endoplasmic reticulum (ER) stress-inducing substances, namely hydrogen peroxide (H2O2) and dithiothreol (DTT) (Fig. 6). H2O2, a potent oxidizing agent, imposes severe oxidative stress on the cellular system, while DTT induces ER stress by disrupting the environment necessary for oxidative protein conjugation. Both substances hbf1Δ The strain showed a slight increase in resistance. This implies that the presence of Hbf1 increases vulnerability to oxidative conditions, and therefore, the removal of Hbf1 can strengthen the fungus against such stress.

[0112] Additionally, we investigated the effect of Hbf1 overexpression on melanin formation, a key protective mechanism of Cryptococcus neoformans. Melanin plays a crucial role not only in protecting bacterial cells from UV and strong ionizing radiation but also in mitigating oxidative damage. Our results indicated that melanin production was significantly reduced when Hbf1 was overexpressed (Fig. 7). Given that melanin plays a vital role in the pathogenicity of Cryptococcus neoformans, this reduction may impair the bacteria's ability to survive under hostile environmental conditions during infection of a host organism.

[0113] Unexpectedly, Hbf1 deletion increased resistance to oxidative stress, whereas overexpression prevented the effective utilization of melanin to protect against such 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 the survival but also for the pathogenicity of 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 to suppress Cryptococcus infection by regulating its expression or function.

[0115] 4. Analysis of the intracellular localization of Hbf1

[0116] To investigate the intracellular localization of Hbf1 in Cryptococcus neoformans, an mRuby3 fluorescent tag was attached to the C-terminus of the Hbf1 protein. hbf1Δ::HBF1 A -mRuby3 reporter strain was constructed. This genetic modification was evaluated to see if the fusion protein maintains biological activity under stress conditions (Fig. 8). This strain showed that resistance to oxidative stress and endoplasmic reticulum (ER) stress was restored to wild-type levels, confirming that the Hbf1-mRuby fusion protein maintains full function.

[0117] Afterwards, standard conditions and HBF1 We focused on elucidating the intracellular localization of Hbf1 under host-like conditions (HMC) that strongly induce expression. Under standard and untreated conditions, Hbf1 was distributed throughout the cytoplasm with weak fluorescence intensity. However, under HMC conditions, Hbf1 was relocated to specific intracellular sites, indicating that it is localized to specific regions under host-like conditions. This targeted relocation suggests the possibility that Hbf1 performs specialized functions related to stress responses or cellular signaling pathways.

[0118] Further analysis using fluorescence microscopy confirmed that the brightness of the fluorescence signal increased under HMC conditions compared to untreated cells (Fig. 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 cells' response to host-like conditions. Furthermore, the timing of the fluorescence increase peaking within 30 minutes of HMC treatment suggests that Hbf1 is an early response protein, which is likely regulated by a Hob1-dependent transcription mechanism. These temporal expression and localization patterns indicate that Hbf1 plays a crucial role in the immediate cellular response to environmental stress.

[0119] These observations highlight the complex regulation and critical functional roles of Hbf1 within Cryptococcus neoformans, particularly its response to environmental challenges that mimic host conditions. This extended analysis provides significant insights into the cellular behavior and fungal biology of Hbf1, contributing to the understanding of the molecular mechanisms by which Cryptococcus neoformans adapts and survives in hostile host environments.

[0121] 5. Analysis of the Blood-Brain Barrier (BBB) ​​Crossing by Hbf1 Variant Cryptococcus neoformans

[0122] This study investigated whether Hbf1 is essential for crossing the BBB. This conclusion is based on the identification of Hbf1 as an important downstream target in transcriptome analysis. The in vitro BBB model, first developed by Vu et al., utilizes human brain microvascular endothelial cells (HBMECs) cultured on a transwell membrane and mimics the physical separation between blood and brain compartments. This model, in previous studies mpr1Δ It was verified using mutants, non-pathogenic Saccharomyces cerevisiae (S288C strain), and pathogenic Cryptococcus neoformans (H99 strain). We adopted a similar transwell-based system to evaluate whether Hbf1 can promote BBB penetration.

[0123] As a result, wild-type (WT) Cryptococcus neoformans successfully crossed the BBB within 24 hours of culture. In contrast, hbf1Δ The mutation significantly reduced the ability to cross the BBB, highlighting the potential role of Hbf1 in this process. Subsequently, to determine whether Hbf1 directly affects BBB permeability, we used an Hbf1 complementary strain ( hbf1Δ::HBF1 ) and mutant strains with overexpression of the Hbf1 gene (P H3 :HBF1 ...was evaluated. The complementary strain restored BBB penetration ability to wild-type levels, whereas the overexpression mutant strain did not exceed wild-type levels (Fig. 10). These results indicate that while Hbf1 is essential for BBB penetration, overexpression does not proportionally increase BBB permeability. RNA-seq transcriptome analysis confirmed that HBF1 was upregulated 4.55-fold under HMC conditions, observing the functional effects under these conditions. This correlation highlights that Hbf1 plays an important role in crossing the human BBB.

Claims

Claim 1 Cryptococcus neoformans ( C. neoformans A step of contacting a candidate substance with ); and of the Cryptococcus HBF1 A method for screening inhibitors of the blood-brain barrier (BBB) ​​of Cryptococcus neoformans, comprising: a step of measuring the expression level of the mRNA of a gene or the protein thereof, or measuring the activity of the protein thereof; and a step of screening the candidate substance as a blood-brain barrier crossing inhibitor if the measured expression level or activity is reduced compared to a control group that has not come into contact with the candidate substance. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete

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