Centromere binding protein cbf1, its encoding gene and use thereof

Abstract

The present application belongs to the field of biotechnology and medicine, and relates to a centromere binding protein Cbf1, a coding gene thereof and application. Specifically, the present application provides a Cryptococcus virulence related protein, which is a centromere binding protein Cbf1. Meanwhile, the present application also provides application of the centromere binding protein Cbf1 in preparation of a medicine, CBF1 Gene knockout leads to reduction of tolerance of the strain to 37 DEG C, oxidation and cell wall pressure, and reduction of melanin production, so that the protein can be used as a drug target and applied to research and development of a new drug for treating diseases caused by Cryptococcus.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to the important role of a centromere-binding protein Cbf1 and its encoding gene in the pathogenicity of Cryptococcus grate. Background Technology

[0002] Cryptococcosis is an invasive infection caused by fungi of the genus Cryptococcus, including Cryptococcus neoformans. Cryptococcus neoformans ) and Cryptococcus grease ( Cryptococcus gattii Cryptococcal meningitis primarily affects the lungs and central nervous system, causing approximately 150,000 deaths globally each year, making it one of the leading causes of life-threatening fungal meningitis. With the widespread use of antifungal drugs, traditional antifungal treatments face the challenge of drug resistance. Compared to Cryptococcus neoformans, Cryptococcus gattii can infect immunocompetent hosts, and its unique pathogenic and drug resistance mechanisms are not yet fully understood, necessitating in-depth research to develop effective prevention and control strategies.

[0003] Currently, clinical treatments for Cryptococcal infections are limited to polyenes, azoles, pyrimidines, and echinocandins. Azides inhibit lanosterol biosynthesis by suppressing lanosterol 14α-demethylase encoded by ERG11 in the ergosterol biosynthesis pathway. Amphotericin B liposomes combined with 5-fluorocytosine are often used to treat the induction phase. Fluconazole is the first-line drug for the consolidation and maintenance phases, but long-term use can lead to resistance and treatment failure. Echinocandins disrupt the integrity of fungal cell walls by inhibiting the production of the key cell wall component (1,3)-β-D-glucan; the pyrimidine analogue flucytosine acts as an antimetabolite, blocking DNA synthesis.

[0004] However, in recent years, the widespread use of antifungal drugs has led to the rapid emergence of drug-resistant strains of Cryptococcus grate, so finding new drug targets has always been a research focus.

[0005] In *Saccharomyces cerevisiae*, Cbf1 is a positive regulator of methionine (Met) biosynthesis genes. It activates transcription by binding to E-box elements in the promoter regions of these genes, including MET2, MET3, MET5, MET6, MET10, MET14, MET16, MET25, and SAM2. The absence of Cbf1 prevents yeast from growing in methionine-deficient media (Met-phenotype).

[0006] Cbf1 also binds to centromere DNA element I (CDEI) in yeast. Cbf1 binding helps improve chromosome segregation fidelity and reduce the frequency of chromosome aneuploidy. Cbf1 also participates in regulating other non-methionine-related biological processes in Saccharomyces cerevisiae, such as respiration, vitamin metabolism, and autophagy, indicating that it is a pleiotropic global regulator.

[0007] However, there is currently no information regarding CBF1 Research on the role of genes and their encoded proteins in Cryptococcus. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides more therapeutic targets for Cryptococcus by identifying proteins associated with its pathogenicity, thus enabling its application in the treatment of diseases caused by Cryptococcus.

[0009] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a centromere-binding protein Cbf1, said centromere-binding protein Cbf1 comprising: The amino acid sequence as shown in SEQ ID NO.1; or, The sequence described in SEQ ID NO.1 is an amino acid sequence that has undergone substitution, deletion, or addition of one or more amino acid residues and is related to the virulence of Cryptococcus gutterinica.

[0010] This invention identified a Cryptococcus virulence-related protein, the Cbf1 protein (encoded by the gene CGB_D7380W in Cryptococcus grate), whose deletion could significantly affect the tolerance of Cryptococcus grate to high temperatures, cell wall stress, and oxidative stress, and reduce melanin production. Therefore, this invention constructed a [specific protein name - missing from original text] based on the wild-type Cryptococcus grate WM276 strain. CBF1 Gene deletion mutants were identified, and these strains were then studied.

[0011] The sequence of SEQ ID NO.1 is as follows: MPDPELSPIDPALQQPTLNSIQEAHDSAGVNPLNEFAQQVLGREADAENLLDFGRAHSILGEEQTFGDLLQAERRKDGEDEDDVGADRHMEHHQHGHEHVHEGHMEGQGEDVHYMAESVEQVMDPLPGRARG VRQAKKRRVGDDVVDGDGQIIDPLEYAKSRKDNHREVESRRRQAIADGIAAEIAQLLPSPPTPKEGKGQLLKRAVTYIHELLGKIARSVEEVAQVEQEKQKLRLMSDHAPYALNLPGGRRRIWQQAVILNWRG This invention tested the growth ability of wild-type strains and deletion mutants under cell wall pressure and oxidative stress conditions at 37°C. The results showed that the growth of the deletion mutants was inhibited compared to the wild-type under these conditions, and the mutants also exhibited a significantly reduced ability to produce melanin. Therefore, this invention provides a Cryptococcus protein Cbf1, which can serve as a potential target for developing novel treatment methods and drugs.

[0012] Secondly, the centromere-binding protein Cbf1, as described in the first aspect, is involved. CBF1 Genes, the ones mentioned CBF1 Genes include: The nucleotide sequence shown in SEQ ID NO.2; or, A nucleotide sequence that has more than 90% homology with SEQ ID NO.2 and is associated with the virulence of Cryptococcus grate.

[0013] The sequence of SEQ ID NO.2 is as follows: atgccagaccccgagctatcgcccatagacccagccctccagcaaccgaccctcaactccatacaggaagcccacgactcggcaggtgtcaatcctctcaacgagttcgcccagcaggtactcggaagagaggccgatgctgagaacctcctcgactttgggcgtgcccactcaatactcggagaggagcagacattc ggagatctactgcaggctgaaagaagaaaagatggagaagatgaagacgatgttggtgctgacaggcatatggagcatcatcaacatggtcatgaacatgtccacgaagggcacatggagggtcaaggagaggatgtgcattacatggctgagagtgtagaacaagtcatggatccgcttccagggcgcgctagaggag tacgccaagcaaagaagagaagggtcggcgacgatgtagttgacggtgatggccaaataatagacccgctggagtacgccaaaagccgaaaagataacc acagagaagtagaaagccggcggaggcaagctatagcggatggaatcgctgagattgctcaactccttcctagtcctccaaccccaaaagaagggaaagg acaactcctcaaacgggctgttacatatattcacgagttatactgggaaagattgcacgaagtgtggaggaagttgcgcaagttgagcaagaaaagcaaaa gctccggctgatgagcgatcacgctccatacgctttgaatcttcctggcggcaggcggaggatttggcagcaagcagtaattttgaattggagagggtaa A nucleotide sequence that shares more than 90% homology with SEQ ID NO.2 and is associated with the virulence of Cryptococcus grate is as shown in SEQ ID NO.3, which contains an intron sequence, as follows: atgccagaccccgagctatcgcccatagacccagccctccagcaaccgaccctcaactccatacaggaagcccacgactcggcaggtgtcaatcctctcaacgagttcgcccagcaggtactcggaagagaggccgatgctgagaacctcctcgactttgggcgtgcccactcaatactcggagaggagcagacattcggagatctactgcaggctgaaagaagaaaagatggagaagatgaagacgatgttggtgctgacaggcatatggagcatcatcaacatggtcatgaacatgtccacgaagggcacatggagggtcaaggagaggatgtgcattacatggctgagagtgtagaacaagtcatggatccgcttccagggcgcgctagaggagtacgccaagcaaagaagagaagggtcggcgacgatgtagttgacggtgatggccaaataatagacccgctggagtacgccaaaagccgaaaagataaccac gtgcgtcctccgcctttcttccagtg ctctggccctgacatcttcgcatgtag agagaagtagaaagccggcggaggcaagctatagcggatggaatcgctgagattgctcaactccttcctagtcctccaaccccaaaagaagggaaaggacaactcctcaaacgggctgttacatatattcacgagttactgggaaagattgcacgaagtgtggaggaagttgcgcaagttgagcaagaaaagcaaaagctccgg gtgggttgagctcattggtatgacatatccgaactaatgaaaaatagatggacattgcacatcttgaagcgcg gttag ctgatgagcgatcacgctccatacgctttgaatcttcctggcggcaggcggaggatttggcagcaagcagtaattttgaattggagagggtaa Among them, the intron sequence is the sequence shown by the underlined part, specifically gtgcgtcctccgcctttcttccagtg ctctggccctgacatcttcgcatgtag (SEQ ID NO.4) and gtgggttgagctcattggtatgacatatccga actaatgaaaaatagatggacattgcacatcttgaagcgcggttag (SEQ ID NO.5).

[0014] Thirdly, the present invention also provides an inhibitor of centromere-binding protein Cbf1, said inhibitor inhibiting the expression of centromere-binding protein Cbf1 as described in the first aspect.

[0015] Fourthly, the present invention also provides a kit for knocking out the centromere-binding protein Cbf1 as described in the first aspect.

[0016] This invention can be performed using the TRACE method. CBF1 For gene deletion, the specific primers used in this method are shown in SEQ ID NO. 6~11.

[0017] Fifthly, the present invention also provides a method for reducing the toxicity of Cryptococcus grease, the method comprising: Targeting centromere-binding protein Cbf1, the expression of Cbf1 is inhibited or the protein encoding Cbf1 is knocked out. CBF1 The steps of gene generation.

[0018] This invention discovered that the centromere-binding protein Cbf1 deletion mutant exhibits decreased tolerance to high temperature, cell wall stress, and oxidative stress, reduced melanin production, and weakened pathogenicity against the larvae of the large wax moth. Therefore, inhibiting or knocking out the expression of the centromere-binding protein Cbf1 is effective. CBF1 The gene can produce Cryptococcus grate, which has lower toxicity and has potential applications in subsequent drug preparation or screening.

[0019] The use of Cbf1 in the preparation of a drug that can treat diseases caused by Cryptococcus.

[0020] In a sixth aspect, the present invention provides the use of centromere-binding protein Cbf1 or the gene encoding said centromere-binding protein Cbf1 in the preparation of medicaments for the prevention or treatment of cryptococcal diseases.

[0021] As a preferred technical solution of the present invention, the application includes drug screening.

[0022] The above technical solution has the following advantages or beneficial effects: This invention focuses on the Cbf1 gene of Cryptococcus neoformans and involves the deletion of its coding gene in Cryptococcus grate. CBF1 Gene deletion can reduce Cryptococcus's tolerance to 37°C, cell wall stress, and oxidative stress, decrease melanin production, and weaken its pathogenicity to the larvae of the wax moth. By targeting Cbf1, drugs that reduce the pathogenicity of Cryptococcus can be obtained, and the obtained drugs can improve the treatment effect of cryptococcosis. Attached Figure Description

[0023] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0024] Figure 1 Provided according to embodiments of the present invention CBF1 The effect of gene deletion on the growth of Cryptococcus gutterinica at 30℃ and 37℃ is shown in the figure, with the left figure at 30℃ and the right figure at 37℃.

[0025] Figure 2 Provided according to embodiments of the present invention CBF1 The effect of gene deletion on Cryptococcus gutterinica under cell wall pressure conditions of 1.5 M KCl and 0.03% SDS is shown in the figure. The left figure is for 1.5 M KCl and the right figure is for 0.03% SDS.

[0026] Figure 3 Provided according to embodiments of the present invention CBF1 The effect of gene deletion on Cryptococcus gutterinii under oxidative stress conditions.

[0027] Figure 4 Provided according to embodiments of the present invention CBF1 The result of gene deletion reducing melanin production in Cryptococcus gattii (Gastrodinella lataniae) Figure 5 Provided according to embodiments of the present invention CBF1 The result of gene deletion leading to reduced pathogenicity of Cryptococcus gutterusol against larvae of the large wax moth. Detailed Implementation

[0028] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product manual.

[0029] In this article, Cryptococcus is a pathogenic bacterium that can cause disease. Diseases caused by Cryptococcus are commonly referred to as cryptococcosis, which is a lung or disseminated infectious disease caused by Cryptococcus, primarily causing pneumonia and meningitis, but also infecting the skin, bones, or internal organs. By using Cbf1 of this invention as a drug target, drugs effective in treating cryptococcosis can be obtained and applied in pharmaceutical production and the treatment of cryptococcosis.

[0030] Example 1 CBF1 Deletion of genes in Cryptococcus grate This invention uses the TRACE method. CBF1The specific procedure for gene deletion is as follows: Will CBF1 Extend the upstream and downstream sequences of the gene by 2000 bp each as reference sequences. Design primers Left F, Left R, Right F, and Right R (the reverse primer Left R for the upstream fragment and the forward primer Right F for the downstream fragment each carry a sequence of approximately 20 bp matching resistance fragments to facilitate subsequent overlap-PCR) to amplify fragments of 1-1.5 kb each upstream and downstream of the gene, which will be used as homologous fragments for gene deletion homologous recombination.

[0031] The two fragments, along with the resistant fragment NAT (Nourseothricin), were used as templates for overlap-PCR.

[0032] Then, using this overlap-PCR product as a template, the TRACE system was used to analyze... CBF1 The gene was deleted (as described in Yumeng Fan and Xiaorong Lin. 2018. Multiple Applications of a TransientCRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcusneoformans Species Complex. Genetics. 2018 Apr; 208(4): 1357–1372).

[0033] Received CBF1 Deletion strains cbf1Δ express.

[0034] The primers used (SEQ ID NO. 6~11) are shown in Table 1 below: Table 1

[0035] Example 2 CBF1 The effect of gene deletion on the growth of Cryptococcus at 37°C.

[0036] Wild-type strains. cbf1Δ After serial dilution, the samples were inoculated onto YPD plates and incubated at 37°C. It was found that at 37°C, compared to the wild type, [the following results were observed]. cbf1Δ The growth of [the organism] is significantly weakened, such as [the following]. Figure 1 As shown.

[0037] Experimental results show that compared with the wild type, CBF1The deletion of the gene severely restricts the growth of Cryptococcus at 37°C.

[0038] Example 3 CBF1 The influence of genes on the growth of Cryptococcus under cell wall stress conditions.

[0039] Wild-type strains. cbf1Δ After serial dilution, the samples were inoculated onto YPD plates containing 1.5 M KCl or 0.03% SDS and incubated at 30°C. Compared to the wild type, [the results showed that] cbf1Δ The growth of [the organism] is significantly weakened, such as [the following]. Figure 2 As shown.

[0040] Experimental results show that compared with the wild type, CBF1 The deletion of the gene reduces Cryptococcus's tolerance to cell wall stress.

[0041] Example 4 CBF1 The effect of genes on the growth of Cryptococcus under oxidative conditions.

[0042] Wild-type strains. cbf1Δ After serial dilution and inoculation onto YPD plates supplemented with hydrogen peroxide, it was found that compared with the wild type, cbf1Δ The growth of [the organism] is significantly weakened, such as [the following]. Figure 3 As shown.

[0043] Experimental results show that compared with the wild type, CBF1 The deletion of the gene severely inhibits the growth of Cryptococcus under oxidative stress conditions.

[0044] Example 5 CBF1 The influence of genes on melanin production in Cryptococcus.

[0045] Wild-type strains. cbf1Δ When inoculated onto melanin-inducing plates, compared to wild-type, [the results were observed]. cbf1Δ Melanin production is significantly reduced, such as Figure 4 As shown.

[0046] Experimental results show that compared with the wild type, CBF1 The deletion of the gene reduces the production of melanin in Cryptococcus.

[0047] Example 6 In the model of the large wax moth larva, CBF1 The influence of genes on the pathogenicity of Cryptococcus.

[0048] Using PBS as a negative control and WM276 as a positive control, and... cbf1ΔThe mutant strains were injected into the larvae of the model organism *Gnaphalium affine* for pathogenicity testing. cbf1Δ-1, cbf1Δ-2, cbf1Δ-3 (This is a set of three parallel experiments).

[0049] Compared with the wild-type group (positive control), cbf1Δ The mortality rate of the strain against the larvae of the large wax moth was significantly lower than that of the wild-type group, such as... Figure 5 As shown, the proof CBF1 Genes can significantly affect the virulence of Cryptococcus grate.

[0050] This invention mainly studies the important protein Cbf1 of Cryptococcus grate, and found that its deletion can lead to a decrease in the tolerance of Cryptococcus grate WM276 strain to high temperature, cell wall pressure and oxidative stress, a reduction in melanin production, and a weakening of pathogenicity to the larvae of the large wax moth.

[0051] Therefore, according to the present invention, Cryptococcus grate Cbf1 is the target of the drug. The drug can target Cbf1, thereby reducing the pathogenicity of Cryptococcus, and can thus be used to treat diseases caused by Cryptococcus.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Centromere-binding protein Cbf1, characterized in that, The centromere-binding protein Cbf1 includes: The amino acid sequence as shown in SEQ ID NO.1; or, The sequence described in SEQ ID NO.1 is an amino acid sequence that has undergone substitution, deletion, or addition of one or more amino acid residues and is related to the virulence of Cryptococcus gutterinica.

2. Encoding the centromere-binding protein Cbf1 as described in claim 1 CBF1 Genes, characterized by, The CBF1 Genes include: The nucleotide sequence shown in SEQ ID NO.2; or, A nucleotide sequence that has more than 90% homology with SEQ ID NO.2 and is associated with the virulence of Cryptococcus grate.

3. A repressor of centromere-binding protein Cbf1, said repressor inhibiting the expression of centromere-binding protein Cbf1 as described in claim 1.

4. A kit for knocking out the centromere-binding protein Cbf1 as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit contains specific primers as shown in SEQ ID NO. 6~11.

6. A method for reducing the virulence of Cryptococcus grebindus, characterized in that, The method includes: Targeting centromere-binding protein Cbf1, the expression of Cbf1 is inhibited or the protein encoding Cbf1 is knocked out. CBF1 The steps of gene generation.

7. Centromere-binding protein Cbf1 or encoding the centromere-binding protein. CBF1 The application of the gene in the preparation of drugs for the prevention or treatment of cryptococcal diseases.

8. The application according to claim 7, characterized in that, The applications include drug screening.

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