Candida albicans BUB2 gene deletion attenuated strain, construction method and application thereof

Abstract

The application discloses a kind of Candida albicans BUB2 Gene deletion attenuated strain, its construction method and application belong to the field of genetic engineering technology.The attenuated strain is Candida albicans BUB2 deletion strain with BUB2 gene not expressed, its construction method uses CRISPR / Cas9 system, with HIS1 as screening marker, by knocking out box and sgRNA, Cas9 co-transforms Candida albicans wild type strain, replaces two alleles of BUB2 gene to obtain.The phenotype experiment verifies that BUB2 deletion strain is highly sensitive to hydrogen peroxide, cadmium chloride and congo red;Hyphal induction experiment shows that the hyphal production capacity of deletion strain significantly decreases, and the hyphal length significantly shortens;Biofilm formation experiment shows that the biofilm formation capacity of deletion strain is about 41.4% of wild type;Galleria mellonella and mouse infection model confirm that the virulence of BUB2 deletion strain significantly decreases, and the survival rate of infection group is much higher than that of wild type group.The application provides a new action target for clinical treatment of Candida albicans infection, and can be used for preparing or screening anti-Candida albicans infection drugs, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to Candida albicans. BUB2 Gene deletion attenuated strains, their construction methods and applications. Background Technology

[0002] Candida albicans, also known as Candida albicans yeast, is generally found in a symbiotic state in the oral cavity, upper respiratory tract, intestines, and vagina of healthy individuals, and is usually non-pathogenic. However, factors such as antibiotic-induced ecological imbalance and iatrogenic disruption of the mucosal or skin barrier can induce Candida albicans to transition from a symbiotic to a pathogenic state, causing acute, subacute, or invasive fungal infections, leading to severe illness in the hematologic and respiratory systems, with a high mortality rate. Statistics show that Candida albicans infections account for approximately 70% of all Candida infections. The pathogenic mechanisms of Candida albicans are generally considered to include bacterial adhesion and invasion, morphological transformation, biofilm formation, nutrient acquisition, and pH response. However, recent findings also indicate that Candida albicans' DNA damage response and metal ion homeostasis are closely related to its pathogenicity. Currently, the increasing number of immunocompromised individuals, the limitations of clinical antifungal drugs, and the persistent problem of fungal resistance mean that Candida albicans remains a significant fungal pathogen threatening human health. Therefore, in-depth analysis of the pathogenic mechanism of Candida albicans and the development of novel drug targets are of great guiding significance for the clinical treatment of fungal diseases and for solving the problem of resistance to traditional antifungal drugs.

[0003] Cell cycle regulation in *Candida albicans* plays a crucial role in normal cell proliferation. Bub2, in particular, is involved in responding to cell cycle and spindle abnormalities, potentially affecting morphological transition and pathogenicity. Although in *Saccharomyces cerevisiae*, BUB2 As a GAP protein (GTPase activator), Bub2 is involved in regulating mitotic exit. While Candida albicans and Saccharomyces cerevisiae share several similarities in their cell cycles, they differ in the transcriptional characteristics of cell cycle regulators, the function and necessity of conserved proteins, and differences in cell cycle progression. This makes Bub2 a promising area for further exploration. Exploring the pathogenicity of Candida albicans and developing drugs targeting Bub2 is of significant guiding importance for the clinical treatment of Candida albicans. Summary of the Invention

[0004] Addressing the shortcomings of existing technologies and solving the problems of the increasing number of immunocompromised individuals, the limitations of clinical antifungal drugs, and fungal resistance, this invention provides a solution for Candida albicans. These challenges include the unclear understanding of molecules involved in cell cycle and spindle assembly in Candida albicans and their relationship with virulence, as well as the growing prominence of limitations and fungal resistance in clinical antifungal drugs. BUB2Gene-deleted attenuated strains, their construction methods, and applications are used to reduce the yeast-hyphae morphological conversion rate of Candida albicans, thereby weakening the virulence of Candida albicans and preparing drugs for treating Candida albicans infections.

[0005] To address the problems in the existing technology, the technical solution adopted by this invention is as follows: A lack BUB2 The method for constructing an attenuated Candida albicans strain includes the following steps: using the CRISPR / Cas9 system, with the HIS1 gene as a selection marker, the knockout cassette, sgRNA, and Cas9 are co-transformed into a wild-type Candida albicans strain, and the gene is knocked out via homologous recombination. BUB2 Two alleles of the gene were selected to obtain homozygous deletion strains in which the BUB2 gene was not expressed.

[0006] Preferably, the knockout cassette is a DNA fragment with a HIS1 selection marker obtained by PCR amplification.

[0007] Preferably, it is constructed by the above-described construction method, and it is... BUB2 A Candida albicans strain with BUB2 deletion that does not express the gene.

[0008] Preferably, the BUB2 Gene non-expression is achieved by knocking out the spindle assembly checkpoint gene BUB2.

[0009] The above-mentioned missing BUB2 The application of attenuated strains of Candida albicans with genetic mutations in the preparation of drugs for treating Candida albicans infections.

[0010] Preferably, the drug is in the form of BUB2 Genes are the target.

[0011] The above-mentioned missing BUB2 Application of genetically modified attenuated Candida albicans strains in screening for therapeutic drugs for Candida albicans.

[0012] Preferably, the Candida albicans treatment drug is administered via RNA interference or gene recombination. BUB2 It can be prepared by not expressing the gene; or by preparing a BUB2 protein antagonist. BUB2 The protein is prepared by a method that prevents the gene-expressed protein from functioning.

[0013] Explanation of the principle: This invention utilizes a CRISPR / Cas9-mediated gene knockout system to amplify the Cas9 gene via in vitro PCR and target... BUB2 The sgRNA was obtained, and PCR amplification was performed using long primers with homologous arms to generate the sgRNA containing homologous arms. HIS1The repair DNA fragment was selected by transforming all PCR products (Cas9, sgRNA, and repair DNA) into Candida albicans wild-type strain SN152. Transformants were plated on histidine-deficient plates for selection. The transformants were then used... BUB2 Colony PCR was performed using TeF and TeR primers surrounding the gene to obtain two [samples / samples]. BUB2 Homozygous deletion strains in which all alleles have been replaced; BUB2 The deletion of genes leads to a significant impairment in hyphal formation in the deleted strains, and this is observed in both the large wax borer infection model and the mouse model. BUB2 The deletion strains also exhibit significant pathogenic defects, and various pieces of evidence suggest that... BUB2 It has the potential to be an excellent target for targeted therapy of Candida albicans infection. Beneficial effects

[0014] Compared with existing technologies, one is missing BUB2 The construction and application of attenuated Candida albicans strains have the following beneficial effects: In the preparation or screening of therapeutic drugs for Candida albicans, this invention uses... BUB2 Genes that are therapeutic target genes, enabling BUB2 Drugs that do not express genes; or drugs that... BUB2 The protein expressed by the gene is the antagonist; or... BUB2 The gene, along with other genes, is used as a target gene for silencing. The attenuated Candida albicans strain of the present invention has a significantly reduced mycelial growth capacity compared with wild-type Candida albicans, and the toxicity of Candida albicans is also significantly reduced in the infection experiments of giant wax moth and mice. 3. A deficiency in this invention BUB2 The application of attenuated Candida albicans strains in virulence regulation through detection BUB2 The effect of gene deletion on Candida albicans hyphal formation; detection BUB2 The virulence of Candida albicans was missing; the missing... BUB2 The role of attenuated Candida albicans strains in virulence regulation provides a theoretical basis for the treatment of Candida albicans infection and offers direction for the preparation or screening of drugs for treating Candida albicans infection. Attached Figure Description

[0015] Figure 1 In Embodiment 1 of this application BUB2 Construction and result detection of the deletion strain; where A is a schematic diagram of the construction, through... HIS1 replace BUB2 B represents the two alleles; B is a PCR detection diagram of the transformant colonies. Figure 2 In Embodiment 2 of this application BUB2The phenotypic results of Candida albicans growth on YPD medium due to the absence of the gene.

[0016] Figure 3 In Embodiment 3 of this application BUB2 The results of regulating Candida albicans mycelial growth are shown in the following figures: A shows the mycelial and colony morphology in liquid serum and RPMI 1640 mycelial induction medium; B shows the mycelial length statistics in liquid mycelial induction medium; and C shows the mycelial and colony morphology in liquid serum and RPMI 1640 mycelial induction medium.

[0017] Figure 4 In Embodiment 4 of this application BUB2 The result of regulating Candida albicans biofilm formation is shown in the figure.

[0018] Figure 5 In Embodiment 5 of this application BUB2 In the virulence diagram of Candida albicans strains, A represents the wax moth model and B represents the mouse model. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following embodiments are conventional methods commonly known to those skilled in the art and do not constitute a limitation on the present invention in any way.

[0020] BUB2 The nucleotide sequence of the gene is shown in SEQ ID NO.1: atggagcaagaattactcctcaaaggcagaaagaaagactctattgagcagtttattagtggctcccagctatttcttgagaatggtttgtcgcagcttcgatacatggtactagtagaaggattatcaacacctgaaggatacgatcagtgtccataccgctcatatgtatggagcatactatgtcgagttcccccattcccaactgaacagtatttgcaattgctagaaacctcaagtcagaccctcccagatgaattactaatcaaaataaaaaatgatacttttcgtacttttgatgaacgataaaaaattccatctgaaagtgaacgaggaatctttgattaggatactatcgtgtattggaataactacggaagttggctatgtattgtacaaggtttaaatgttttattggcacccatcgcatacgtttgtcacaaaagtgaacctcaggcatttgcaatcttgcacaat ttgataaccaagcaaatcccattatatattactcctaacatggaaggagtccacacgggtttgcaattggttgacgcagttttgaaaataatagatcctgtcttgagtgactatctagactctaagtttttaaaggcaaagatatacgctttcccttcgatactaactttatgtgcgtgtactccgcctttgaaatcagttttgaaactatgggactttctatttgcgtatggcactcatataaatgtgctctttgttgtggcacaattaataatatgtcgttcgagcattttggaaagtgaacaaccaatgaaaatcttgagaacttggccaaatttagaagaaaatgaaatcataaagttgagtctcagtttcatacctgaactaccagagaagctctatgatttaatagctagacatggttacgatagaaaagttcctaaagaattggcaagatttttagaagaaaaa BUB2The amino acid sequence of the gene such as SEQ ID Shown in NO.2: MEQELLLKGRKKDSIEQFISGSQLFLENGLSQLRYMVLVEGLSTPEGYDQCPYRSYVWSILCRVPPFPTEQYLQLLETSSQTLPDELLIKIKNDTFRTLMNDKKFHSKVNEESLIRILSCIGITTEVGYVQGLNVLLAPIAYVCHKSEPQAFAI LHNLITKQIPLYITPNMEGVHTGLQLVDAVLKIIDPVLSDYLDSKFLKAKIYAFPSILTLCACTPPLKSVLKLWDFLFAYGTHINVLFVVAQLIICRSSILESEQPMKILRTWPNLEENEIIKLSLSFIPELPEKLYDLIARHGYDRKVPKELARFLEEK.

[0021] Example 1:

[0022] A lack BUB2 Construction of an attenuated Candida albicans strain by knocking out the spindle assembly checkpoint gene. BUB2 The preparation process is as follows: Step 1: To construct Candida albicans BUB2 Gene-attenuated strains were used with CRISPR technology. HIS1 Filter markers were knocked out BUB2 Two alleles.

[0023] like Figure 1 As shown, PCR amplification carries HIS1 The knockout box, along with sgRNA and Cas9-transformed wild-type Candida albicans strain SN152 (a gift from Professor Malcolm Whiteway of Canada), replaced... BUB2 Gene; PCR identification revealed that lanes 1-15 were transformants converted into the knockout system, and lane 16 was the wild-type control. Results were obtained through... BUB2 -Te-F and BUB2 -Te-R can amplify to about 1.0 kb bands in the wild type, but in the correct... BUB2 A band of approximately 1.5 kb can be amplified from the deletion strain. For example... Figure 1 As shown, transformers 7, 8, 12, and 13 are knockouts. BUB2 The correct transformant of the gene.

[0024] A lack BUB2 Attenuated strains of Candida albicans with the gene deleted BUB2 The attenuated strain of Candida albicans is BUB2 Candida albicans that does not express genes BUB2 Deletion strain.

[0025] Missing BUB2 Attenuated strains of Candida albicans can be produced through RNA interference or gene recombination. BUB2 Genes are not expressed; or through preparation BUB2 Protein antagonists, BUB2 The protein expressed by the gene does not function.

[0026] Example 2: BUB2 Effects of gene deletion on Candida albicans phenotype To prove Candida albicans BUB2 The function was tested in this embodiment. BUB2 The growth experiment of the deletion strain on different phenotypic plate media, specifically, the steps are as follows: Wild-type strains of Candida albicans and BUB2 The missing strain is Candida albicans. BUB2 The gene-attenuated strain was cultured at 30°C with shaking (200 rpm) overnight. The overnight culture was then serially diluted 10-fold to obtain 10... 0 10 -1 10 -2 10 -3 10 -4 Five dilution gradients were used. 3 μL of bacterial suspension at each dilution gradient was pipetted vertically onto YPD plates and YPD plates supplemented with 3 mL H₂O₂, 400 μM CdCl₂, and 1.6 mg / mL Congo red, respectively. The plates were then incubated at 30°C for 2 days. Results are as follows: Figure 2 As shown, by analyzing wild-type strains of Candida albicans and BUB2 Phenotypic experiments of the deletion strains showed that BUB2 The deletion strain is highly sensitive to hydrogen peroxide, cadmium chloride, and Congo red.

[0027] Example 3: BUB2 Effects of gene deletion on Candida albicans hyphal formation To prove Candida albicans BUB2 To investigate the impact of the absence on mycelial growth, this embodiment conducted liquid and solid mycelial induction experiments.

[0028] Specifically, wild-type strains of Candida albicans and BUB2 The missing strain is Candida albicans. BUB2The attenuated strain was cultured overnight at 30°C with shaking (200 rpm) until saturation. The next day, it was transferred at a 1 / 20 ratio to liquid RPMI 1640 and 15% FBS (serum diluted to 15% with liquid YPD medium), and cultured at 37°C with shaking for 2 hours. The bacterial cells were collected and photographed for observation. Furthermore, the original bacterial culture was diluted 10 times... -5 The diluted concentrations were transferred to liquid RPMI 1640 and 20% FBS solid medium, respectively, and incubated at 37°C for 5 days. The morphology of the solid mycelium was then photographed and observed.

[0029] The results are as follows Figure 3 As shown, after 2 hours of transfer to mycelial induction medium, all wild-type strains were able to form distinct mycelia, while BUB2 The deletion strain exhibits fewer budding cells and shorter hyphal length; for example, in liquid RPMI 1640 medium, the wild-type hyphae are approximately 33.3 μm long, while... BUB2 The mycelial length of the deletion strain was only 6.0 μm. However, on solid RPMI 1640 and 15% FBS (with 2% agar powder added) media, the wild-type strain could form wrinkled colonies normally, while... BUB2 The missing strain cannot form wrinkled colonies, indicating that Candida albicans... BUB2 The absence of these features affected mycelial growth.

[0030] Example 4: Effect of BUB2 deficiency on Candida albicans biofilm formation To demonstrate the effect of Candida albicans BUB2 deficiency on biofilm formation, a biofilm formation experiment was conducted in this embodiment.

[0031] Wild-type Candida albicans strains and the BUB2 deletion strain (i.e., attenuated Candida albicans BUB2 gene strain) were cultured overnight at 30°C with shaking until saturation. The next day, the bacterial culture that had reached the stationary phase was diluted to OD595=1.0 and added to 24-well plates. The plates were incubated at 37°C for 48 hours. The bacterial culture in the wells was discarded, and unbound bacterial cells were washed away with sterile PBS. The plates were then air-dried at room temperature. 0.4% crystal violet was added to each well, and the plates were incubated in the dark for 45 minutes. Unbound crystal violet was washed away with ddH2O. 95% anhydrous ethanol was added, and the plates were incubated at room temperature for 45 minutes for decolorization. The OD595 of the decolorized solution was measured.

[0032] The results are as follows Figure 4 As shown in the figure, this diagram represents the WT strain and BUB2 A statistical graph showing the OD595 measured in the decolorized biofilm solution of the deletion strain; BUB2 The absorbance of the deletion strain at 595 nm was approximately 41.4% of that of the wild-type group.

[0033] Example 5: BUB2 The virulence of Candida albicans is significantly reduced when it is missing. To prove BUB2 To investigate the impact of the deficiency on toxicity regulation, this embodiment uses the large wax moth and mice for toxicological experiments.

[0034] Wild strains of Candida albicans and BUB2 The missing strain is Candida albicans. BUB2 The genetically attenuated strain was cultured overnight at 30°C with shaking (200 rpm) until it reached the stationary phase. Cells were collected and counted, and the bacterial concentration was adjusted to 1×10⁻⁶. 7 10 μL of bacterial solution was drawn up using a micro-syringe and injected into the caudal peduncle of the larvae of the large wax moth. The cells were then cultured at 37°C and the survival rate was observed. Cells that did not respond to external stimuli were considered dead.

[0035] The results are as follows Figure 5 As shown in Figure A, the large wax moth infected with the wild-type strain began to die on day 1, with an average survival rate of 26.7% by day 7 and a median survival of 5 days; while the infected... BUB2 The missing strains of the large wax moth still partially survived until the end of the 7-day observation period, with an average survival rate of 80%.

[0036] And the wild-type strain of Candida albicans and BUB2 Similar results were observed when the deletion strain was injected into mice, such as... Figure 5 As shown in Figure B: all mice infected with the wild-type strain died by day 8, with a median survival of 7 days; while those infected with... BUB2 Mice with the deletion strain maintained a 100% survival rate until the end of the experiment. These results fully demonstrate... BUB2 It plays an important role in the regulation of Candida albicans virulence, and its absence leads to a significant decrease in cytotoxicity.

[0037] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A lack BUB2 A method for constructing an attenuated Candida albicans strain, characterized in that, Includes the following steps: Using the CRISPR / Cas9 system and the HIS1 gene as a selection marker, the knockout cassette, sgRNA, and Cas9 were co-transformed into wild-type Candida albicans strains. Two alleles of the BUB2 gene were knocked out through homologous recombination, and homozygous deletion strains that did not express the BUB2 gene were obtained through screening.

2. The construction method according to claim 1, characterized in that, The knockout cassette is obtained through PCR amplification and contains... HIS1 Selected DNA fragments with screening tags.

3. A lack BUB2 A genetically modified attenuated strain of Candida albicans, characterized by: Constructed by the construction method described in claim 1 or 2 BUB2 Candida albicans that does not express genes BUB2 Deletion strain.

4. The missing item as described in claim 3 BUB2 A genetically modified attenuated strain of Candida albicans, characterized by: The BUB2 Gene non-expression is achieved by knocking out the spindle assembly checkpoint gene. BUB2 It was achieved.

5. The deficiency as described in claim 3 or 4 BUB2 The application of attenuated strains of Candida albicans with genetic mutations in the preparation of drugs for treating Candida albicans infections.

6. The application according to claim 5, characterized in that, The drug BUB2 Genes are the target.

7. The deficiency as described in claim 3 or 4 BUB2 Application of genetically modified attenuated Candida albicans strains in screening for therapeutic drugs for Candida albicans.

8. The application according to claim 7, characterized in that, The Candida albicans treatment drug uses RNA interference or gene recombination to... BUB2 It is prepared by gene non-expression; or by preparation BUB2 Protein antagonists, BUB2 The protein is prepared by a method that prevents the gene-expressed protein from functioning.

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