A Candida albicans attenuated strain lacking the PRO1 gene, its construction method and application

Abstract

This invention discloses an attenuated Candida albicans strain lacking the PRO1 gene, its construction method, and its applications, belonging to the field of genetic engineering technology. Both alleles of the PRO1 gene in the attenuated strain are knocked out, preventing the expression of functional Δ¹-pyrrolline-5-carboxylic acid synthase. The invention also discloses a method for constructing this attenuated strain using a CRISPR / Cas9 system, replacing the PRO1 gene with a HIS1 selection marker. Experiments show that this attenuated strain exhibits reduced hyphal growth, significantly decreased biofilm formation, increased susceptibility to oxidative stress, and significantly reduced pathogenicity in a large wax moth infection model. The attenuated strain described in this invention can be used to prepare a live attenuated Candida albicans vaccine or as a tool for screening antifungal drugs that mimic the PRO1 deletion effect, providing a new approach for the prevention and treatment of Candida albicans infections.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a missing... PRO1 Gene-generated attenuated Candida albicans 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] The rate-limiting step in proline biosynthesis is mainly composed of Δ 1 5-pyrrolidone-5-carboxylic acid synthase (P5CS, commonly found in fungi by...) PRO1 The enzyme P5C, encoded by a gene, catalyzes the conversion of glutamate to P5C. In plants, this enzyme is central to de novo proline synthesis, and its activity is closely related to responses to stresses such as drought and salinity. Plants lacking P5C synthase cannot rapidly accumulate proline under stress, which not only weakens cellular osmotic regulation but may also lead to an imbalance in the distribution of reducing power (NADPH) in chloroplasts used to scavenge reactive oxygen species, affecting the protection of the photosynthetic system and making the plant more susceptible to oxidative damage. In the pathogenic fungus *Cryptococcus neoformans*, the biosynthetic capacity of proline has been shown to be related to its colonization ability in the host's lungs. In *Candida albicans*, it has been found that... PRO1 The absence of proline severely impairs its growth and survival in oligotrophic environments and may affect its hyphal development and biofilm formation. However, overall, how proline biosynthesis in Candida albicans precisely regulates its virulence and the molecular network of its interaction with the host remains to be further elucidated. Summary of the Invention

[0004] This application addresses the shortcomings of existing technologies, resolving issues such as the increasing number of immunocompromised individuals, the limitations of clinical antifungal drugs, fungal resistance, the unclear molecular mechanisms involved in copper homeostasis regulation in Candida albicans and their relationship with virulence, and the growing prominence of limitations and fungal resistance in clinical antifungal drugs. It provides a solution to these problems. PRO1 The construction and application of attenuated Candida albicans strains were studied 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 achieve the above objectives, this application provides the following technical solution: An attenuated strain of Candida albicans lacking the PRO1 gene, wherein both alleles of the PRO1 gene in the attenuated strain have been knocked out.

[0006] Preferably, the attenuated strain is obtained by replacing two alleles of the PRO1 gene with selection markers through homologous recombination mediated by the CRISPR / Cas9 system.

[0007] Preferably, the screening marker is the HIS1 gene.

[0008] A method for constructing an attenuated Candida albicans strain lacking the PRO1 gene includes the following steps: PCR amplification of a knockout cassette with a selection marker; co-transforming the knockout cassette, an sgRNA expression cassette targeting the PRO1 gene, and a Cas9 nuclease into a wild-type Candida albicans strain; and screening with a selection medium to obtain a Candida albicans PRO1-deficient strain.

[0009] Preferably, the screening marker is the HIS1 gene, and the screening medium is an SD-His plate.

[0010] The application of any of the above-mentioned Candida albicans attenuated strains lacking the PRO1 gene in the preparation of attenuated live vaccines.

[0011] principle: This technology utilizes the CRISPR / Cas9-mediated gene knockout system to PCR amplify the Cas9 gene and the targeted gene using the PV093 plasmid as a template. PRO1 The sgRNA was obtained, and the pFA-HA-HIS1 plasmid with homologous arms was used as a template for PCR amplification to generate the sgRNA. HIS1 Selection of marker-tagged repair DNA fragments: All PCR products (Cas9, sgRNA, and repair DNA) were collected from wild-type Candida albicans strains. Transformants were plated on histidine-deficient plates for selection. Transformants were then used... PRO1 Colony PCR was performed using TeF and TeR primers surrounding the gene to obtain two [samples / samples]. PRO1Homozygous deletion strains in which all alleles have been replaced; PRO1 Gene deletion leads to resistance in media containing DNA-damaging agents such as MMS and HU, sensitivity in media containing oxidative stress agents such as H2O2, CdCl2, and ZnCl2, and sensitivity to media containing CuSO4 and Congo Red. However, in the *Gnaphalium affine* infection model, PRO1 The deletion strains also exhibit significant pathogenic defects, and various pieces of evidence suggest that... PRO1 It has the potential to be an excellent target for targeted therapy of Candida albicans infection.

[0012] Beneficial effects: This application has a deficiency. PRO1 The construction and application of attenuated Candida albicans strains with genetic modification have the following advantages compared with existing technologies: 1. The Candida albicans treatment drug of the present invention is based on... PRO1 Genes that are therapeutic target genes, enabling PRO1 Drugs that do not express genes, or those that... PRO1 Drugs that antagonize the protein expressed by the gene; or drugs that... PRO1 Drugs that silence genes along with other genes, acting as target genes, in order to... PRO1 Using genes as targets, drugs that prevent the expression of these genes are screened or prepared to reduce the yeast-hyphae morphology conversion rate of Candida albicans, thereby weakening the virulence of Candida albicans and treating Candida albicans infection. This provides new agents and directions for the treatment of Candida albicans infection. 2. The embodiments of the present invention verified that the attenuated strain of Candida albicans had a significantly reduced mycelial growth capacity compared with the wild-type Candida albicans, and the toxicity of Candida albicans was also significantly reduced in the infection experiments of giant wax moth and mice. 3. A deficiency in this invention PRO1 The application of attenuated Candida albicans strains in virulence regulation through detection PRO1 The effect of gene deletion on Candida albicans hyphal formation; detection PRO1 The virulence of Candida albicans was missing; the missing... PRO1 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

[0013] Figure 1 This is a diagram showing the construction and results of the PRO1 deletion strain in Example 1 of this application; where A is a schematic diagram of the construction, in which two alleles of PRO1 are replaced by HIS1; and B is a PCR detection diagram of the transformant colonies. Figure 2 The phenotypic result for the missing PRO1 in Embodiment 2 of this application; Figure 3 The image shows the results of PRO1 regulating Candida albicans hyphae generation in Example 3 of this application. In the image, A shows the hyphae and colony morphology in liquid and solid serum and Spider hyphae induction medium, and B shows the biofilm formation ability of the PRO1-deficient strain. Figure 4 In Embodiment 4 of this application PRO1 The role of Candida albicans in regulating virulence. Detailed Implementation

[0014] 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. Example

[0016]

[0017] A lack PRO1 Construction of an attenuated Candida albicans strain by knocking out the proline synthesis gene. PRO1 The preparation process is as follows: Step 1: To construct Candida albicans PRO1 Gene-attenuated strains, this application utilizes CRISPR method to utilize HIS1 Filter markers were knocked out PRO1 Two alleles.

[0018] like Figure 1 As shown, PCR amplification carries HIS1 The knockout cassette, the PRO1-targeting sgRNA, and the Cas9-expressing DNA fragment were co-transformed together with wild-type Candida albicans strain SN152 (a gift from Professor Malcolm Whiteway of Canada) using the lithium acetate method to replace... PRO1 Genes; transformants were plated on SD-His plates and incubated at 30°C for 2 days. Transformants were then picked for streak purification and genotyping was performed using colony PCR. PCR identification revealed that lane 1 was the wild-type control, and lanes 2-8 were... PRO1 Different clones of the deletion strain, results were obtained through PRO1- TeF and PRO1 -TeR can amplify to about 1.5kb bands in the wild type, but in the correct... PRO1 A band of approximately 1.8 kb can be amplified from the deletion strain. For example... Figure 1 As shown, transformers 2, 3, 6, and 8 are the correctly knocked-out transformers.

[0019] A lack PRO1 Attenuated strains of Candida albicans with the gene deleted PRO1 The attenuated strain of Candida albicans is PRO1 Candida albicans that does not express genes PRO1 Deletion strain.

[0020] Example 2: The PRO1 gene affects the phenotype of Candida albicans. To demonstrate the function of Candida albicans PRO1, this example tested the phenotype of PRO1-deficient strains on culture media stimulated by different oxidative stress reagents.

[0021] Wild-type Candida albicans strains SN152 and PRO1 deletion strain were cultured overnight at 30°C with shaking (200 rpm) for approximately 18 hours. The overnight cultures were then serially diluted 10-fold to 10⁻⁶. -4 Take 3 μL of the stock solution, 10 -1 10 -2 10 -3 10 -4 The diluted solution was spotted onto YPD (1% yeast extract, 2% peptone, 2% glucose, and 2% agar powder) plates containing 0.015% MMS, 35 mM HU, 8 mM CuSO4, 2.5 mM H2O2, 600 μM CdCl2, 4 mM ZnCl2, and 1.5 mM Congo Red, and incubated at 30°C for 2 days.

[0022] The results are as follows Figure 2 As shown, PRO1 It exhibits resistance in MMS and HU media containing DNA damage reagents, and sensitivity in media containing oxidative stress reagents H₂O₂, CdCl₂, and ZnCl₂, as well as sensitivity in media containing CuSO₄ and Congo Red. These results indicate... PRO1 Genes are involved in many cellular functions.

[0023] Example 3 PRO1 Effects of gene deletion on Candida albicans hyphal formation To prove Candida albicans PRO1 To investigate the impact of the absence on mycelial growth, this embodiment conducted liquid and solid mycelial induction experiments.

[0024] Detection PRO1 Effect of missing components on Candida albicans hyphal generation: Candida albicans wild-type strain SN152 and PRO1 The deletion strain is Candida albicans. PRO1The attenuated strain was cultured overnight at 30°C with shaking until saturation. The next day, it was transferred at a 1 / 20 ratio to liquid Spider medium (peptone, 10g; mannitol, 10g; dimethyl hydrogen phosphate, 2g; agar powder, 13.5g, diluted with double-distilled water to 1000mL) and 20% FBS medium (serum diluted in 20% liquid YPD), respectively, and cultured at 37°C with shaking for 2 hours. The bacterial cells were collected and photographed for observation. Wild-type Candida albicans strain SN152 and... PRO1 The deletion strain is Candida albicans. PRO1 The attenuated strain was cultured overnight at 30°C with shaking until saturation. 4 μL of the bacterial culture was spotted onto solid Spider and 20% FBS medium and placed in an incubator at 37°C for 3-4 days. The culture was then photographed and observed. Detection of Candida albicans PRO1 The effect of deficiency on biofilm formation: wild-type Candida albicans strain and PRO1 The deletion strain was inoculated into YNB medium and cultured overnight at 30°C on a shaker (200 rpm) for about 18 hours. The OD value was adjusted to 1.0 with liquid YNB medium. 2 ml of bacterial culture was added to each sample into a 24-well plate and incubated at 37°C for 48 hours. Each well was washed three times with 1 mL PBS. After standing at room temperature for 45 min, 385 μL of 0.4% crystal violet was added and stained for 45 min in the dark. The plate was washed three times with 1 mL of sterile water. Then, 700 μL of 95% ethanol was added for destaining, and the absorbance was measured at a wavelength of 595 nm.

[0025] The results are as follows Figure 3 The results showed that all wild-type strains could form distinct hyphae, while PRO1 The solid hyphae formed by the deletion strain have a longer diameter; the hyphae of both strains are basically the same length in liquid serum medium. PRO1 Compared with the wild type, the deletion strain showed a significant decrease in biofilm formation ability, only about 57.3% of the wild type level.

[0026] Example 4: PRO1 The virulence of Candida albicans is significantly reduced when it is missing. To prove PRO1 To investigate the impact of the deficiency on toxicity regulation, this embodiment uses the large wax moth (300mg, 30 moths per group) for toxicological experiments.

[0027] Wild strains of Candida albicans and PRO1 The deletion strain is Candida albicans. PRO1 The genetically attenuated strain was cultured overnight to the stationary phase. Cells were collected and counted using a hemocytometer, and the bacterial concentration was adjusted to 1×10⁻⁶. 7 / mL, 10μL of bacterial solution was drawn with a micro-syringe and injected into the caudal peduncle of the large wax moth larva, and then cultured at 37℃ to observe the survival rate. Those that did not respond to external stimuli were considered dead.

[0028] The results are as follows Figure 4 As shown, all the large wax moths infected with the wild-type strain SN152 died by the third day, with an average survival period of 1 day, while those infected with... PRO1 The survival rate of the missing *Heliotropium indicum* reached 50% by the end of the 7-day observation period, with an average survival time of 6.5 days, which was statistically significant compared to the wild-type group (p < 0.0001). These results fully demonstrate... PRO1 It plays an important role in the regulation of Candida albicans virulence, and its absence leads to a significant decrease in cytotoxicity.

[0029] 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 PRO1 A genetically modified attenuated strain of Candida albicans, characterized by: Both alleles of the PRO1 gene in the attenuated strain were knocked out.

2. The missing item as described in claim 1 PRO1 A genetically modified attenuated strain of Candida albicans, characterized by: The attenuated strain underwent homologous recombination mediated by the CRISPR / Cas9 system, utilizing selection markers to replace... PRO1 It is obtained from two alleles of a gene.

3. The missing item as described in claim 2 PRO1 A genetically modified attenuated strain of Candida albicans, characterized by: The selection marker is the HIS1 gene.

4. A method for constructing the missing part of claim 1 PRO1 The method for obtaining attenuated Candida albicans strains of genes is characterized by, Includes the following steps: PCR amplification of a knockout cassette with a selection marker was performed. The knockout cassette, an sgRNA expression cassette targeting the PRO1 gene, and a Cas9 nuclease were co-transformed into a wild-type strain of Candida albicans. The strain was then screened on a selection medium to obtain a Candida albicans PRO1-deleted strain.

5. The method according to claim 4, characterized in that, The selection marker is the HIS1 gene, and the selection medium is SD-His plate.

6. The deficiency as described in any one of claims 1 to 3 PRO1 Application of gene-modified Candida albicans attenuated strains in the preparation of attenuated live vaccines.

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