Verification method and application of ADH2 gene in systemic candida albicans infection treatment

Abstract

The invention discloses a verification method and application of an ADH2 gene in systemic Candida albicans infection treatment, and belongs to the technical field of microbial medicine and anti-infection treatment. The verification method comprises the following steps: constructing an ADH2 single allele knockout strain and an ADH2 double allele knockout strain by taking wild type candida albicans SC5314 as a starting strain, and evaluating the effectiveness of an ADH2 target spot through body and appearance verification, in-vitro metabolism verification, in-vitro drug sensitivity verification and immunosuppression mouse in-vivo pathogenicity and curative effect verification. In the application aspect, the medicine for treating systemic Candida albicans infection is prepared by regulating and controlling the function of the ADH2 gene in the Candida albicans, the abnormal metabolism of a biological membrane caused by function imbalance of the ADH2 can be corrected, and the sensitivity of the Candida albicans to fluconazole is enhanced or the toxicity of the Candida albicans is reduced. The invention provides a new intervention way for the drug resistance problem of the antifungal drug, and provides technical support for research and development of the antifungal drug.

Description

Technical Field

[0001] This invention relates to the field of microbial medicine and anti-infective therapy technology, specifically to... ADH2 Validation methods and applications of genes in the treatment of systemic Candida albicans infection. Background Technology

[0002] Candida albicans ( Candida albicans It is a common opportunistic fungal pathogen in clinical practice, which can cause a series of infections from colonization of the skin and mucous membranes to blood dissemination. In immunocompromised individuals, this fungus is prone to causing systemic candidiasis, which accounts for 40%-60% of hospital-acquired fungal infections and may be life-threatening.

[0003] Currently, azole antifungal drugs (such as fluconazole) are the first-line drugs for treating Candida albicans infections. With the widespread clinical use of these drugs, the proportion of Candida albicans strains resistant to fluconazole has been rising continuously, from 5.2% in 2010 to 12.7% in 2023. In some regions, the proportion of multidrug-resistant strains has exceeded 20%, which has limited clinical treatment options.

[0004] The pathogenicity of Candida albicans depends on a variety of virulence-related phenotypes, such as adhesion, yeast-hyphae transformation, and biofilm formation. These phenotypes are closely related to cellular metabolic homeostasis and redox balance, and together constitute the molecular basis for its invasion of tissues, evasion of immune clearance, and drug resistance.

[0005] The alcohol dehydrogenase (ADH) family is a key protein family regulating carbohydrate metabolism and redox homeostasis in Candida albicans, and the functions of its members are closely related to the survival and pathogenicity of the fungus. Among them, ADH1 The function of genes has accumulated a lot of data in clinical research and application. Changes in their expression levels will simultaneously affect the expression of genes related to the resistance of Candida albicans to fluconazole and virulence. These are currently known factors associated with fungal metabolism and pathogenicity.

[0006] But belonging to the same ADH family ADH2 The role of genes in Candida albicans has not yet been clearly understood. Whether they participate in the regulation of virulence phenotypes such as adhesion and hyphae formation, whether they can affect cellular energy metabolism processes, and their association with antifungal drug sensitivity are all currently lacking research conclusions that can support clinical applications. The relevant mechanisms cannot provide a reference for treatment.

[0007] In current clinical treatment, antifungal drug resistance has become a major obstacle to the treatment of systemic Candida albicans infection. Existing therapeutic targets mostly focus on single pathological processes, making it difficult to overcome the resistance barrier. Therefore, there is an urgent need to explore new therapeutic targets that can be used for clinical translation, and to establish corresponding target efficacy verification technologies to fill the gaps in existing treatment strategies and provide new intervention directions for systemic Candida albicans infection. Summary of the Invention

[0008] This invention addresses the problems of high antifungal drug resistance rates in the clinical treatment of systemic Candida albicans infections, the inability of existing targets to simultaneously intervene in fungal virulence and metabolism, and the lack of new targets and validation technologies that can be clinically translated. The aim is to reveal... ADH2 The study investigates the regulatory role of genes in the pathogenicity and fluconazole susceptibility of Candida albicans, providing insights into their application as drug targets in the treatment of systemic Candida albicans infections. It also establishes a complete in vitro-in vivo validation methodology, offering a new direction for antifungal drug development.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: ADH2 A method for validating the role of a gene in the treatment of systemic Candida albicans infection, the method comprising the following steps: S1 model construction: Using wild-type Candida albicans SC5314 as the starting strain, the model was constructed using fusion PCR and homologous recombination techniques. ADH2 Uniallelic knockout strains and ADH2 Diallelic knockout strain; verified by PCR using specific primer pairs. ADH2 The correctness of the gene integration site was verified using the wild-type Candida albicans SC5314 as the experimental control group. S2 in vitro morphological verification: Comparison of the above in vitro... ADH2 Uniallelic knockout strains ADH2 Phenotypic differences between biallelic knockout strains and wild-type Candida albicans in at least one aspect of growth kinetics, adhesion ability, hyphal formation ability, and biofilm metabolic activity; with wild-type strains as controls, differences were determined by statistical methods, and p<0.05 was considered significant. S3 In vitro metabolic verification: Comparison of the above in vitro ADH2 Uniallelic knockout strains ADH2 Diallelic knockout strains and wild-type Candida albicans showed differences in intracellular ATP levels, mitochondrial membrane potential, and other parameters. NAD+ / NADH Differences in at least one aspect of metabolic indicators in the ratios; using wild-type strains as a control, differences were determined by statistical methods, with p<0.05 considered statistically significant; S4 In vitro drug susceptibility testing: Evaluate and compare the results in vitro. ADH2 Uniallelic knockout strains ADH2The sensitivity of biallelic knockout strains and wild-type Candida albicans to fluconazole; S5 In vivo pathogenicity and efficacy verification: Using an immunosuppressed mouse model of systemic Candida infection, the pathogenicity and efficacy of S5 were evaluated and compared in vivo. ADH2 Uniallelic knockout strains ADH2 Differences in pathogenicity between biallelic knockout strains and wild-type Candida albicans, and differences in the therapeutic effects of fluconazole on infections.

[0010] Further, in step S2, the statistical method is a t-test or one-way ANOVA; the mycelial formation ability is verified by observing the mycelial morphology after the cells are inoculated into the mycelial induction medium; the biofilm metabolic activity is determined by the XTT reduction method.

[0011] Furthermore, in step S3, the statistical method is a t-test or one-way ANOVA; the detection result of mitochondrial membrane potential is expressed as the red / green fluorescence ratio.

[0012] Further, in step S4, the sensitivity of the strain to fluconazole is assessed by spot method; the spot method includes preparing a gradient dilution of the strain, spotting it onto a culture medium containing fluconazole and a drug-free control culture medium respectively, and observing the colony growth after incubation.

[0013] Further, in step S5, the method for constructing the immunosuppressed mouse systemic Candida infection model is as follows: female ICR mice are immunosuppressed by intraperitoneal injection of cyclophosphamide, followed by intraperitoneal injection of Candida albicans suspension to establish infection.

[0014] Furthermore, fluconazole was administered via intraperitoneal injection, starting 24 hours after infection and continued continuously. After the last treatment, the fungal load in the mice's kidneys was measured and the pathological damage to the kidney tissue was observed.

[0015] ADH2 The application of genes in the treatment of systemic Candida albicans infection, wherein the application is verified using any of the methods described above.

[0016] Furthermore, the application is as follows: by regulating Candida albicans... ADH2 The function of the gene, and the preparation of drugs for treating systemic Candida albicans infections; ADH2 Gene function regulation targets ADH2 Functional imbalance, the imbalance including ADH2 Insufficient gene expression or ADH2 Abnormal activity of the gene-encoded product; the effects of the drug include: through... ADH2 Gene regulation of energy metabolism and redox homeostasis in Candida albicans, and correction of... ADH2Functional imbalances lead to abnormal biofilm metabolism, which reduces excessive biofilm formation, enhances the sensitivity of Candida albicans to fluconazole, and reduces the virulence of Candida albicans.

[0017] Furthermore, the function of regulating the ADH2 gene includes: targeting ADH2 Candida albicans strains with insufficient gene expression or abnormally reduced activity of their encoded products, recovered ADH2 Gene expression level or ADH2 The activity of gene-encoded products.

[0018] Furthermore, the specific manifestations of the drug's regulation of energy metabolism and redox homeostasis in Candida albicans are as follows: targeting ADH2 Dysfunctional Candida albicans strains reduce intracellular ATP levels, alter mitochondrial membrane potential, and decrease... NAD+ / NADH Ratio; where ADH2 The mitochondrial membrane potential is increased in biallelic knockout strains. ADH2 The mitochondrial membrane potential is reduced in monoallelic knockout strains.

[0019] Furthermore, the drug correction ADH2 The specific manifestations of abnormal biomembrane metabolism caused by functional imbalance are: targeting ADH2 Dysfunctional Candida albicans strains regulate biofilm metabolic activity, thereby reducing excessive biofilm formation.

[0020] Furthermore, the mechanism by which the drug enhances the sensitivity of Candida albicans to fluconazole is as follows: targeting... ADH2 In Candida albicans strains with functional imbalances, restoring the normal function of the ADH2 gene can downregulate drug resistance-related genes. CDR1 , CDR2 , MDR1 and ERG11 The level of expression.

[0021] Furthermore, the specific manifestations of the drug in reducing the virulence of Candida albicans are as follows: targeting... ADH2 Dysfunctional Candida albicans strains have weakened adhesion ability or inhibited hyphal formation ability, thereby reducing pathogenicity after infecting the host.

[0022] Beneficial effects: 1. This invention regulates... ADH2 Gene function, can downregulate drug resistance-related genes CDR1 , CDR2 , MDR1 and ERG11 The expression level of [the substance] can be increased to enhance the sensitivity of Candida albicans to fluconazole, providing a new intervention approach to address the antifungal drug resistance crisis and alleviating the current situation of limited clinical treatment options.

[0023] 2. The present invention is based onADH2 By targeting genes, the energy metabolism and redox homeostasis of Candida albicans can be regulated, and virulence-related phenotypes such as adhesion, hyphae formation, and biofilm formation can be simultaneously affected. This enables synergistic regulation of multiple links in the fungal metabolism-virulence-drug resistance, providing technical support for the development of antifungal drugs beyond traditional targets.

[0024] 3. This invention not only clearly defines... ADH2 Genes can serve as novel therapeutic targets for systemic Candida albicans infection. A comprehensive technical system has been established, covering model construction, in vitro phenotypic verification, in vitro metabolic verification, in vitro drug sensitivity verification, and in vivo pathogenicity and efficacy verification. Through comparison of gene knockout strains with wild-type strains, combined with molecular biology, cell biology, and animal experimental methods, effective verification can be achieved. ADH2 The effectiveness and application value of the target provide standardized technical support for its clinical translation and antifungal drug development, and complement existing treatment strategies. Attached Figure Description

[0025] Figure 1 for ADH2 Schematic diagram of the effects of gene knockout on the growth, adhesion, hyphae formation and biofilm formation of Candida albicans; Figure 2 for ADH2 Schematic diagram of the effects of gene knockout on energy metabolism and redox balance in Candida albicans; Figure 3 for ADH2 Schematic diagram of the effect of gene knockout on fluconazole sensitivity in Candida albicans; Figure 4 for ADH2 A schematic diagram illustrating the effect of gene knockout on the pathogenicity of Candida albicans and the efficacy of fluconazole in a mouse model of systemic infection. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments.

[0027] Example 1: Construction and Validation of ADH2 Gene Functionally Regulated Strains Using the wild-type Candida albicans strain SC5314 as the starting strain, fusion PCR and homologous recombination techniques were employed to construct... ADH2 Uniallelic knockout strains (adh2 Δ / ADH2) and ADH2 Biallelic knockout strain (adh2 Δ / Δ). Specific steps include: designing and synthesizing upstream and downstream homologous arms for homologous recombination and primers for selecting marker genes; obtaining gene knockout fragments via fusion PCR; transforming them into SC5314 competent cells; and selecting positive clones using antibiotic-resistant culture medium. Stepwise...ADH2 Alleles were knocked out. PCR verification was performed using primers specific to the integration site, and DNA sequencing confirmed correct construction. All strains were routinely cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose), with 2% agar added to the solid medium.

[0028] The size of the PCR product was verified by agarose gel electrophoresis to ensure accurate integration of the gene knockout or complement fragments. The successfully constructed strains were then expanded and cryopreserved for subsequent experiments.

[0029] Example 2: ADH2 Effects of gene regulation on the phenotype of Candida albicans Growth kinetics analysis: Strains in the exponential growth phase (SC5314, ...) were analyzed. adh2 Δ / ADH2 , adh2 Δ / Δ) Collect, wash, and resuspend in fresh YPD medium to adjust initial concentration. OD600 The value was 0.04. The cells were incubated at 30°C with shaking at 200 rpm, and measurements were taken at 0, 4, 6, 8, 10, 12, 24, and 30 hours. OD600 The values ​​were used to plot the growth curve. The results showed that... adh2 Δ / ADH2 The strain grows fastest during the logarithmic growth phase. adh2 Δ / Δ is the second slowest, followed by the wild-type SC5314. Figure 1 A). Towards the stable period, adh2 Δ / ADH2 , adh2 The cell density of Δ / Δ was higher than that of wild-type SC5314.

[0030] Adhesion ability assay: Microplate assay was used. 12-well plates were pre-coated with fetal bovine serum (FBS) overnight. Cells from each strain in the logarithmic growth phase were collected, resuspended in Spider medium to 1×10^7 cells / mL, and 1 mL of bacterial suspension was added to each well. The plates were incubated at 37°C for 2 hours. Unadhered cells were gently washed with PBS to remove any cells. Fresh Spider medium was added, and the plates were cultured for another 24 hours before observation. Slide staining was also performed: the bacterial suspension was added to sterile slides and incubated at 37°C for 2 hours. The slides were washed three times with PBS, fixed with 95% ethanol for 10 min, stained with lactophenol cotton blue for 5 min, rinsed with distilled water, air-dried, and observed under a microscope. Results showed that, compared to SC5314, adh2 Δ / Δ and adh2 Δ / ADH2 The adhesion ability of both is weakened. Figure 1 B). Further analysis of the expression of adhesion-related genes showed that, adh2 Δ / ADH2 In strainsALS1 Gene expression upregulation ALS3 Gene expression downregulation adh2 Δ / Δ strains ALS1 and ALS3 Gene expression was significantly lower than that of SC5314 ( Figure 1 C). This indicates that ADH2 Through coordination ALS1 and ALS3 The dynamic balance of gene expression regulates the adhesion process of Candida albicans.

[0031] Mycelial formation ability assessment: Each strain (1×10^6 cells / mL) was inoculated into YPD, YPD+10% FBS, Spider, and BHI mycelial induction media, respectively. After incubation at 37°C for 2 hours, mycelial morphology was observed under a microscope. Simultaneously, serial dilutions of the strain were spotted onto the above solid media and SLAD agar plates, and colony morphology was observed after incubation at 37°C for 3 days. The results showed that, under induction conditions, strain SC5314 exhibited strong mycelial formation ability, while... adh2 Δ / Δ and adh2 Δ / ADH2 The mycelial formation ability is impaired, especially in SLAD medium. adh2 Δ / ADH2 It grows almost exclusively in the form of yeast. adh2 Δ / Δ only forms short pseudohyphae ( Figure 1 D, 1E). Hyphae-related genes HWP1 The expression level was highest in SC5314, while... adh2 Δ / Δ and adh2 Δ / ADH2 The average was significantly lowered ( Figure 1 F), which indicates that ADH2 It may be possible through regulation HWP1 Expression and nitrogen limitation response regulate hyphal morphogenesis.

[0032] Biofilm metabolic activity assay: The XTT reduction method was used. Overnight cultured bacterial strains were collected, washed, and resuspended in RPMI 1640 medium to 1×10^6 cells / mL. 100 μL of the bacterial suspension was added to a 96-well plate pre-coated with FBS and incubated at 37°C for 90 minutes to allow adsorption. Unattached cells were washed away with PBS, and fresh RPMI 1640 medium was added. The plates were incubated at 37°C for 48 hours to form a biofilm. After washing with PBS, 150 μL of XTT solution was added to each well and incubated at 37°C in the dark for 4 hours. 70 μL of the supernatant was transferred to a new 96-well plate, and the absorbance was measured using a microplate reader. The results showed that… adh2 Δ / Δ and adh2 Δ / ADH2 The biomembrane metabolic activities of both were significantly higher than those of SC5314 ( Figure 1G).

[0033] Example 3: ADH2 Effects of gene regulation on energy metabolism and redox homeostasis in Candida albicans Intracellular ATP level assay: Strains in logarithmic growth phase were collected, adjusted to 1×10^6 cells / mL, and incubated at 30°C for 30 minutes. ATP content was measured using the BacTiter-Glo kit (Promega) according to the manufacturer's instructions. Chemiluminescence values ​​were detected using a Thermo Scientific microplate reader, and ATP concentration was calculated based on the standard curve (10 pM-100 nM). Results showed that compared to SC5314, adh2 Δ / Δ and adh2 Δ / ADH2 ATP levels decreased ( Figure 2 A).

[0034] Mitochondrial membrane potential detection: After overnight culture, the concentration of each strain was adjusted to 2.0 × 10^6 CFU / mL and cultured at 35°C with shaking at 200 rpm for 6 hours. The fluorescence intensity (excitation wavelength 490 nm, emission wavelength red 590 nm / green 530 nm) was detected using a microplate reader according to the instructions of the fungal ΔΨm detection kit. Mitochondrial membrane potential is expressed as the red / green fluorescence ratio. The results showed that... adh2 Δ / ADH2 The mitochondrial membrane potential was significantly reduced, while adh2 The mitochondrial membrane potential is abnormally elevated (Δ / Δ). Figure 2 B); Intracellular reactive oxygen species (ROS) level determination: Cells of various strains in logarithmic growth phase were treated using a fungal ROS detection kit (green fluorescent probe) according to the manufacturer's instructions. Fluorescence intensity was detected using a microplate reader (excitation 490 nm, emission 530 nm). Results showed that compared to SC5314, adh2 Δ / Δ and adh2 Δ / ADH2 ROS levels decreased ( Figure 2 C).

[0035] NAD + / NADH ratio determination: Cells from each strain in the logarithmic growth phase were collected and washed with PBS. The chemiluminescence value was detected using a multi-functional microplate reader following the instructions of the NAD / NADH-Glo™ assay kit (Promega), and NAD ratio was calculated. + / NADH ratio. The results show... adh2 Δ / ADH2 , adh2 Δ / Δ NAD + The / NADH ratios were significantly lower than those of SC5314 (Figure 2 D).

[0036] Expression analysis of key enzymes in the glycolysis pathway: RT-qPCR detection revealed that... adh2 Δ / ADH2 In strains HXK1 , PGI1 , PGK1 The expression is upregulated, while CDC19 No significant changes were observed in expression; adh2 Δ / Δ strains only HXK1 Slightly increased ( Figure 2 E). This indicates ADH2 The changes in function affect energy metabolism and redox balance, and may be achieved through different regulatory mechanisms.

[0037] Example 4: ADH2 Effects of gene regulation on fluconazole susceptibility in Candida albicans Spot susceptibility testing: Strains cultured overnight were collected, washed, resuspended in PBS, and adjusted to 1.0 × 10^6 cells / mL. Serial 10-fold dilutions were performed, with 4 μL of each dilution spotted onto YPD plates (control) and YPD plates containing 2 μg / mL fluconazole. Colony growth was observed after incubation at 30°C for 48 hours. Results showed that fluconazole significantly inhibited the growth of SC5314, while... adh2 Δ / Δ and adh2 Δ / ADH2 The inhibition is relatively weak ( Figure 3 A), indicating ADH2 The genetic alteration reduced the strain's sensitivity to fluconazole.

[0038] Analysis of drug resistance-related gene expression: RT-qPCR was used to detect the expression of drug resistance-related genes in various strains. CDR1, CDR2, MDR1 and ERG11 Gene expression levels. Total RNA was extracted from cells in the logarithmic growth phase, reverse transcribed into cDNA, and then subjected to real-time quantitative PCR. Using 18S rRNA as an internal reference gene, the relative gene expression level was calculated using the 2^(-ΔΔCt) method. The results showed that, in adh2 Δ / ADH2 In the strain, efflux pump genes CDR1, CDR2, MDR1 and target genes ERG11 The expression level was significantly upregulated (2-4 times higher than SC5314), while adh2 The Δ / Δ strains showed no significant difference compared to the wild type. Figure 3 B). This reveals ADH2 One of the potential molecular mechanisms regulating fluconazole sensitivity is through influencing the expression of resistance-related genes.

[0039] Example 5: ADH2In vivo validation of gene regulation in a systemic Candida infection model Establishment and treatment of mouse infection model: Forty female ICR mice (6-8 weeks old, 18-22 g) were purchased. All animal experiments were conducted in accordance with relevant ethical guidelines. The experimental design process is as follows: Figure 4 As shown in Figure A, mice underwent immunosuppression by intraperitoneal injection (ip) of cyclophosphamide (100 mg / kg / day) for 4 consecutive days. On the 3rd day of immunosuppression, systemic infection was established by intraperitoneal injection of 200 μL of Candida albicans suspension (5 × 10^7 cells / mL). Twenty-four hours post-infection, mice were treated with intraperitoneal injection of fluconazole (2 mg / kg / day) for 4 consecutive days; the control group received an equal volume of the solvent. The survival status and body weight of the mice were monitored throughout the treatment. Mice nearing death were euthanized, and survival curves were plotted.

[0040] Kidney fungal load and histopathological analysis: 24 hours after the last fluconazole treatment, surviving mice were sacrificed. One kidney was harvested, sterilely homogenized, and spread on SDA medium. Colony-forming units (CFU) were counted to quantify the fungal load. The other kidney was harvested, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned to 4 μm thickness for HE and PAS staining. Inflammatory infiltration, fungal colonization, and pathological damage in the kidney tissue were observed under a microscope.

[0041] In vivo pathogenicity and efficacy assessment: Survival analysis showed that, compared with the uninfected control group, the infection rate was significantly higher. adh2 Mice with Δ / Δ or SC5314 showed significantly reduced survival rates. Figure 4 B). Kidney fungal load measurement and histopathological observation indicated infection. adh2 Δ / Δ or SC5314 can both cause high fungal load, severe tissue damage, extensive inflammatory infiltration, and significant fungal colonization in the kidneys of mice. Figure 4 (C, 4D). Fluconazole treatment improved the survival rate of SC5314-infected mice, reduced fungal load, and alleviated kidney pathological damage (lesions tended to be multiple small lesions); however, in... adh2 In the Δ / Δ infection group, fluconazole showed limited therapeutic effect, and the degree of kidney damage in mice was more severe. Figure 4 (B, 4C, 4D). This indicates... ADH2 The absence of this substance enhances the pathogenicity of Candida albicans in vivo, while significantly weakening the therapeutic effect of fluconazole.

[0042] The above embodiments confirm that ADH2 The study investigated the key role of genes in regulating the pathogenicity, metabolism, and fluconazole susceptibility of Candida albicans and verified the feasibility of intervention targeting these genes. [[ID= By influencing energy metabolism, redox homeostasis, virulence phenotype, and drug resistance gene expression, it becomes a key node connecting metabolism and pathogenicity. [Targeting...] ​The regulatory strategies provided important targets for the development of novel antifungal therapies.

[0043] This embodiment also provides a ​ The application of genes in the treatment of systemic Candida albicans infection, wherein the application is performed using the above-mentioned verification method, and the type of application is one of the drugs or reagents.

[0044] In summary, this invention discloses... ​ The regulatory role of genes and the establishment of corresponding verification systems provide new targets and technical support for the treatment of systemic Candida albicans infection. The scope of protection of this invention is defined by the appended claims. All equivalent substitutions or improvements made based on the technical solutions and inventive concepts of this invention fall within the scope of protection of this invention.

Claims

1. ADH2 A method for validating genes in the treatment of systemic Candida albicans infection, characterized in that, The verification method includes the following steps: S1 model construction: Using wild-type Candida albicans SC5314 as the starting strain, the model was constructed using fusion PCR and homologous recombination techniques. ADH2 Uniallelic knockout strains and ADH2 Diallelic knockout strain; verified by PCR using specific primer pairs. ADH2 To verify the correctness of the gene integration site, wild-type Candida albicans SC5314 was used as the experimental control group. S2 in vitro morphological verification: Comparison of the above in vitro... ADH2 Uniallelic knockout strains ADH2 Phenotypic differences between biallelic knockout strains and wild-type Candida albicans in at least one aspect of growth kinetics, adhesion ability, hyphal formation ability, and biofilm metabolic activity; with wild-type strains as controls, differences were determined by statistical methods, and p<0.05 was considered significant. S3 In vitro metabolic verification: Comparison of the above in vitro ADH2 Uniallelic knockout strains ADH2 Diallelic knockout strains and wild-type Candida albicans showed differences in intracellular ATP levels, mitochondrial membrane potential, and other parameters. NAD+ / NADH Differences in at least one aspect of metabolic indicators in the ratios; using wild-type strains as a control, differences were determined by statistical methods, with p<0.05 considered statistically significant; S4 In vitro drug susceptibility testing: Evaluate and compare the results in vitro. ADH2 Uniallelic knockout strains ADH2 The sensitivity of biallelic knockout strains and wild-type Candida albicans to fluconazole; S5 In vivo pathogenicity and efficacy verification: Using an immunosuppressed mouse model of systemic Candida infection, the pathogenicity and efficacy of S5 were evaluated and compared in vivo. ADH2 Uniallelic knockout strains ADH2 Differences in pathogenicity between biallelic knockout strains and wild-type Candida albicans, and differences in the therapeutic effects of fluconazole on infections.

2. As described in claim 1 ADH2 A method for validating genes in the treatment of systemic Candida albicans infection, characterized in that, In step S2, the statistical method is either a t-test or a one-way ANOVA; the mycelial formation ability is verified by observing the mycelial morphology after the cells are inoculated into the mycelial induction medium; and the biofilm metabolic activity is determined by the XTT reduction method.

3. As described in claim 1 ADH2 A method for validating genes in the treatment of systemic Candida albicans infection, characterized in that, In step S3, the statistical method is either a t-test or a one-way ANOVA; the detection result of mitochondrial membrane potential is expressed as the red / green fluorescence ratio.

4. As described in claim 1 ADH2 A method for validating genes in the treatment of systemic Candida albicans infection, characterized in that, In step S4, the sensitivity of the strain to fluconazole is assessed by spot method; the spot method includes preparing a gradient dilution of the strain, spotting it onto a culture medium containing fluconazole and a drug-free control culture medium respectively, and observing the colony growth after incubation.

5. The method according to claim 1 ADH2 A method for validating genes in the treatment of systemic Candida albicans infection, characterized in that, In step S5, the method for constructing the immunosuppressed mouse systemic Candida infection model is as follows: female ICR mice are immunosuppressed by intraperitoneal injection of cyclophosphamide, followed by intraperitoneal injection of Candida albicans suspension to establish infection.

6. The method according to claim 5 ADH2 A method for validating genes in the treatment of systemic Candida albicans infection, characterized in that, Fluconazole was administered via intraperitoneal injection, starting 24 hours after infection and continued continuously. After the last treatment, the fungal load in the mice's kidneys was measured and the pathological damage to the kidney tissue was observed.

7. ADH2 The application of genes in the treatment of systemic Candida albicans infection is characterized by, The application employs the verification method described in any one of claims 1 to 6.

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