Application of HAP43 gene expression inhibitor in preparation of medicine for treating candidiasis

Abstract

The invention discloses an application of an HAP43 gene expression inhibitor in preparation of a drug for treating candidiasis. The invention proves that the HAP43 gene can enhance the toxicity and drug resistance of the candida albicans by up-regulating the expression of the PCK1 gene, and the expression of the PCK1 gene can be down-regulated by inhibiting the expression of the HAP43 gene, so that the formation of a candida albicans biofilm is prevented, a new thought and a target site are provided for preventing and treating the formation of the candida albicans biofilm and the formation of the drug resistance of the biofilm, and the application of the HAP43 gene to the preparation of the candida albicans biofilm is promoted. And a theoretical basis is provided for developing high-efficiency and low-toxicity antifungal drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of a HAP43 gene expression inhibitor in the preparation of a drug for treating candidiasis albicans. Background Art

[0002] Candida is one of the most important causes of opportunistic fungal diseases in clinic, which can cause skin and systemic infections. 80% of Candida infections are related to Candida albicans. Currently, the commonly used antifungal drugs in clinic include polyenes, triazoles, echinocandins, acrylamides and flucytosine. However, the resistance to traditional antifungal drugs can increase by 1000 times after biofilm formation, which may be related to the expression of certain related genes induced by Candida albicans. At the same time, biofilm can continuously spread Candida albicans, leading to persistent infection. Therefore, the research on inhibiting biofilm formation has important clinical significance for the treatment of Candida albicans-related infections.

[0003] Existing studies have shown that the loss of the HAP43 gene enhances the sensitivity of Candida albicans to fluconazole (FLC), and iron-rich may regulate the formation of FLC resistance in Candida albicans through the HAP43 gene, so the HAP43 gene is related to the fluconazole resistance of Candida albicans. However, in existing studies, there is no research on the specific mechanism of action of HAP43 and PCK1 genes in biofilm formation and biofilm resistance formation. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a drug for treating candidiasis by using a HAP43 gene expression inhibitor in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Provided is the use of a HAP43 gene expression inhibitor in the preparation of a drug for treating candidiasis albicans.

[0007] Furthermore, the HAP43 gene expression inhibitor can regulate the expression of the PCK1 gene.

[0008] Furthermore, the HAP43 gene can enhance the virulence of Candida albicans by upregulating the expression of the PCK1 gene.

[0009] Furthermore, the HAP43 gene can enhance the drug resistance of Candida albicans by upregulating the expression of the PCK1 gene.

[0010] Furthermore, the HAP43 gene can enhance the formation of Candida albicans biofilm by upregulating the expression of PCK1.

[0011] Furthermore, the drug for treating candidiasis also includes pharmaceutically acceptable excipients and / or carriers.

[0012] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0013] The present invention proves that the HAP43 gene can enhance the virulence and drug resistance of Candida albicans by up-regulating the expression of the PCK1 gene, and inhibiting the expression of the HAP43 gene can down-regulate the expression of the PCK1 gene, thereby preventing the formation of Candida albicans biofilm, providing new ideas and target sites for the prevention and treatment of Candida albicans biofilm formation and biofilm drug resistance formation, and providing a theoretical basis for the development of highly effective and low-toxic antifungal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 HAP43Δ / Δ+PCK1 is shown OE The identification results are as follows; A is the gel run after the vector was digested with AgeI and PCK1 was recovered and connected with abclone 2x assembly mix; B is PCK1 amplification (p band 1500bp is positive); C is the connection of the target fragment and the vector at a molar ratio of 1:10 (after introducing PCK1 into the vector, the vector + PCK1 is larger than the empty vector and the gel run speed is slower, so the correctness can be verified by plasmid gel run); D is the PCR identification verification picture after transfection.

[0015] Figure 2 The OD492 values ​​of biofilms of strain SN152 and HAP43Δ / Δ+PCK1 are shown. OE The OD492 values ​​of the biofilms of the strains were similar, with no significant difference.

[0016] Figure 3 The results show that the HAP43 gene deletion strain has similar biofilm proliferation activity to the parent strain SN152 when the glucose concentration is 200 μM without the addition of fluconazole. A is the OD492 value of the XTT experiment of glucose / 2-D-deoxyglucose and fluconazole combined to the biofilm of SN152 strain; B is the OD492 value of glucose / 2-D-deoxyglucose and fluconazole combined to the biofilm of HAP43Δ / Δ+PCK1 OE Figure 1 shows the OD492 value of the XTT experiment on the biofilm of the strain; C is the OD492 value of the XTT experiment on the biofilm of the HAP43Δ / Δ strain using glucose / 2-D-deoxyglucose and fluconazole in combination; D is the OD492 value of the XTT experiment on the biofilm of the PCK1Δ / Δ strain using glucose / 2-D-deoxyglucose and fluconazole in combination.

[0017] Figure 4The figure shows the bacterial load in the liver and kidney tissues of mice after systemic fungal infection; A is the average log10 value of the bacterial load in the kidneys on the second day after infection with each strain; B is the average log10 value of the bacterial load in the liver on the second day after infection with each strain. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0019] The present invention constructs HAP43Δ / Δ+PCK1 OE (HAP43 knockout strain overexpressing PCK1), and compared HAP43Δ / Δ+PCK1 by inducing biofilm formation and XTT assay. OE and SN152 strain biofilm proliferation activity to verify whether the HAP43 gene affects the biofilm formation of Candida albicans by regulating the PCK1 gene.

[0020] Experimental reagents: plasmid extraction kit, Tian Enze; PCR amplification kit, Tiangen (Beijing); anhydrous ethanol, Tianjin Oriental Chemical Plant; isopropanol, Tianjin Oriental Chemical Plant; peptone, OXOID; sodium dodecyl sulfate (SDS), Beijing Solebold Technology Co., Ltd.; chloroform, Sinopharm Chemical Reagent Co., Ltd. (Shanghai); ZymoYeastStar-GenomicDNA Kit LB medium, ZYMO RESEARCH Biotechnology Co., Ltd. (California, USA); DNA marker, Shanghai Weidi Biotechnology Co., Ltd.; PCR product recovery kit, Shanghai Yanjin Biotechnology Co., Ltd.; endonuclease, Shanghai Bioengineering; RNase A, Shanghai Bioengineering; T4 DNA ligase, Shanghai Shanbao Biotechnology Co., Ltd.; FastTaq polymerase, Thermo Fisher Scientific; YPD solid culture medium, Thermo Fisher Scientific; PBS powder, Thermo Fisher Scientific; 50XTAE, Wuhan Sewell Biotechnology Co., Ltd.; salmon sperm DNA (SSDNA), Beijing Solebold Technology Co., Ltd.; fluorescent brightener (CFW), Thermo Fisher Scientific; peptone, Shanghai Bioengineering; glucose powder, OXOID; agar powder, Shanghai Bioengineering; yeast extract, Beijing Xikai Innovation Technology Co., Ltd.

[0021] 1. Verification Experiment 1

[0022] 1.1 Strain HAP43Δ / Δ+PCK1 OE Construction

[0023] 1) PCR was performed using the PCK1 gene synthesis sequence as a template. The system was as follows: Mix 45 μL, forward primer (10 μM, designed using Primer 5 software) 1 μL, reverse primer (10 μM, designed using Primer 5 software) 1 μL, template 50 ng, and deionized water to 50 μL;

[0024] The reaction system was vortexed and mixed, and then centrifuged and loaded into a PCR instrument. The DNA amplification reaction procedure was as follows:

[0025] ①98℃ 2min

[0026] ②98℃ 10sec

[0027] ③55℃ 10sec

[0028] ④72℃ 10sec / kb

[0029] ⑤72℃ 5min

[0030] ②-④ Repeat 25-30 cycles.

[0031] 2) Separate the PCR products by 1% agarose gel electrophoresis, cut out the bands of the desired size under a gel cutter, and recover them with the Omega gel recovery kit. When cutting the gel, pay attention to cutting only the gel containing the target band to reduce the ineffective volume of the gel. The operation should be as fast as possible and the UV irradiation time should be short. Then, digest the PET-28a vector and the obtained PCR fragment twice using the following system:

[0032] The PCR reaction system was: 10× buffer 4 μL, DNA (vector / PCR product) 1 μg, endonuclease I 1 μL, endonuclease II 1 μL, and deionized water to 40 μL;

[0033] The reaction system was kept at 37°C for 3-5 hours.

[0034] 3) Separate the products after enzyme digestion by 1% agarose gel electrophoresis, cut out the vector and target gene fragments by gel cutting instrument, and recover them by Omega gel recovery kit.

[0035] 4) Connect the target fragment and the vector at a molar ratio of 1:10. The system is as follows: 10× buffer 1μL, cloning vector 0.03pmol, target fragment 0.3pmol, T4 ligase 1μL, deionized water to 10μL;

[0036] The reaction conditions were 4°C overnight or room temperature (25°C) for 4-6 hours.

[0037] 5) The ligation product was transformed into E. coli using the heat shock method, plated on a resistance plate, and cultured in a 37°C incubator overnight.

[0038] 6) Pick a suitable single colony from the plate and culture it. Use the following system to perform bacterial solution PCR to identify positive clones; the PCR identification system is 10× buffer 2.5μL, dNTPs 2μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, bacterial solution 2μL, Taq enzyme 10.25μL, and deionized water to 25μL;

[0039] The reaction system was amplified by PCR according to the following steps:

[0040] ①94℃10min

[0041] ②98℃30s

[0042] ③55℃30s

[0043] ④72℃1min / kb

[0044] ⑤72℃10min

[0045] ⑥16℃ insulation

[0046] ②-④ Set 30 cycles. Separate PCR products by 1% agarose gel electrophoresis containing EB, observe and record under UV, and retain positive clones.

[0047] 7) The strain can be stored in 30% glycerol at -20℃ for short term storage and -70℃ for long term storage.

[0048] 8) Transfect into Candida albicans HAP43Δ / Δ and perform identification PCR (existing technology, which will not be described in detail in this application).

[0049] 1.2 Biofilm formation experiment

[0050] After the activated Candida albicans was washed three times with PBS, the bacterial solution was diluted to a concentration of 1x10 6 cells / mL, and then spread it into a 96-well plate, 200μL per well. Then put the 96-well plate in a 37℃ constant temperature incubator and culture it for 90 minutes, which is recorded as the adhesion stage. After 90 minutes of culture, take out the 96-well plate, discard the supernatant, wash it with sterile PBS, then add RPMI1640 culture medium, and place it in a 37℃ constant temperature incubator and culture it for 24 hours.

[0051] 1.3 XTT experiment comparison of strain HAP43Δ / Δ+PCK1 OE Biofilm metabolic activity of the parent strain SN152

[0052] ① Checkerboard experiment of fluconazole and glucose in RPMI1640 medium: fluconazole concentration ranged from 0 μg / mL to 64 μg / mL, and glucose concentration ranged from 0 μM to 200 μM.

[0053] ② Checkerboard experiment of fluconazole and 2-deoxy-D-glucose in RPMI1640 medium: the concentration of fluconazole was 0 μg / mL to 64 μg / mL, and the concentration of 2-deoxy-D-glucose was 0 μM to 32 μM.

[0054] Experimental methods:

[0055] (1)① For SN152 strain: the first 96-well plate was set up with 8 columns and 8 rows. 180 μL of RPMI 1640 culture medium and 20 μL of 256 μg / mL FLC solution were added to well 1 of each row and mixed thoroughly. 100 μL of bacterial solution was added to wells 2 to 8 for serial dilution so that the final drug concentrations in each well were 256, 128, 64, 32, 16, 8, 4, and 0 μg / mL, respectively. The second 96-well plate was set up with 8 columns and 8 rows and the final glucose concentrations were 800, 400, 200, 100, 50, 25, and 0 μM, respectively. The third 96-well plate was set up with 8 rows and 10 columns. The tenth column was the blank control and the ninth column was the control group. 100 μL of bacterial solution was added to each well. 50 μL of each well of the first 96-well plate and the second 96-well plate were transferred to the third 96-well plate. 100 μL of PBS; for HAP43 gene-deficient bacteria and PCK1 gene-deficient bacteria: add 180 μL of RPMI 1640 culture medium and 20 μL of 80 μg / mL FLC solution to well 1 in each row, mix thoroughly, and perform serial dilutions in wells 1 to 8 so that the final drug concentrations in each well are 8, 4, 2, 1, and 0 μg / mL, respectively. Other steps are the same as above.

[0056] ② For SN152 strain: Set up the first 96-well plate with 8 columns and 7 rows, and add RPMI to the No. 1 well in each row. 180 μL of 1640 culture medium and 20 μL of 2560 μg / mL FLC solution were mixed thoroughly, and 100 μL of bacterial solution was added to wells 2 to 8 for serial dilution, so that the final drug concentrations in each well were 256, 128, 64, 32, 16, 8, 4, and 0 μg / mL, respectively. The second 96-well plate was set up with 8 columns and 7 rows, and the final 2-DG concentrations were set to 128, 64, 32, 16, 8, 4, and 0 μM, respectively. The third 96-well plate was set up with 10 columns and 7 rows, with the tenth column as the blank control and the ninth column as the control group. 100 μL of bacterial solution was added to each well, and 50 μL of each well of the first 96-well plate and the second 96-well plate were transferred to the third 96-well plate respectively. 100 μL PBS was added to the control group on the basis of the bacterial solution. For HAP43 gene-deficient bacteria and PCK1 gene-deficient bacteria: RPMI was added to well 1 in each row. 180 μL of 1640 culture medium and 20 μL of 80 μg / mL FLC solution were mixed thoroughly, and serial dilutions were performed in wells 1 to 8 so that the final drug concentrations in each well were 8, 4, 2, 1, and 0 μg / mL, respectively. Other steps were the same as above.

[0057] (2) Cultivate biofilm formation (same as 1.2).

[0058] (3) Biofilm XTT reduction experiment to measure OD492 value (conventional technical means).

[0059] 2. Experimental Results 1

[0060] 2.1 Construction of HAP43Δ / Δ+PCK1 OE Results Identification

[0061] refer to Figure 1 , proving that the present invention has successfully constructed HAP43Δ / Δ+PCK1 OE .

[0062] 2.2HAP43 gene can regulate biofilm formation by upregulating the expression of PCK1 gene

[0063] refer to Figure 2 The results of XTT reduction experiments showed that the average OD492 of three replicate experiments of SN152 strain biofilm and HAP43Δ / Δ+PCK1 OE The average OD492 values ​​of the biofilms of the strains were similar, with no significant difference (P>0.05), indicating that HAP43Δ / Δ+PCK1 OE Similar to the biofilm proliferation activity of SN152 strain, that is, after overexpressing the PCK1 gene, the biofilm of the HAP43 gene-deficient strain recovered the biofilm metabolic activity of the parent strain SN152, indicating that the HAP43 gene can regulate biofilm formation by upregulating the expression of PCK1.

[0064] 2.3 The biofilm proliferation activity of the HAP43 gene-deficient strain in a high-sugar environment is similar to that of the parent strain SN152

[0065] refer to Figure 3 The results showed that glucose can weaken the inhibitory effect of fluconazole on biofilm activity when different concentrations of glucose and fluconazole were used together to treat four strains (SN152 parent strain, HAP43 gene knockout strain, PCK1 gene knockout strain, and HAP43 gene knockout strain overexpressing PCK1). Moreover, as the concentration of glucose increased, the proliferation activity of the biofilm of the four strains increased accordingly. On the contrary, the results were opposite when different concentrations of 2-DG (glycolysis inhibitor) and fluconazole were used together to treat the four strains.

[0066] In the repeated experiment, the OD value of the XTT experiment of the biofilm of the HAP43 gene-deficient strain was similar to the OD value of the parent bacteria SN152 when fluconazole was not added and the glucose concentration was 200 μM, and the difference was not statistically significant (P>0.05), indicating that the biofilm proliferation activity of the HAP43 gene-deficient strain was similar to that of the parent bacteria SN152, that is, the biofilm proliferation activity of the HAP43 gene-knockout strain was restored to the proliferation activity of the parent bacteria SN152 biofilm after overexpressing PCK1 or increasing the glucose concentration to 200 μM. The PCK1 gene plays an important role in promoting gluconeogenesis. Overexpression of PCK1 will inevitably promote gluconeogenesis, that is, increase sugar concentration, further confirming that HAP43 can regulate biofilm formation through PCK1.

[0067] 3. Verification Experiment 2

[0068] A mouse model of systemic infection with Candida albicans was constructed using the parental strain SN152, the HAP43 gene knockout strain overexpressing the PCK1 gene, the HAP43 gene knockout strain, and the PCK1 gene knockout strain. The infection status of the strains in mice was observed, and the CFU counts of liver and kidney tissues were detected to preliminarily explore whether the HAP43 gene can regulate the virulence of Candida albicans through the PCK1 gene.

[0069] SPF C57BL / 6J mice (♀, 6 weeks old) were purchased from Shanghai Slake Experimental Animal Co., Ltd. All mice were first fed for one week in an environmental experiment, maintaining appropriate temperature and appropriate light intensity, 12 h light and dark alternation, free access to food and water, and the experiment was conducted after the mice were stable. The animal experiments in this study have been approved by the Ethics Committee of Tongji University.

[0070] 3.1 Strain activation

[0071] The parent strains SN152, HAP43Δ / Δ+PCK1 OEUse a sterile inoculation loop to pick a single clone colony on a sterile clean bench, spread it on a sterile YPD plate, place it in a constant temperature fungal incubator, and culture it at 30°C for 48h. Store the activated strains in a -4°C refrigerator in preparation for the next experiment.

[0072] 3.2 Establishment of a mouse systemic fungal infection model

[0073] The parent strains SN152, HAP43Δ / Δ+PCK1 OE A small amount of glycerol-frozen bacteria from -80℃ were taken from strains, HAP43Δ / Δ strains, and PCK1Δ / Δ strains and inoculated into YPD solid medium, and cultured at 30℃ for 48h; one colony was picked and placed in 1mL liquid YPD medium, and shaken at 30℃ for 16h to maintain the fungus in the late exponential growth stage. Centrifuge at 1,000×g for 5min, wash three times with 0.9% saline until the supernatant is colorless, discard the supernatant, and then adjust the bacterial concentration to 1×10 with 0.9% saline. 6 The mice were divided into an experimental group and a control group. The control group was injected with parent bacteria SN152. The experimental group was divided into three groups, each of which was injected with HAP43Δ / Δ+PCK1 via the tail vein of the mice. OE The strain, HAP43Δ / Δ strain and PCK1Δ / Δ strain were added at 0.1 mL / 10 g each to cause systemic fungal infection in C57BL / 6J mice (4 mice / group). The infection status of the mice was observed.

[0074] 3.3 Kidney viable colony count (CFU) detection

[0075] 48 hours after modeling, mice (4 mice / group) were killed, kidneys were removed, weighed, and homogenized with 5 mL of 0.9% saline. The homogenate was then diluted with 0.9% saline for 10 and 10 min. 2 Multiple dilutions were obtained by analogy. 100 μL of each was spread on a YPD plate, cultured in a fungal incubator at 30°C for 48 hours, and then the number of colonies was counted. The minimum number of colonies grown on the YPD plate was 20. The number of viable colonies (CFU) in the kidney was calculated, plotted as log10 CFU / g, and a t-test was performed. Calculation formula: CFU / g = number of colonies × dilution factor × 50 / kidney weight (g).

[0076] 3.4 Liver colony count (CFU) detection

[0077] The method is the same as 3.3.

[0078] 3.5 Statistical analysis

[0079] Each experiment was repeated at least three times, and Graphpad 6.0 and Clamfit 10.0 software were used to process and measure data. SPSS21.0 and Sigmaplot 10.0 software were used for statistical analysis and drawing. The measured data followed a normal distribution and were expressed as mean ± standard deviation. The comparison between two groups was performed using the t-test, and the comparison between multiple groups was performed using one-way analysis of variance.

[0080] IV. Experimental Results 2

[0081] refer to Figure 4 The viable bacterial count of the target organs of Candida albicans infection, kidney and liver, was converted to log10 CFU / g. The average log10 value of the bacterial load on the second day of infection of the kidney by the parent strain was 5.82 CFU / g, while the average log10 value of the tissue bacterial load of the kidney infected by the HAP43 knockout strain overexpressing the PCK1 gene was 5.84 CFU / g, the average log10 value of the tissue bacterial load of the kidney infected by the HAP43 knockout strain overexpressing the PCK1 gene was 5.61 CFU / g, and the average log10 value of the tissue bacterial load of the kidney infected by the PCK1 knockout strain was 5.60 CFU / g. The results showed that the tissue bacterial load of the kidney infected by the HAP43 knockout strain overexpressing the PCK1 gene (on the second day after infection) was not significantly different from that of the parent strain (P>0.05), while the tissue bacterial load of the kidney infected by the HAP43 knockout strain and the PCK1 knockout strain (on the second day after infection) was significantly reduced compared with the parent strain (P<0.0001). The average log10 value of bacterial load in the liver on the second day of infection by the parental bacteria was 5.18 CFU / g, while the average log10 value of bacterial load in the liver tissue infected by the HAP43 knockout strain overexpressing the PCK1 gene was 5.16 CFU / g, the average log10 value of bacterial load in the liver tissue infected by the HAP43 knockout strain was 4.83 CFU / g, and the average log10 value of bacterial load in the kidney tissue infected by the PCK1 knockout strain was 4.82 CFU / g. The results showed that the bacterial load in the liver tissue infected by the HAP43 knockout strain overexpressing the PCK1 gene (on the second day after infection) was not significantly different from that of the parental bacteria (P>0.05), while the bacterial load in the liver tissue infected by the HAP43 knockout strain and the PCK1 knockout strain (on the second day after infection) was decreased compared with the parental bacteria (P<0.0001).

[0082] The above experimental results prove that the Candida albicans HAP43 gene can regulate the virulence of Candida albicans through PCK1.

[0083] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of HAP43 gene expression inhibitor in the preparation of drugs for treating candidiasis.

2. The use according to claim 1, characterized in that: The HAP43 gene expression inhibitor can regulate the expression of the PCK1 gene.

3. The use according to claim 2, characterized in that: The HAP43 gene can enhance the virulence of Candida albicans by upregulating the expression of the PCK1 gene.

4. The use according to claim 2, characterized in that: The HAP43 gene can enhance the drug resistance of Candida albicans by upregulating the expression of the PCK1 gene.

5. The use according to claim 4, characterized in that: The HAP43 gene can enhance the formation of Candida albicans biofilm by upregulating the expression of the PCK1 gene.

6. The use according to claim 1, characterized in that: The drug for treating candidiasis albicans further comprises pharmaceutically acceptable auxiliary materials and / or carriers.

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