法尼醇对白念珠菌生物膜cAMP-PKA信号通路的影响及耐药相关性

doi:10.13591/j.cnki.kqyx.2025.05.001

摘要/Abstract

摘要：

目的探究法尼醇对白念珠菌生物膜cAMP-PKA信号通路相关分子的调控作用及其与耐药相关性。方法将白念珠菌标准株、氟康唑耐药株、白念珠菌野生株及RAS1基因高表达株生物膜培养至不同时相(6、12、24、36 h),法尼醇处理后使用XTT减低法检测氟康唑抑制各组生物膜50%活性的最低药物浓度(sessile minimal inhibitory concentration 50%,SMIC₅₀),利用qPCR技术和Western blot检测白念珠菌标准株、氟康唑耐药株cAMP-PKA信号通路RAS1、CYR1、PDE2基因、蛋白表达及白念珠菌野生株、RAS1基因高表达株RAS1基因表达。结果 ①与标准株相比,白念珠菌耐药株6、12、24 h生物膜SMIC₅₀较高;RAS1无明显差异(P>0.05),CYR1在6、24 h生物膜中表达明显增加(P<0.01),PDE2在6 h生物膜中表达下降(P<0.01)。②法尼醇处理后,标准株和耐药株生物膜的耐药性降低。与未进行法尼醇处理相比,标准株和耐药株RAS1在各时相生物膜表达减少(P<0.01);CYR1在6、24、36 h生物膜中表达减少,在12 h生物膜中表达增加(P<0.01);PDE2在12 h生物膜中表达升高(P<0.01)。③与野生株相比,RAS1基因高表达株12、24 h生物膜SMIC₅₀较高,12、24、36 h生物膜中RAS1基因表达明显更高(P<0.01)。④法尼醇处理后,野生株和RAS1基因高表达株耐药性降低。与未进行法尼醇处理相比,野生株和RAS1基因高表达株12、24 h生物膜中RAS1基因表达下降(P<0.01)。结论法尼醇可以通过调控白念珠菌cAMP-PKA通路中相关耐药分子RAS1、CYR1、PDE2的表达影响白念珠菌生物膜对氟康唑的敏感性,调控效果在生物膜不同形成阶段具有差异。

关键词: 白念珠菌, 生物膜, 法尼醇, cAMP-PKA, 耐药

Abstract:

Objective To explore the regulatory role of farnesol in Candida albicans (C. albicans) biofilm cAMP-PKA signaling pathway and its correlation with drug resistance. Methods Standard, fluconazole-resistant, wild and high RAS1 gene expression strains of C. albicans were cultured to different phases of the biofilm (6,12,24,36 h), and the sessile minimal inhibitory concentration 50%(SMIC₅₀) of fluconazole were determined by XTT reduction after farnesol treatment. The regulatory effects of farnesol on the expression of genes related to the cAMP-PKA signaling pathway in standard and fluconazole-resistant strains of C. albicans, such as RAS1, CYR1, PDE2 were examined using qPCR; the effects of farnesol on the protein expression of the pathway were analyzed by Western blot. RAS1 gene expression of the wild and high RAS1 gene expression strains was measured by qPCR. Results ① Compared with the standard strain, resistant strains of C. albicans had higher levels of biofilm SMIC₅₀ at 6, 12 and 24 h; there was no significant difference in RAS1 expression (P>0.05), while CYR1 expression increased significantly at 6 and 24 h in the biofilm (P<0.01), and PDE2 expression decreased at 6 h in the biofilm (P<0.01). ②After treatment with farnesol, the resistance of the biofilm of the standard strain and drug-resistant strain decreased. Compared with no treatment with farnesol, the expression of RAS1 in the biofilm of the standard strain and drug-resistant strain decreased at all time points (P<0.01); CYR1 expression decreased in the biofilm at 6, 24 and 36 h, and increased in the biofilm at 12 h (P<0.01); PDE2 expression increased in the 12 h biofilm (P<0.01). ③Compared with the wild strain, the high expression strain of RAS1 gene showed higher SMIC₅₀ in the biofilm at 12 and 24 h, and significantly higher expression of RAS1 gene in the biofilm at 12, 24 and 36 h (P<0.01). ④After treatment with farnesol, the resistance of wild-type strains and high expression strains of RAS1 gene decreased. Compared with the untreated group, the expression of RAS1 gene in the biofilm of wild-type and RAS1 gene high expression strain decreased at 12 and 24 h (P<0.01). Conclusion Farnesol can affect the sensitivity of C. albicans biofilm to fluconazole by regulating the expression of resistance molecules RAS1, CYR1 and PDE2 in the cAMP-PKA pathway. The regulatory effect varies at different stages of biofilm formation.

Key words: Candida albicans, biofilm, farnesol, cAMP-PKA, drug resistance

中图分类号:

R781.54

王阳, 陈雪旖, 俞沈君, 曹雪姣, 魏昕, 杨璇. 法尼醇对白念珠菌生物膜cAMP-PKA信号通路的影响及耐药相关性[J]. 口腔医学, 2025, 45(5): 321-327.

WANG Yang, CHEN Xueyi, YU Shenjun, CAO Xuejiao, WEI Xin, YANG Xuan. The effect of farnesol on the cAMP-PKA signaling pathway of Candida albicans biofilms and its correlation with drug resistance[J]. Stomatology, 2025, 45(5): 321-327.

图/表 6

表1

表2

图1

图2

表3

图3

参考文献 37

[1]	Ponde NO, Lortal L, Ramage G, et al. Candida albicans biofilms and polymicrobial interactions[J]. Crit Rev Microbiol, 2021, 47(1): 91-111.
[2]	Pereira R, Dos Santos Fontenelle RO, de Brito ES, et al. Biofilm of Candida albicans: Formation, regulation and resistance[J]. J Appl Microbiol, 2021, 131(1): 11-22. doi: 10.1111/jam.14949 pmid: 33249681
[3]	File B, Sobel R, Becker M, et al. Fluconazole-resistant Candida albicans vaginal infections at a referral center and treated with boric acid[J]. J Low Genit Tract Dis, 2023, 27(3): 262-265.
[4]	Sachivkina N, Senyagin A, Podoprigora I, et al. Enhancement of the antifungal activity of some antimycotics by farnesol and reduction of Candida albicans pathogenicity in a quail model experiment[J]. Vet World, 2022, 15(4): 848-854.
[5]	Hall RA, Turner KJ, Chaloupka J, et al. The quorum-sensing molecules farnesol/homoserine lactone and dodecanol operate via distinct modes of action in Candida albicans[J]. Eukaryot Cell, 2011, 10(8): 1034-1042.
[6]	Zhao LX, Li DD, Hu DD, et al. Effect of tetrandrine against Candida albicans biofilms[J]. PLoS One, 2013, 8(11): e79671.
[7]	She XD, Zhang LL, Peng JW, et al. Mitochondrial complex Ⅰ core protein regulates cAMP signaling via phosphodiesterase Pde2 and NAD homeostasis in Candida albicans[J]. Front Microbiol, 2020, 11: 559975.
[8]	Singkum P, Muangkaew W, Suwanmanee S, et al. Suppression of the pathogenicity of Candida albicans by the quorum-sensing molecules farnesol and tryptophol[J]. J Gen Appl Microbiol, 2020, 65(6): 277-283.
[9]	Yapıcı M, Gürsu BY, Dağ İ. In vitro antibiofilm efficacy of farnesol against Candida species[J]. Int Microbiol, 2021, 24(2): 251-262.
[10]	钱芳, 魏昕, 许雯倩, 等. XTT减低法检测法尼醇对白念珠菌生物被膜的抑制作用[J]. 口腔生物医学, 2014, 5(2): 82-85.
[11]	张琴琴, 马鸣, 花荣, 等. 法尼醇对白念珠菌生物膜葡聚糖的影响及白念珠菌耐药相关性[J]. 口腔医学, 2023, 43(6): 488-493.
[12]	章珍珍, 夏金萍, 马鸣, 等. 白念珠菌RAS1基因高表达菌株的构建及鉴定[J]. 口腔生物医学, 2016, 7(2): 72-75.
[13]	Lamoth F, Lewis RE, Kontoyiannis DP. Investigational antifungal agents for invasive mycoses: A clinical perspective[J]. Clin Infect Dis, 2022, 75(3): 534-544. doi: 10.1093/cid/ciab1070 pmid: 34986246
[14]	Shafiei M, Peyton L, Hashemzadeh M, et al. History of the development of antifungal azoles: A review on structures, SAR, and mechanism of action[J]. Bioorg Chem, 2020, 104: 104240.
[15]	Pristov KE, Ghannoum MA. Resistance of Candida to azoles and echinocandins worldwide[J]. Clin Microbiol Infect, 2019, 25(7): 792-798.
[16]	Rai LS, Wijlick LV, Bougnoux ME, et al. Regulators of commensal and pathogenic life-styles of an opportunistic fungus-Candida albicans[J]. Yeast, 2021, 38(4): 243-250.
[17]	Ho J, Camilli G, Griffiths JS, et al. Candida albicans and candidalysin in inflammatory disorders and cancer[J]. Immunology, 2021, 162(1): 11-16.
[18]	Arastehfar A, Shaban T, Zarrinfar H, et al. Candidemia among Iranian patients with severe COVID-19 admitted to ICUs[J]. J Fungi (Basel), 2021, 7(4): 280.
[19]	Ramage G, Vandewalle K, Wickes BL, et al. Characteristics of biofilm formation by Candida albicans[J]. Rev Iberoam Micol, 2001, 18(4): 163-170. pmid: 15496122
[20]	Massey J, Zarnowski R, Andes D. Role of the extracellular matrix in Candida biofilm antifungal resistance[J]. FEMS Microbiol Rev, 2023, 47(6): fuad059.
[21]	Xia JP, Qian F, Xu WQ, et al. In vitro inhibitory effects of farnesol and interactions between farnesol and antifungals against biofilms of Candida albicans resistant strains[J]. Biofouling, 2017, 33(4): 283-293.
[22]	Castilla R, Passeron S, Cantore ML. N-acetyl-D-glucosamine induces germination in Candida albicans through a mechanism sensitive to inhibitors of cAMP-dependent protein kinase[J]. Cell Signal, 1998, 10(10): 713-719. pmid: 9884022
[23]	Jothi R, Hari Prasath N, Gowrishankar S, et al. Bacterial quorum-sensing molecules as promising natural inhibitors of Candida albicans virulence dimorphism: An In silico and in vitro study[J]. Front Cell Infect Microbiol, 2021, 11: 781790.
[24]	Davis-Hanna A, Piispanen AE, Stateva LI, et al. Farnesol and dodecanol effects on the Candida albicans Ras1-cAMP signalling pathway and the regulation of morphogenesis[J]. Mol Microbiol, 2008, 67(1): 47-62. doi: 10.1111/j.1365-2958.2007.06013.x pmid: 18078440
[25]	Huang GH, Huang Q, Wei YJ, et al. Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans[J]. Mol Microbiol, 2019, 111(1): 6-16.
[26]	Zeng GS, Neo SP, Pang LM, et al. Comprehensive interactome analysis for the sole adenylyl cyclase Cyr1 of Candida albicans[J]. Microbiol Spectr, 2022, 10(6): e0393422.
[27]	Chen SY, Xia JP, Li CX, et al. The possible molecular mechanisms of farnesol on the antifungal resistance of C. albicans biofilms: The regulation of CYR1 and PDE2[J]. BMC Microbiol, 2018, 18(1): 203.
[28]	Min K, Jannace TF, Si HY, et al. Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans[J]. PLoS Pathog, 2021, 17(8): e1009861.
[29]	Lindsay AK, Deveau A, Piispanen AE, et al. Farnesol and cyclic AMP signaling effects on the hypha-to-yeast transition in Candida albicans[J]. Eukaryot Cell, 2012, 11(10): 1219-1225. doi: 10.1128/EC.00144-12 pmid: 22886999
[30]	Shapiro RS, Zaas AK, Betancourt-Quiroz M, et al. The Hsp90 co-chaperone Sgt1 governs Candida albicans morphogenesis and drug resistance[J]. PLoS One, 2012, 7(9): e44734.
[31]	Cao YY, Cao YB, Xu Z, et al. cDNA microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol[J]. Antimicrob Agents Chemother, 2005, 49(2): 584-589.
[32]	Weber S, Zeller M, Guan KM, et al. PDE2 at the crossway between cAMP and cGMP signalling in the heart[J]. Cell Signal, 2017, 38: 76-84. doi: S0898-6568(17)30178-X pmid: 28668721
[33]	Sadek MS, Cachorro E, El-Armouche A, et al. Therapeutic implications for PDE2 and cGMP/cAMP mediated crosstalk in cardiovascular diseases[J]. Int J Mol Sci, 2020, 21(20): 7462.
[34]	Morais Vasconcelos Oliveira J, Conceição Oliver J, Latércia Tranches Dias A, et al. Detection of ERG11 Overexpression in Candida albicans isolates from environmental sources and clinical isolates treated with inhibitory and subinhibitory concentrations of fluconazole[J]. Mycoses, 2021, 64(2): 220-227. doi: 10.1111/myc.13208 pmid: 33176021
[35]	Paul S, Kannan I, Mohanram K. Extensive ERG11 mutations associated with fluconazole-resistant Candida albicans isolated from HIV-infected patients[J]. Curr Med Mycol, 2019, 5(3): 1-6.
[36]	Bhattacharya S, Sae-Tia S, Fries BC. Candidiasis and mechanisms of antifungal resistance[J]. Antibiotics (Basel), 2020, 9(6): 312.
[37]	Banerjee A, Pata J, Sharma S, et al. Directed mutational strategies reveal drug binding and transport by the MDR transporters of Candida albicans[J]. J Fungi (Basel), 2021, 7(2): 68.

基因	序列(5'→3')
RAS1	F:GGTAAATCCGCTTTAACCATTC R:GCCAGATATTCTTCTTGTCCAG
CYR1	F:CCGACTACTGGCTTACCAACTC R:GCTACATCATTCTCTGCAACTTC
PDE2	F:GTGACGAGACCGTTGAGAGTAT R:ATCGGGTTGTATTTCAAGTATT
β-actin	F:GACCAAGACATCAAGGTATCAT R:GTGTTCAATTGGGTATCTCAAG

组别	6 h	12 h	24 h	36 h
A组	512	1 024	1 024	>1 024
B组	32	128	256	1 024
C组	>1 024	>1 024	>1 024	>1 024
D组	64	256	512	>1 024

组别	6 h	12 h	24 h	36 h
E组	>1 024	1 024	1 024	>1 024
F组	1 024	512	512	>1 024
G组	>1 024	>1 024	>1 024	>1 024
H组	128	256	512	>1 024