基于pH信号通路的白头翁汤正丁醇提取物对白念珠菌生物膜作用机制的研究
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摘要
该文基于pH信号通路探讨白头翁汤正丁醇提取物(butyl alcohol extract of Baitouweng Decoction,BAEB)对白念珠菌生物膜的影响。以扫描电镜观察pH突变株生物膜形态结构; CLSM测量pH突变株生物膜厚度;酶标仪检测pH突变株生物膜活性;流式细胞仪检测pH突变株生物膜损伤; qRT-PCR法检测pH突变株生物膜相关基因的表达。结果显示,对生物膜结构,PHR1基因缺失造成结构缺陷,PHR1回补则基质较多; BAEB干预对该2株无明显影响。RIM101基因缺失或回补无明显结构损伤,但BAEB干预后,这2种菌株生物膜均被明显抑制。对生物膜厚度,PHR1基因缺失或PHR1回补的厚度降低;BAEB干预后,该2株厚度变化不明显。而RIM101基因缺失或回补对生物膜厚度影响不大;加入BAEB后,该2株生物膜厚度明显变薄。对生物膜活性,PHR1基因缺失、PHR1回补和RIM101基因缺失均造成活性降低,RIM101回补无明显变化;BAEB干预后,PHR1基因缺失、PHR1回补、RIM101缺失与RIM101回补的生物膜活性与干预前相比显著降低。对生物膜损伤程度,PHR1基因缺失、PHR1回补和RIM101基因缺失、RIM101回补均出现不同程度损伤;加入BAEB干预后,PHR1缺失或PHR1回补的损伤率差异并不大,但RIM101缺失或RIM101回补的损伤率显著升高。对基因表达水平,除HSP90基因上调外,PHR1缺失、PHR1回补、RIM101缺失、RIM101回补的ALS3,SUN41,HWP1,UME6和PGA10基因均出现了不同倍数的下调趋势。该研究表明,pH信号通路中PHR1和RIM101基因突变能提高菌株对于抗真菌药物BAEB的敏感性,从而抑制白念珠菌生物膜形成及相关基因的表达。
This study aimed to investigate the effect of butyl alcohol extract of Baitouweng Decoction( BAEB) on Candida albicans biofilms based on pH signal pathway. The morphology of biofilms of the pH mutants was observed by scanning electron microscope. The biofilm thickness of the pH mutants was measured by CLSM. The biofilm activity of the pH mutants was analyzed by microplate reader.The biofilm damage of the pH mutants was detected by flow cytometry. The expression of pH mutant biofilm-related genes was detected by qRT-PCR. The results showed that the deletion of PHR1 gene resulted in the defect of biofilm,but there were more substrates for PHR1 complementation. BAEB had no significant effect on the two strains. RIM101 gene deletion or complementation did not cause significant structural damage,but after BAEB treatment,the biofilms of both strains were significantly inhibited. For the biofilm thickness,PHR1 deletion or complementation caused the thickness to decrease,after BAEB treatment,the thickness of the two strains did not change significantly. However,RIM101 gene deletion or complementation had little effect on the thickness,and the thickness of the two strains became thinner after adding BAEB. For biofilm activity,PHR1 deletion or complementation and RIM101 deletion resulted in decreased activity,RIM101 complementation did not change significantly; BAEB significantly inhibited biofilm activity of PHR1 deletion,PHR1 complemetation,RIM101 deletion and RIM101 complemetation strains. For the biofilm damage,PHR1 gene deletion or complementation,RIM101 gene deletion or complementation all showed different degrees of damage; after adding BAEB,the damage rate of PHR1 deletion or complementation was not significantly different,but the damage rate of RIM101 deletion or complementation was significantly increased. Except to the up-regulation of HSP90 gene expression,ALS3,SUN41,HWP1,UME6 and PGA10 genes of PHR1 deletion,PHR1 complementation,RIM101 deletion,and RIM101 complementation strains showed a downward expression trend. In a word,this study showed that mutations in PHR1 and RIM101 genes in the pH signaling pathway could enhance the sensitivity of the strains to the antifungal drug BAEB,thus inhibiting the biofilm formation and related genes expression in C. albicans.
This study aimed to investigate the effect of butyl alcohol extract of Baitouweng Decoction( BAEB) on Candida albicans biofilms based on pH signal pathway. The morphology of biofilms of the pH mutants was observed by scanning electron microscope. The biofilm thickness of the pH mutants was measured by CLSM. The biofilm activity of the pH mutants was analyzed by microplate reader.The biofilm damage of the pH mutants was detected by flow cytometry. The expression of pH mutant biofilm-related genes was detected by qRT-PCR. The results showed that the deletion of PHR1 gene resulted in the defect of biofilm,but there were more substrates for PHR1 complementation. BAEB had no significant effect on the two strains. RIM101 gene deletion or complementation did not cause significant structural damage,but after BAEB treatment,the biofilms of both strains were significantly inhibited. For the biofilm thickness,PHR1 deletion or complementation caused the thickness to decrease,after BAEB treatment,the thickness of the two strains did not change significantly. However,RIM101 gene deletion or complementation had little effect on the thickness,and the thickness of the two strains became thinner after adding BAEB. For biofilm activity,PHR1 deletion or complementation and RIM101 deletion resulted in decreased activity,RIM101 complementation did not change significantly; BAEB significantly inhibited biofilm activity of PHR1 deletion,PHR1 complemetation,RIM101 deletion and RIM101 complemetation strains. For the biofilm damage,PHR1 gene deletion or complementation,RIM101 gene deletion or complementation all showed different degrees of damage; after adding BAEB,the damage rate of PHR1 deletion or complementation was not significantly different,but the damage rate of RIM101 deletion or complementation was significantly increased. Except to the up-regulation of HSP90 gene expression,ALS3,SUN41,HWP1,UME6 and PGA10 genes of PHR1 deletion,PHR1 complementation,RIM101 deletion,and RIM101 complementation strains showed a downward expression trend. In a word,this study showed that mutations in PHR1 and RIM101 genes in the pH signaling pathway could enhance the sensitivity of the strains to the antifungal drug BAEB,thus inhibiting the biofilm formation and related genes expression in C. albicans.
引文
[1] Davis D. Adaptation to environmental pH in Candida albicans and its relation to pathogenesis[J].Curr Genet,2003,44(1):1.
[2] Davis D,Edwards J E Jr,Mitchell A P,et al. Candida albicans RIM101 pH response pathway is required for host pathogen interaction[J].Infect Immun,2000,68(10):5953.
[3] Davis D,Wilson R B,Mitchell A P. RIM101-dependent and independent pathways govern pH response in Candida albicans[J].Mol Cell Biol,2000,20(3):971.
[4] Mitchell A P. Dimorphism and virulence in Candida albicans[J].Curr Opin Microbiol,1998,1(6):687.
[5] Cornet M, Gaillardin C. pH signaling in human fungal pathogens:a new target for antifungal strategies[J]. Eukaryot Cell,2014,13(3):342.
[6] Staniszewska M,Bondaryk M,Swoboda-Kopec E,et al. Candida albicans morphologies revealed by scanning electron microscopy analysis[J]. Braz J Microbiol,2013,44(3):813.
[7] X Wu,R R Santos,J Finkgremmels. Cadmium modulates biofilm formation by Staphylococcus epidermidis[J]. Int J Environ Res Public Health,2015,12(3):2881.
[8]张梦翔,夏丹,施高翔,等.白头翁汤正丁醇提取物对碱性pH条件下白念珠菌VVC临床临床株形态转化的影响[J].中国中药杂志,2015,40(4):712.
[9] Maurya I K,Pathak S,Sharma M,et al. Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species(ROS)and disruption of cell wall against Candida albicans[J]. Peptides,2011,32(8):1732.
[10]颜贵明,施高翔,邵菁,等.黄芩苷联合氟康唑对白念珠菌生物膜的抑制作用研究[J].中国真菌学杂志,2015,10(3):141.
[11]张梦翔,陆克乔,夏丹,等.白头翁汤正丁醇提取物对白念珠菌VVC临床株体外生物膜形成的抑制作用[J].中国真菌学杂志,2015,10(6):323.
[12]张晓芬,张超云.白头翁汤加味综合治疗复发性念珠菌性阴道病[J].中国实验方剂学杂志,2012,18(9):279.
[13]李明春,梁勇,魏东盛,等.白念珠菌RIM101途径与环境pH的应答反应[J].微生物学报,2007,47(2):366.
[14] Gomez-Raja J,Davis D A. Theβ-arrestin-like protein Rim8 is hyperphosphorylate and complexes with Rim21 and Rim101 to promote adaptation to neutral-alkaline pH[J]. Eukaryot Cell,2012,11(5):683.
[15] Selvig K,Alspaugh J A. pH response pathways in fungi:adapting to host-derived and environmental signals[J]. Mycobiology,2011,39(4):249.
[16] Davis D A. How human pathogenic fungi sense and adapt to pH:the link to virulence[J]. Curr Opin Microbiol, 2009, 12(4):365.
[17] Wang H,Liang Y,Zhang B,et al. Alkaline stress triggers an immediate calcium fluctuation in Candida albicans mediated by Rim101p and Crz1p transcription factors[J]. FEMS Yeast Res,2011,11(5):430.
[18] Taff H T,Nett J E,Zarnowski R,et al. A Candida biofilm-induced pathway for matrix glucan delivery:implications for drug resistance[J].PLoS Pathog,2012,8(8):e1002848.
[19] Asleson C M,Bensen E S,Gale C A,et al. Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p[J].Mol Cell Biol,2001,21(4):1272.
[20] Shen J,Cowen L E,Griffin A M,et al. The Candida albicans pescadillo homolog is required for normal hypha-to-yeast morphogenesis and yeast proliferation[J]. Proc Natl Acad Sci USA,2008,105(52):20918.
[2] Davis D,Edwards J E Jr,Mitchell A P,et al. Candida albicans RIM101 pH response pathway is required for host pathogen interaction[J].Infect Immun,2000,68(10):5953.
[3] Davis D,Wilson R B,Mitchell A P. RIM101-dependent and independent pathways govern pH response in Candida albicans[J].Mol Cell Biol,2000,20(3):971.
[4] Mitchell A P. Dimorphism and virulence in Candida albicans[J].Curr Opin Microbiol,1998,1(6):687.
[5] Cornet M, Gaillardin C. pH signaling in human fungal pathogens:a new target for antifungal strategies[J]. Eukaryot Cell,2014,13(3):342.
[6] Staniszewska M,Bondaryk M,Swoboda-Kopec E,et al. Candida albicans morphologies revealed by scanning electron microscopy analysis[J]. Braz J Microbiol,2013,44(3):813.
[7] X Wu,R R Santos,J Finkgremmels. Cadmium modulates biofilm formation by Staphylococcus epidermidis[J]. Int J Environ Res Public Health,2015,12(3):2881.
[8]张梦翔,夏丹,施高翔,等.白头翁汤正丁醇提取物对碱性pH条件下白念珠菌VVC临床临床株形态转化的影响[J].中国中药杂志,2015,40(4):712.
[9] Maurya I K,Pathak S,Sharma M,et al. Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species(ROS)and disruption of cell wall against Candida albicans[J]. Peptides,2011,32(8):1732.
[10]颜贵明,施高翔,邵菁,等.黄芩苷联合氟康唑对白念珠菌生物膜的抑制作用研究[J].中国真菌学杂志,2015,10(3):141.
[11]张梦翔,陆克乔,夏丹,等.白头翁汤正丁醇提取物对白念珠菌VVC临床株体外生物膜形成的抑制作用[J].中国真菌学杂志,2015,10(6):323.
[12]张晓芬,张超云.白头翁汤加味综合治疗复发性念珠菌性阴道病[J].中国实验方剂学杂志,2012,18(9):279.
[13]李明春,梁勇,魏东盛,等.白念珠菌RIM101途径与环境pH的应答反应[J].微生物学报,2007,47(2):366.
[14] Gomez-Raja J,Davis D A. Theβ-arrestin-like protein Rim8 is hyperphosphorylate and complexes with Rim21 and Rim101 to promote adaptation to neutral-alkaline pH[J]. Eukaryot Cell,2012,11(5):683.
[15] Selvig K,Alspaugh J A. pH response pathways in fungi:adapting to host-derived and environmental signals[J]. Mycobiology,2011,39(4):249.
[16] Davis D A. How human pathogenic fungi sense and adapt to pH:the link to virulence[J]. Curr Opin Microbiol, 2009, 12(4):365.
[17] Wang H,Liang Y,Zhang B,et al. Alkaline stress triggers an immediate calcium fluctuation in Candida albicans mediated by Rim101p and Crz1p transcription factors[J]. FEMS Yeast Res,2011,11(5):430.
[18] Taff H T,Nett J E,Zarnowski R,et al. A Candida biofilm-induced pathway for matrix glucan delivery:implications for drug resistance[J].PLoS Pathog,2012,8(8):e1002848.
[19] Asleson C M,Bensen E S,Gale C A,et al. Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p[J].Mol Cell Biol,2001,21(4):1272.
[20] Shen J,Cowen L E,Griffin A M,et al. The Candida albicans pescadillo homolog is required for normal hypha-to-yeast morphogenesis and yeast proliferation[J]. Proc Natl Acad Sci USA,2008,105(52):20918.
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