白念珠菌不同生物状态下ALS4、ALS9基因mRNA的表达
摘要
目的：通过对白念珠菌酵母状态和芽管状态,以及浮游状态和生物膜状态下ALS4、ALS9基因mRNA表达水平的比较,探讨这两种基因在白念珠菌表型转换及生物膜形成中可能发挥的作用,为进一步研究白念珠菌的发病机制提供理论依据。
方法：选取3株标准株白念珠菌和10株临床株白念珠菌进行实验研究。白念珠菌酵母状态的培育和芽管状态的诱导以及浮游状态和生物膜状态的制备：GBS培养基,25℃振荡培养48h,收获酵母状态；pH7.0的RPMI-1640液体培养基,37℃震荡培养3h,收获芽管状态；体外6孔细胞培养板涂布小牛血清,RPMI-1640液体培养基,37℃静置培养48h,收获生物膜状态。液氮反复冻融加Trizol法提取不同生长状态白念珠菌的总RNA, RNA的纯度经紫外分光光度计A260/280比值估计,RT-PCR两步法扩增ALS4、ALS9基因mRNA,及内对照ACT1基因mRNA,扩增产物琼脂糖凝胶电泳,凝胶成像系统拍照。采用QuantityOne电泳分析软件对电泳条带进行分析,应用SPSS医学统计软件包两样本均数配对资料的t检验对实验数据进行统计学处理(以P<0.05为显著性标准)。
结果：标准株白念珠菌芽管状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均明显高于酵母状态,临床株白念珠菌芽管状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均明显高于酵母状态；标准株白念珠菌生物膜状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均高于浮游状态,临床株白念珠菌生物膜状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均高于浮游状态。
结论：ALS4基因和ALS9基因可能在白念珠菌的酵母相向菌丝相转换和体外生物膜形成中起重要作用。
Objective To evaluate the role of Candida albicans ALS gene in transformation and biofilm formation, to provide theoretical basis for Candida albicans pathogenesis so that compare the expression of ALS gene mRNA level of Candida albicans between yeast and hyphal, between plantonic and biofilm.
Methods Three C.albicans standard and ten clinical strain were induced to form yeast form in the condition cultured in GBS culture medium for 48h at 25℃, at planktonic condition. Germ tubes were induced by cultured in RPMI-1640 medium for 3h at 37℃, pH value at 7.0 with the shake speed of 150rpm. Biofilm models in vitro were formed on 6-well cell culture plate in RPMI-1640 media., after 48h incubation, the biofilm-grown cells were harvest. Liquid nitrogen and Trizol method were performed to isolate total RNA from the four different biological status separately. The purity of RNA was measured by absorption at 260/280nm with spectrophotometer. A semi-quantitative RT-PCR two-step method was developed to determine ALS4 and ALS9 genes against the housekeeping gene ACT 1.The identity of cloned fragments was confirmed with agarose gel electrophoresis. Gene expression leves were assayed by gel documentation system and Quantity One software. The paired test was used to analyze the experimental data.
Result The RT-PCR results showed that the expression levels of ALS4 mRNA (P <0.05) and ALS9mRNA (P<0.05) of the standard strains were higher in germ tube compared with yeast form. Expression levels of ALS4 mRNA (P<0.05) and ALS9 mRNA (P<0.05) of the clinical strains were higher in germ tube compared with yeast form. Expression levels of ALS4 mRNA (P<0.05) and ALS9mRNA (P<0.05) of the standard strains in biofilm increased compare with planktonic cells.There were also increases in biofilm cells compared with the planktonic cells in the expression levels of ALS4mRNA(P<0.05) and ALS9mRNA (P<0.05) of the clinical strain.
Conclusion The ALS4 and ALS9 gene may play an important role in Candida albicans transformation from yeast to germ tube and in biofilm formation in vitro.
方法：选取3株标准株白念珠菌和10株临床株白念珠菌进行实验研究。白念珠菌酵母状态的培育和芽管状态的诱导以及浮游状态和生物膜状态的制备：GBS培养基,25℃振荡培养48h,收获酵母状态；pH7.0的RPMI-1640液体培养基,37℃震荡培养3h,收获芽管状态；体外6孔细胞培养板涂布小牛血清,RPMI-1640液体培养基,37℃静置培养48h,收获生物膜状态。液氮反复冻融加Trizol法提取不同生长状态白念珠菌的总RNA, RNA的纯度经紫外分光光度计A260/280比值估计,RT-PCR两步法扩增ALS4、ALS9基因mRNA,及内对照ACT1基因mRNA,扩增产物琼脂糖凝胶电泳,凝胶成像系统拍照。采用QuantityOne电泳分析软件对电泳条带进行分析,应用SPSS医学统计软件包两样本均数配对资料的t检验对实验数据进行统计学处理(以P<0.05为显著性标准)。
结果：标准株白念珠菌芽管状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均明显高于酵母状态,临床株白念珠菌芽管状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均明显高于酵母状态；标准株白念珠菌生物膜状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均高于浮游状态,临床株白念珠菌生物膜状态的ALS4基因mRNA(P<0.05)和ALS9基因mRNA(P<0.05)相对表达水平均高于浮游状态。
结论：ALS4基因和ALS9基因可能在白念珠菌的酵母相向菌丝相转换和体外生物膜形成中起重要作用。
Objective To evaluate the role of Candida albicans ALS gene in transformation and biofilm formation, to provide theoretical basis for Candida albicans pathogenesis so that compare the expression of ALS gene mRNA level of Candida albicans between yeast and hyphal, between plantonic and biofilm.
Methods Three C.albicans standard and ten clinical strain were induced to form yeast form in the condition cultured in GBS culture medium for 48h at 25℃, at planktonic condition. Germ tubes were induced by cultured in RPMI-1640 medium for 3h at 37℃, pH value at 7.0 with the shake speed of 150rpm. Biofilm models in vitro were formed on 6-well cell culture plate in RPMI-1640 media., after 48h incubation, the biofilm-grown cells were harvest. Liquid nitrogen and Trizol method were performed to isolate total RNA from the four different biological status separately. The purity of RNA was measured by absorption at 260/280nm with spectrophotometer. A semi-quantitative RT-PCR two-step method was developed to determine ALS4 and ALS9 genes against the housekeeping gene ACT 1.The identity of cloned fragments was confirmed with agarose gel electrophoresis. Gene expression leves were assayed by gel documentation system and Quantity One software. The paired test was used to analyze the experimental data.
Result The RT-PCR results showed that the expression levels of ALS4 mRNA (P <0.05) and ALS9mRNA (P<0.05) of the standard strains were higher in germ tube compared with yeast form. Expression levels of ALS4 mRNA (P<0.05) and ALS9 mRNA (P<0.05) of the clinical strains were higher in germ tube compared with yeast form. Expression levels of ALS4 mRNA (P<0.05) and ALS9mRNA (P<0.05) of the standard strains in biofilm increased compare with planktonic cells.There were also increases in biofilm cells compared with the planktonic cells in the expression levels of ALS4mRNA(P<0.05) and ALS9mRNA (P<0.05) of the clinical strain.
Conclusion The ALS4 and ALS9 gene may play an important role in Candida albicans transformation from yeast to germ tube and in biofilm formation in vitro.
引文
[1]王鲁,叶庆佾,唐书谦.白念珠菌芽管特异性抗原的研究.中华皮肤科杂志.1994,27(3)：136-138.
[2]Berardinelli S.Opheim DJ. New germ tube induction medium for the identification of Candida albicans. Clin Microbiol.1985,22(5):861-862.
[3]Green CB, Cheng G, Chandra J, et al. RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candi-diasis and in model biofilms. Microbiology.2004,150(Pt 2):267-275.
[4]Kuhn DM, Chandra J, Mukherjee PK, et al. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect Immun.2002, 70(2):878-888.
[5]潘峥,魏昕,刘卫红.改进Trizol法提取白色念珠菌总RNA初探.临床口腔医学杂志,2007,23(9)：531-533.
[6]Qi QG, Hu T, Fu CH, et al. Comparison of ALS gene mRNA between sessile and planktonic Candida albicans of oral cavities. Hua Xi Kou Qiang Yi Xue Za Zhi. 2005,23(3):233-236.
[7]苑天红,王名永,吴升伟等。白念珠菌二相性与毒力关系的实验研究.中国皮肤性病学杂志.200418(10)580-581.
[8]Lo H, Kohler J R, DiDomenico B, et al. Nonfilamentous C. albicans mutants are avirulent. Cell.1997,90(5):939-949.
[9]Kumamoto CA. Candida biofilms. CurrOp in Microbial.2002,5(6):608-611.
[10]Lois L. Hoyer. The ALS gene family of Candida albicans. Trends in Microbiology. 2001,9(4):176-180.
[11]Green CB, Marretta SM, Cheng G, et al. RT-PCR analysis of Candida albicans ALS gene expression in a hyposalivatory rat model of oral candidiasis and in HIV-positive human patients. Med Mycol.2006,44(2):103-111.
[12]Hoyer LL, Green CB, Oh SH, et al. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family-a sticky pursuit. Med Mycol.2008,46(1): 1-15.
[13]O'Connor L, Lahiff S, Casey F, et al. Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler. Mol Cell Probes.2005,19(3):153-162.
[14]Fu Y, Rieg G, Fonzi WA, et al. Expression of the Candida albicans gene ALS1 in Saccharomyces cerevisiae induces adherence to endothelial and epithelial cells. Infect Immun.1998,66(4):1783-1786.
[15]Fu Y, Ibrahim AS, Sheppard DC, et al. Candida albicans Alslp:an adhesin that is a downstream effector of the EFG1 filamentation pathway. Mol Microbiol.2002, 44(1):61-72.
[16]Ibrahim AS, Spellberg BJ, Avenissian V, et al. Vaccination with recombinant N-terminal domain of Alslp improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity. Infect Immun.2005,73(2):999-1005.
[17]Fratti RA, Belanger PH, SanatiH, et al. The effect of the new triazole, voriconazole(UK2109,496), on the interactions of Candida albicans and Candida krus-eiwith endothelial cells. J Chemother.1998,10(1):7-16.
[18]黎斌,王鲁,蒋戈等.白念珠菌孢子相、菌丝相胞壁提取物SDS-PAGE分析中华皮肤科杂志.2003,36(11)：656-657.
[19]Zhao X, Oh SH, Yeater KM, et al. Analysis of the Candida albicans Als2p and Als4p adhesins suggests the potential for compensatory function within the Als family. Microbiology.2005,151(Pt 5):1619-1630.
[20]Nobile CJ, Schneider HA, Nett JE, et al. Complementary adhesion function in C. albicans biofilm formation. Curr Biol.2008,18(14):1017-1024.
[21]Sheppard DC, Yeaman MR, Welch WH, et al. Functional and structural diversity in the Als protein family of Candida albicans. Biol Chem.2004,279(29):30480-30489.
[22]Zhao X, Oh S-H, Hoyer LL. Unequal contribution of ALS9 alleles to adhesion between Candida albicans and vascular endotheial cells.Microbiology.2007,153(Pt 7):2342-2350.
[23]Watnick P, Kolter R. Biofilm, city of microbes. Bacteriol.2000,182(10):2675-2679.
[24]Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida albicans biofilms. Med Microbiol.1999,48(7):671-679.
[25]Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal Pathogen Candida albicans:development, architecture, and drug resistance. Baciteriol.2001, 183(18):5385-5394.
[26]Nail is H, Kucharikova S, Ricicova M, et al. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms:identif-ication of model-dependent and-independent gene expression. BMC Microbiol.2010, 10(1):114.
[1]Haynes K. Virulence in Candida species. Trends Microbiol.2001,9(12):591-596.
[2]秦启贤.临床真菌学[M].上海：复旦大学出版社,2001,58-59.
[3]Redding S, Bhatt B, Rawls HR, et al. Inhibition of Candida albicans biofilm formation on denture material. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009,107(5):669-672.
[4]Brown AJ, Gow NA. Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol.1999,7(8):333-338.
[5]Sommeborn A, Bockmubh DP, GeradsM et al. Protein kinaseA encoded by TPK2 re-gulated dimorphism of Candida albicans. MolMicrobiol.2000,35(2):386-396.
[6]Lo H, Kohler JR, DiDomenico B, et al. Nonfilamentous C. albicans mutants are avirulent. Cell.1997,90(5):939-949.
[7]Pendrak ML, Roberts DD. Hemoglobin is an effective inducer of hyphal diffe-rentiation in Candida albicans. Med Mycol.2007,45(1):61-71.
[8]Bailey DA, Feldmann PJ, BoveyM,et al. The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins. Bacteriol.1996,178(18):5353-5360.
[9]Hoyer LL, Scherer S, Shatzman AR, et al. Candida albicans ALS1:Domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif. Mol Microbiol.1995,15(1):39-54.
[10]Hoyer LL, Payne TL, BellM,et al. Candida albicans ALS3 and insights into the nature of the ALS gene family. Curr Genet.1998,33(6):451-459.
[11]石婧,张宏.白念珠菌菌丝相和酵母相ERG11基因部分序列差异性的探讨.中华皮肤科杂志.2004,37：472-474.
[12]郭宁如,吕桂霞,吴绍熙.念珠菌体外黏附上皮细胞的观察.中国医学科学院学报.1994,16(4)：312-315.
[13]Fratti RA, Belanger PH, SanatiH, et al. The effect of the new triazole, voriconazole(UK2109,496), on the interactions of Candida albicans and Candida kruseiwith endothelial cells. Chemother.1998,10(1):7-16.
[14]RodriguesAG, Mardh PA, Pina V, et al. Germ tube formation changes surface hydrophobicity of Candida cells. Infect Dis Obstet Gynecol.1999,7(5):222-226.
[15]Ghannoum MA. Potential role of phospholipases in virulence and fungal path-ogenesis. Clin Microbiol Rev.2000,13(1):122-143.
[16]Taylor BN, Staib P, Binder A. et al. Profile of Candida albicans secreted-aspartic proteinase elicited during vaginal infection. Infect Immun.2005,73(3): 1828-1835.
[17]Kretschmar M, Hube B, Bertsch T, et al. Germ tubes and proteinase activity contribute to virulence of Candida albicans inmurine peritonitis. Infect Immun. 1999,67(12):6637-6642.
[18]Pukkila-Worley R, Peleg AY, Tampakakis E, et al. Candida albicans hyphal formation and virulence assessed using a Caenorhabditis elegans infection model. Eukaryot Cell.2009,8(11):1750-1758.
[19]Chiani P, Bromuro C, Torosantucci A. Defective induction of inter leukin-12 in human monocytes by germtube forms of Candida albicans. Infect Immun.2000,68(10): 5628-5634.
[20]Torosantucci A, Romagnoli G, Chiani P, et al. Candida albicans yeast and germ tube forms interfere differently with human monocyte differentiation into dendritic cells:a novel dimorphism dependent mechanism to escape the hosts immune response. Infect Immun.2004,72 (2):833-843.
[21]Douglas LJ. Candidal biofilms and their role in infection. Trends Microbiol. 2003,11(1):30-36.
[22]Kumamoto CA, VincesMD. Alternative Candida albicans lifestyle:growth on surfaces. Annu RevMicrobial.2005,59:113-133.
[23]Fridkin SK, Jarvis WR. Epdemiology of nosocomial fungal infections. Clin Microbiol Rev.1996,9(4):499-511.
[24]Kennedy MJ. Models for studying the role of fungal attachment in colonization and pathogenesis. Mycopathologia,1990,109(2):123-137.
[25]Kumar CPG, Menon T. Biofilm production by clinical isolates of Candida species. MedicalMycology.2006,44(1):99-101.
[26]Hornby JM, Jensen EC, Li sec AD, et al. Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl Environ Microbiol.2001,67(7): 2982-2992.
[27]Watts HJ, Very AA, Perera THS, et al. Thigmotropism and stretch activated channels in the pathogenic fungus Candida albicans. Microbiology.1998,144(3): 689-695.
[28]Li F, Palecek SP. EAP1, a Candida albicans gene involved in binding human epithelial cells. Eukaryot Cell.2003,2(6):1266-1273.
[29]Kumamoto CA. Candida biofilms. CurrOp in Microbial.2002,5(6):608-611.
[30]Garcia-Sanchez S, Aubert S, Iraqui I, et al. Candida albicans biofilms:a developmental state associated with specific and stable gene expression patterns. Eukaryot Cell.2004,3(2):536-545.
[31]O'Connor L, Lahiff S, Casey F, et al. Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR usinghybridisation probes on the LightCycler. Mol Cell Probes.2005,19(3):153-162.
[32]Mendes A, Mores AU, Carvalho AP. et al. Candida albicans Biofilms Produce More Secreted Aspartyl. Biol. Pharm. Bull.2007,30(9):1813-1815.
[33]AugstenM, Hubner C, NguyenM, et al. Defective hyphal induction of a Candida albicans phosphatidylinisitol 3-phosphate 5-kinase null mutant on solid media does not lead to decreased virulence. Infect Immun.2002,70(8):4462-4470.
[34]Naglik JR, Challacomber SJ, Hube B. Candida albicans secrete aspartyl prote-inases in virulence and pathogenesis. MicrobiolMol Biol Rev.2003,67(3):400-428.
[35]Al-FattaniMA, DouglasLJ. Penetration of Candida biofilms by antifungal agents. Antimicrob. Agents Chemother.2004,48(9):3291-3297.
[36]Reichart PA, Philip sen HP, SchmidtWesthausen A, et al. Pseudomembranous oral candidiasis in HIV infection:ultrastructure findings. Oral PatholMed.1995,24(6): 276-281.
[37]Ramage G, Bachmann S, Patterson TF, et al. Investigation of multidrug efflux pump s in relation to fluconazole resistance in Candida albicans biofilms. Antim-icrob Chemother.2002,49(6):973-980.
[38]Lamfon H, SR Porter, McCullough, et al. Susceptibility of Candida albicans biofilms grown in a constant depth film fermentor to chorhexidine, fluconazole and miconazole:a longitudinal study. Antimicrob Chemother.2004,53(2):383-385.
[39]Lewis RE, DP Kontoyiannis, RO Darouiche, et al. Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model of Candida catheter related bloodstream infection. Antimicrob Agents Chemother.2002,46(11): 3499-3505.
[40]Kuhn DM. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on biop rosthetic surfaces. Infect immune.2000,70 (2):878-888.
[41]Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev.2002,15(2):167-193.
[42]HoyerLL, Hecht JE. The ALS5 gene of Candida albicans and analysis of the Als5p Nterminal domain. Yeast.2001,18(1):9-60.
[43]Hoyer LL. The ALS gene family of Candida albicans. Trends Microbiol. 2001,9 (4):176-180.
[44]Zhao X, Oh SH, Cheng G, et al. ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesion functional comparisons between Als3p and Als1p. Microbiology.2004,150(7):2415-2428.
[45]Hoyer LL, Payne TL, Hecht JE. Identification of Candida albicans ALS2 and ALS4 and localization of Als proteins to the fungal cell surface. Bacteriol.1998,180 (20):5334-5343.
[46]Hoyer LL, Hecht JE. The ALS6 and ALS7 genes of Candida albicans. Yeast.2000, 16(9):847-855.
[47]Zhang N, Harrex AL, Holland BR, et al. Sixty alleles of the ALS7 open reading frame in Candida albicans:ALS7 is a hypermutable contingency locus. Genome Res. 2003,13(9):2005-2017.
[48]Hoyer LL, Green CB, Oh SH, et al. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family a sticky pursuit. Med Mycol.2008,46(1): 1-15.
[49]Fu Y, Rieg G, FonziWA, et al. Expression of the Candida albicans gene ALS1 in Saccharomyces cerevisiae induces adherence to endothelial and epithelial cells. Infect Immun.1998,66(4):1783-1786.
[50]Loza L, Fu Y, Ibrahim AS, et al. Functional analysis of the Candida a lbicans ALS1 gene product. Yeast.2004,21(6):473-482.
[51]Zhao X, Oh SH, Yeater KM, et al. Analysis of the Candida albicans Als2p and Als4p adhesins suggests the potential for compensatory function within the Als family. Microbiology.2005,151(5):1619-1630.
[52]Oh SH, Cheng G, Nuessen JA, et al. Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain. Microbiology.2005,151 (Pt3):673-681.
[53]Otoo HN, Lee KG, Qiu W, et al. Candida albicans Als adhesins have conserved amyloid-forming sequences. Eukaryot Cell.2008,7(5):776-782.
[54]Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans:development, architecture, and drug resistance. Bacteriol.2001, 183(18):5385-5394.
[55]Green CB, Cheng G, Chandra J, et al. RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms. Microbiology.2004,150(Pt 2):267-275.
[56]Nobile CJ, Schneider HA, Nett JE. Complementary adhesin function in C. albicans biofilm formation Curr Biol.2008,18(14):1017-1024.
[57]Zhao X, Oh S-H, Hoyer LL. Unequal contribution of ALS9 alleles to adhesion between Candida albicans and vascular endotheial cells. Microbiology.2007, 153(Pt7):2342-2350.
[2]Berardinelli S.Opheim DJ. New germ tube induction medium for the identification of Candida albicans. Clin Microbiol.1985,22(5):861-862.
[3]Green CB, Cheng G, Chandra J, et al. RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candi-diasis and in model biofilms. Microbiology.2004,150(Pt 2):267-275.
[4]Kuhn DM, Chandra J, Mukherjee PK, et al. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect Immun.2002, 70(2):878-888.
[5]潘峥,魏昕,刘卫红.改进Trizol法提取白色念珠菌总RNA初探.临床口腔医学杂志,2007,23(9)：531-533.
[6]Qi QG, Hu T, Fu CH, et al. Comparison of ALS gene mRNA between sessile and planktonic Candida albicans of oral cavities. Hua Xi Kou Qiang Yi Xue Za Zhi. 2005,23(3):233-236.
[7]苑天红,王名永,吴升伟等。白念珠菌二相性与毒力关系的实验研究.中国皮肤性病学杂志.200418(10)580-581.
[8]Lo H, Kohler J R, DiDomenico B, et al. Nonfilamentous C. albicans mutants are avirulent. Cell.1997,90(5):939-949.
[9]Kumamoto CA. Candida biofilms. CurrOp in Microbial.2002,5(6):608-611.
[10]Lois L. Hoyer. The ALS gene family of Candida albicans. Trends in Microbiology. 2001,9(4):176-180.
[11]Green CB, Marretta SM, Cheng G, et al. RT-PCR analysis of Candida albicans ALS gene expression in a hyposalivatory rat model of oral candidiasis and in HIV-positive human patients. Med Mycol.2006,44(2):103-111.
[12]Hoyer LL, Green CB, Oh SH, et al. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family-a sticky pursuit. Med Mycol.2008,46(1): 1-15.
[13]O'Connor L, Lahiff S, Casey F, et al. Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler. Mol Cell Probes.2005,19(3):153-162.
[14]Fu Y, Rieg G, Fonzi WA, et al. Expression of the Candida albicans gene ALS1 in Saccharomyces cerevisiae induces adherence to endothelial and epithelial cells. Infect Immun.1998,66(4):1783-1786.
[15]Fu Y, Ibrahim AS, Sheppard DC, et al. Candida albicans Alslp:an adhesin that is a downstream effector of the EFG1 filamentation pathway. Mol Microbiol.2002, 44(1):61-72.
[16]Ibrahim AS, Spellberg BJ, Avenissian V, et al. Vaccination with recombinant N-terminal domain of Alslp improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity. Infect Immun.2005,73(2):999-1005.
[17]Fratti RA, Belanger PH, SanatiH, et al. The effect of the new triazole, voriconazole(UK2109,496), on the interactions of Candida albicans and Candida krus-eiwith endothelial cells. J Chemother.1998,10(1):7-16.
[18]黎斌,王鲁,蒋戈等.白念珠菌孢子相、菌丝相胞壁提取物SDS-PAGE分析中华皮肤科杂志.2003,36(11)：656-657.
[19]Zhao X, Oh SH, Yeater KM, et al. Analysis of the Candida albicans Als2p and Als4p adhesins suggests the potential for compensatory function within the Als family. Microbiology.2005,151(Pt 5):1619-1630.
[20]Nobile CJ, Schneider HA, Nett JE, et al. Complementary adhesion function in C. albicans biofilm formation. Curr Biol.2008,18(14):1017-1024.
[21]Sheppard DC, Yeaman MR, Welch WH, et al. Functional and structural diversity in the Als protein family of Candida albicans. Biol Chem.2004,279(29):30480-30489.
[22]Zhao X, Oh S-H, Hoyer LL. Unequal contribution of ALS9 alleles to adhesion between Candida albicans and vascular endotheial cells.Microbiology.2007,153(Pt 7):2342-2350.
[23]Watnick P, Kolter R. Biofilm, city of microbes. Bacteriol.2000,182(10):2675-2679.
[24]Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida albicans biofilms. Med Microbiol.1999,48(7):671-679.
[25]Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal Pathogen Candida albicans:development, architecture, and drug resistance. Baciteriol.2001, 183(18):5385-5394.
[26]Nail is H, Kucharikova S, Ricicova M, et al. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms:identif-ication of model-dependent and-independent gene expression. BMC Microbiol.2010, 10(1):114.
[1]Haynes K. Virulence in Candida species. Trends Microbiol.2001,9(12):591-596.
[2]秦启贤.临床真菌学[M].上海：复旦大学出版社,2001,58-59.
[3]Redding S, Bhatt B, Rawls HR, et al. Inhibition of Candida albicans biofilm formation on denture material. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009,107(5):669-672.
[4]Brown AJ, Gow NA. Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol.1999,7(8):333-338.
[5]Sommeborn A, Bockmubh DP, GeradsM et al. Protein kinaseA encoded by TPK2 re-gulated dimorphism of Candida albicans. MolMicrobiol.2000,35(2):386-396.
[6]Lo H, Kohler JR, DiDomenico B, et al. Nonfilamentous C. albicans mutants are avirulent. Cell.1997,90(5):939-949.
[7]Pendrak ML, Roberts DD. Hemoglobin is an effective inducer of hyphal diffe-rentiation in Candida albicans. Med Mycol.2007,45(1):61-71.
[8]Bailey DA, Feldmann PJ, BoveyM,et al. The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins. Bacteriol.1996,178(18):5353-5360.
[9]Hoyer LL, Scherer S, Shatzman AR, et al. Candida albicans ALS1:Domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif. Mol Microbiol.1995,15(1):39-54.
[10]Hoyer LL, Payne TL, BellM,et al. Candida albicans ALS3 and insights into the nature of the ALS gene family. Curr Genet.1998,33(6):451-459.
[11]石婧,张宏.白念珠菌菌丝相和酵母相ERG11基因部分序列差异性的探讨.中华皮肤科杂志.2004,37：472-474.
[12]郭宁如,吕桂霞,吴绍熙.念珠菌体外黏附上皮细胞的观察.中国医学科学院学报.1994,16(4)：312-315.
[13]Fratti RA, Belanger PH, SanatiH, et al. The effect of the new triazole, voriconazole(UK2109,496), on the interactions of Candida albicans and Candida kruseiwith endothelial cells. Chemother.1998,10(1):7-16.
[14]RodriguesAG, Mardh PA, Pina V, et al. Germ tube formation changes surface hydrophobicity of Candida cells. Infect Dis Obstet Gynecol.1999,7(5):222-226.
[15]Ghannoum MA. Potential role of phospholipases in virulence and fungal path-ogenesis. Clin Microbiol Rev.2000,13(1):122-143.
[16]Taylor BN, Staib P, Binder A. et al. Profile of Candida albicans secreted-aspartic proteinase elicited during vaginal infection. Infect Immun.2005,73(3): 1828-1835.
[17]Kretschmar M, Hube B, Bertsch T, et al. Germ tubes and proteinase activity contribute to virulence of Candida albicans inmurine peritonitis. Infect Immun. 1999,67(12):6637-6642.
[18]Pukkila-Worley R, Peleg AY, Tampakakis E, et al. Candida albicans hyphal formation and virulence assessed using a Caenorhabditis elegans infection model. Eukaryot Cell.2009,8(11):1750-1758.
[19]Chiani P, Bromuro C, Torosantucci A. Defective induction of inter leukin-12 in human monocytes by germtube forms of Candida albicans. Infect Immun.2000,68(10): 5628-5634.
[20]Torosantucci A, Romagnoli G, Chiani P, et al. Candida albicans yeast and germ tube forms interfere differently with human monocyte differentiation into dendritic cells:a novel dimorphism dependent mechanism to escape the hosts immune response. Infect Immun.2004,72 (2):833-843.
[21]Douglas LJ. Candidal biofilms and their role in infection. Trends Microbiol. 2003,11(1):30-36.
[22]Kumamoto CA, VincesMD. Alternative Candida albicans lifestyle:growth on surfaces. Annu RevMicrobial.2005,59:113-133.
[23]Fridkin SK, Jarvis WR. Epdemiology of nosocomial fungal infections. Clin Microbiol Rev.1996,9(4):499-511.
[24]Kennedy MJ. Models for studying the role of fungal attachment in colonization and pathogenesis. Mycopathologia,1990,109(2):123-137.
[25]Kumar CPG, Menon T. Biofilm production by clinical isolates of Candida species. MedicalMycology.2006,44(1):99-101.
[26]Hornby JM, Jensen EC, Li sec AD, et al. Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl Environ Microbiol.2001,67(7): 2982-2992.
[27]Watts HJ, Very AA, Perera THS, et al. Thigmotropism and stretch activated channels in the pathogenic fungus Candida albicans. Microbiology.1998,144(3): 689-695.
[28]Li F, Palecek SP. EAP1, a Candida albicans gene involved in binding human epithelial cells. Eukaryot Cell.2003,2(6):1266-1273.
[29]Kumamoto CA. Candida biofilms. CurrOp in Microbial.2002,5(6):608-611.
[30]Garcia-Sanchez S, Aubert S, Iraqui I, et al. Candida albicans biofilms:a developmental state associated with specific and stable gene expression patterns. Eukaryot Cell.2004,3(2):536-545.
[31]O'Connor L, Lahiff S, Casey F, et al. Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR usinghybridisation probes on the LightCycler. Mol Cell Probes.2005,19(3):153-162.
[32]Mendes A, Mores AU, Carvalho AP. et al. Candida albicans Biofilms Produce More Secreted Aspartyl. Biol. Pharm. Bull.2007,30(9):1813-1815.
[33]AugstenM, Hubner C, NguyenM, et al. Defective hyphal induction of a Candida albicans phosphatidylinisitol 3-phosphate 5-kinase null mutant on solid media does not lead to decreased virulence. Infect Immun.2002,70(8):4462-4470.
[34]Naglik JR, Challacomber SJ, Hube B. Candida albicans secrete aspartyl prote-inases in virulence and pathogenesis. MicrobiolMol Biol Rev.2003,67(3):400-428.
[35]Al-FattaniMA, DouglasLJ. Penetration of Candida biofilms by antifungal agents. Antimicrob. Agents Chemother.2004,48(9):3291-3297.
[36]Reichart PA, Philip sen HP, SchmidtWesthausen A, et al. Pseudomembranous oral candidiasis in HIV infection:ultrastructure findings. Oral PatholMed.1995,24(6): 276-281.
[37]Ramage G, Bachmann S, Patterson TF, et al. Investigation of multidrug efflux pump s in relation to fluconazole resistance in Candida albicans biofilms. Antim-icrob Chemother.2002,49(6):973-980.
[38]Lamfon H, SR Porter, McCullough, et al. Susceptibility of Candida albicans biofilms grown in a constant depth film fermentor to chorhexidine, fluconazole and miconazole:a longitudinal study. Antimicrob Chemother.2004,53(2):383-385.
[39]Lewis RE, DP Kontoyiannis, RO Darouiche, et al. Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model of Candida catheter related bloodstream infection. Antimicrob Agents Chemother.2002,46(11): 3499-3505.
[40]Kuhn DM. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on biop rosthetic surfaces. Infect immune.2000,70 (2):878-888.
[41]Donlan RM, Costerton JW. Biofilms:survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev.2002,15(2):167-193.
[42]HoyerLL, Hecht JE. The ALS5 gene of Candida albicans and analysis of the Als5p Nterminal domain. Yeast.2001,18(1):9-60.
[43]Hoyer LL. The ALS gene family of Candida albicans. Trends Microbiol. 2001,9 (4):176-180.
[44]Zhao X, Oh SH, Cheng G, et al. ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesion functional comparisons between Als3p and Als1p. Microbiology.2004,150(7):2415-2428.
[45]Hoyer LL, Payne TL, Hecht JE. Identification of Candida albicans ALS2 and ALS4 and localization of Als proteins to the fungal cell surface. Bacteriol.1998,180 (20):5334-5343.
[46]Hoyer LL, Hecht JE. The ALS6 and ALS7 genes of Candida albicans. Yeast.2000, 16(9):847-855.
[47]Zhang N, Harrex AL, Holland BR, et al. Sixty alleles of the ALS7 open reading frame in Candida albicans:ALS7 is a hypermutable contingency locus. Genome Res. 2003,13(9):2005-2017.
[48]Hoyer LL, Green CB, Oh SH, et al. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family a sticky pursuit. Med Mycol.2008,46(1): 1-15.
[49]Fu Y, Rieg G, FonziWA, et al. Expression of the Candida albicans gene ALS1 in Saccharomyces cerevisiae induces adherence to endothelial and epithelial cells. Infect Immun.1998,66(4):1783-1786.
[50]Loza L, Fu Y, Ibrahim AS, et al. Functional analysis of the Candida a lbicans ALS1 gene product. Yeast.2004,21(6):473-482.
[51]Zhao X, Oh SH, Yeater KM, et al. Analysis of the Candida albicans Als2p and Als4p adhesins suggests the potential for compensatory function within the Als family. Microbiology.2005,151(5):1619-1630.
[52]Oh SH, Cheng G, Nuessen JA, et al. Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain. Microbiology.2005,151 (Pt3):673-681.
[53]Otoo HN, Lee KG, Qiu W, et al. Candida albicans Als adhesins have conserved amyloid-forming sequences. Eukaryot Cell.2008,7(5):776-782.
[54]Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans:development, architecture, and drug resistance. Bacteriol.2001, 183(18):5385-5394.
[55]Green CB, Cheng G, Chandra J, et al. RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms. Microbiology.2004,150(Pt 2):267-275.
[56]Nobile CJ, Schneider HA, Nett JE. Complementary adhesin function in C. albicans biofilm formation Curr Biol.2008,18(14):1017-1024.
[57]Zhao X, Oh S-H, Hoyer LL. Unequal contribution of ALS9 alleles to adhesion between Candida albicans and vascular endotheial cells. Microbiology.2007, 153(Pt7):2342-2350.