摘要
目的:白念珠菌所致深部感染的发病率明显增高,严重病例难以治疗,死亡率高,部分病例与白念珠菌在体内易形成生物被膜有关。本研究主要对AAF1基因在形成生物被膜中的作用和可能机制进行探讨。
方法:首先使用基因拼接的方法构建质粒pHS1-U3du3-HS2和自主表达AAF1基因的质粒pHS1-GAAU3-HS2。以CAI4为参考菌株(AAF1/AAF1),采用“Ura-blaster”技术,使用质粒pHS1-U3du3-HS2转化结合5-氟乳清酸(5-FOA)压力筛选,将AAF1基因从基因组完全剔除,然后再转入自主表达质粒pHS1-GAAU3-HS2,构建自主表达菌株。通过表型鉴定和不同引物进行PCR扩增鉴定,构建出实验所需的重组菌株。使用体外形成生物被膜模型和动物模型,通过倒置显微镜和扫描电镜比较AAF1基因缺失菌株、AAF1自主表达菌株和参考菌株在形成生物被膜时的形态学差异。最后使用实时定量逆转录PCR方法检测AAF1基因缺失菌株和AAF1基因自主表达菌株两者的HWP1基因和ALS3基因表达水平,并对差异进行比较。
结果:通过基因拼接成功构建了质粒pHS1-U3du3-HS2和pHS1-GAAU3-HS2。成功地将AAF1基因从基因组剔除,构建出菌株M2-2(△aaf1/△aaf1),构建出自主表达AAF1基因的菌株M3(△aaf1/△aaf1+pGAP-AAF1),并通过免疫印迹鉴定。形态学观察不同菌株体外形成的白念珠菌生物被膜发现:菌株M2-2(△aaf1/△aaf1)仅呈稀疏粘附,而菌株M3(△aaf1/△aaf1+pGAP-AAF1)和参考菌株CAI4(AAF1/AAF1)均能形成典型的生物被膜。在白念珠菌形成生物被膜的动物模型上,扫描电镜下显示M2-2(△aaf1/△aaf1)难以形成生物被膜,而M3(△aaf1/△aaf1+pGAP-AAF1)和参考菌株CAI4均形成成熟的生物被膜。实时定量逆转录PCR检测发现:在ALS3基因的表达水平上,M3是M2-2的15.63492倍;在HWP1基因的表达水平上,M3是M2-2的8.817685倍。
结论:利用构建出的质粒pHS1-U3du3-HS2和自主表达AAF1基因的质粒pHS1-GAAU3-HS2,成功地构建出AAF1基因完全剔除菌株M2-2(△aaf1/△aaf1)和自主表达AAF1基因的菌株M3(△aaf1/△aaf1+pGAP-AAF1);体内外的实验表明AAF1基因在白念珠菌形成生物被膜过程中起着重要的作用;这种作用可能是通过细胞壁蛋白Als3和Hwp1来实现的。
Objectives: Infection rates caused by Candida albicans have been incraesingwith exceptionally high mortality. Biofilm formation is a major virulence factor inthe pathogenicity of Candida albcans, and they are difficult to eradicate especiallybecause of their very high antifungal resistance. Although there were some studieshave shed some light, molecular mechanisms that govern biofilm formation andpathogenicity still await full clarification. In this paper the role of AAF1 in biofilmformation will be elplored.
Methods: Firstly, standard in vitro gene splicing techniques were utilized toconstruct plasmid pHS1-U3du3-HS2 uesd in gene disruption strategy asmini-Ura-blaster cassette and plasmid pHS1-GAAU3-HS2 used to overexpressAafl. Using mini-Ura-blaster technology, the△aafl/△aafl homozygous deletionmutant strain M2-2(△aafl/△aafl) was made. Then the strainM3(△aafl/△aafl+pGAP-AAF1) overexpressing Aafl was obtained bytransformed M2-2 with plasmid pHS1-GAAU3-HS2. All Strains were confirmedby PCR using different primers. And Western blot was applied to detect Aaflprotein. Biofilm formation in vitro and in vito by strain CAI4, M2-2 and M3 wereobserved by invert microscope and scanning electron microscope. The reversetranscription and quantitative polymerase chain reaction (qRT-PCR) analysis wasused to compare mRNA abundances of the HWP1 gene and ALS3 gene betweenstrain M3 and strain M2-2 at 48h ofbiofilm development in vitro.
Results: Plasmid pHS1-U3du3-HS2 and plasmid pHS1-GAAU3-HS2constructions were made successivly. The AAF1 gene was disrupted completely and△aafl/△aafl deletion mutant strain M2-2 was made. The complemented strain M3(containing AAF1 ORF) was constructed by transforming M2-2 (△aafl/△aafl)with plasmid pHS1-GAAU3-HS2. And the Aafl protein was confirmed by western blot. The microscope imaging revealed that△aafl/△aafl mutant (strain M2-2)displayed a severe defect in biofilm formation compared to the reference Stain(CAI4), and introduction of a single wild-type AAF1 allele (strain M3) rescued thedefect substantially in vitro biofilm formation model. Biofilm formatiom wasvisualized after 48h by scanning electron microscopy of the intealuminal cathetersurface taken from a rat venous. The wild-type CAI4 and△aafl/△aafl+pGAP-AAF1 complemented strains M3 yielded extensive adherent populations andabundant matrix material. In constrast, the△aafl/△aafl mutant yielded fewadherent cells and was devoid of biofilmmaterial. At 48h of biofilm development invitro,Total RNA was extrated and was measure by qRT-PCR analysis. The foldincrease in mRNA for ALS3 was 15.63492 in△aafl/△aafl+pGAP-AAF1complemented strains M3 as compared with△aafl/△aafl mutant strains M2-2.HWP1 was expressed at 8.817685-fold-higher levels in the complemented strainsM3 (△aafl/△aafl+pGAP-AAF1)than in the△aafl/△aafl mutant strains M2-2.
Conclusions: The constructions of plasmid pHS1-U3du3-HS2,pHS1-GAAU3-HS2 and strains△aafl/△aafl mutant,△aafl/△aafl+pGAP-AAF1 complemented were made correctly.This study demonstrates that therole of AAF1 was critical in Candida albicans biofilm formation in vitro and in vito.And the mechamism may be Als3-mediated and Hwpl-mediated adhered.
引文
1 Bouza E, Munoz P. Epidemiology of candidemia in intensive care units. Int J Antimicrob Agents, 2008,32 Suppl 2:S87-91.
2 Procianoy RS, Silveira RC. Epidemiology of candidemia. J Clin Microbiol,2008,46:1894; author reply 1894.
3 Martin GS, Mannino DM, Eaton S, et al. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med, 2003,348:1546-54.
4 骆俊 吴菊芳 朱德妹 李光辉 张婴元 汪复.上海市华山医院血流感染患者的病原学和临床研究.中华传染病杂志,2006,24.(1):29-34.
5 Guery BP, Arendrup MC, Auzinger G, et al. Management of invasive candidiasis and candidemia in adult non-neutropenic intensive care unit patients: Part Ⅰ. Epidemiology and diagnosis. Intensive Care Med,2009,35:55-62.
6 Hajjeh RA, Sofair AN, Harrison LH, et al. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol, 2004,42:1519-27.
7 Anunnatsiri S, Chetchotisakd P, Mootsikapun P. Fungemia in non-HIV-infected patients: a five-year review. Int J Infect Dis, 2009,13:90-6.
8 Bassetti M, Righi E, Costa A, et al. Epidemiological trends in nosocomial candidemia in intensive care. BMC Infect Dis, 2006,6:21.
9 Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother,2001,45:999-1007.
10 Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol, 2001,183:5385-94.
11 Andes D, Nett J, Oschel P, et al. Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect Immun,2004,72:6023-31.
12 Schinabeck MK, Long LA, Hossain MA, et al. Rabbit model of Candida albicans biofilm infection: liposomal amphotericin B antifungal lock therapy.Antimicrob Agents Chemother, 2004,48:1727-32.
13 Sbarbati A, Fanos V, Bernardi P, et al. Rapid diagnosis of fungal infection of intravascular catheters in newborns by scanning electron microscopy.Scanning, 2001,23:376-8.
14 Chandra J, Mukherjee PK, Leidich SD, et al. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res, 2001,80:903-8.
15 孙秋宁,方凯.念珠菌生物膜的结构、影响因素及其对抗真菌药的敏感性.中国医学科学院学报,2002,24:385-387.
16 d'Enfert C. Biofilms and their role in the resistance of pathogenic Candida to antifungal agents. Curr Drug Targets, 2006,7:465-70.
17 Jabra-Rizk MA, Falkler WA, Meiiler TF. Fungal biofilms and drug resistance.Emerg Infect Dis, 2004,10:14-9.
18 Al-Fattani MA, Douglas LJ. Penetration of Candida biofilms by antifungal agents. Antimicrob Agents Chemother, 2004,48:3291-7.
19 Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev, 2002,15:167-93.
20 Baillie GS, Douglas LJ. Effect of growth rate on resistance of Candida albicans biofilms to antifungal agents. Antimicrob Agents Chemother, 1998,42:1900-5.
21 Mukherjee PK, Chandra J, Kuhn DM, et al. Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of effiux pumps and membrane sterols. Infect Immun, 2003,71:4333-40.
22 Khot PD, Suci PA, Miller RL, et al. A small subpopulation of blastospores in candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and beta-1,6-glucan pathway genes.Antimicrob Agents Chemother, 2006,50:3708-16.
23 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, 2004,3:536-45.
24 Thomas DP, Bachmann SP, Lopez-Ribot JL. Proteomics for the analysis of the Candida albicans biofilm lifestyle. Proteomics, 2006,6:5795-804.
25 Yeater KM, Chandra J, Cheng G, et al. Temporal analysis of Candida albicans gene expression during biofilm development. Microbiology,2007,153:2373-85.
26 Nobile CJ, Schneider HA, Nett JE, et al. Complementary adhesin function in C. albicans biofilm formation. Curr Biol, 2008,18:1017-24.
27 Zhao X, Daniels KJ, Oh SH, et al. Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces. Microbiology,2006,152:2287-99.
28 Nobile CJ, Andes DR, Nett JE, et al. Critical role of Bcrl-dependent adhesinsin C. albicans biofilm formation in vitro and in vivo. PLoS Pathog, 2006,2:e63.
29 Nobile C J, Nett JE, Andes DR, et al. Function of Candida albicans adhesinHwp1 in biofilm formation. Eukaryot Cell, 2006,5:1604-10.
30 Fu Y, Filler SG, Spellberg BJ, et al. Cloning and characterization of CAD1/AAF1, a gene from Candida albicans that induces adherence to endothelial cells after expression in Saccharomyces cerevisiae. Infect Immun,1998,66:2078-84.
31 Li F, Palecek SP. Identification of Candida albicans genes that induce Saccharomyces cerevisiae cell adhesion and morphogenesis. Biotechnol Prog,2005,21:1601-9.
32 Magee PT, Gale C, Berman J, et al. Molecular genetic and genomic approaches to the study of medically important fungi. Infect Immun,2003,71:2299-309.
33 De Backer MD, Magee PT, Pla J. Recent developments in molecular genetics of Candida albicans. Annu Rev Microbiol, 2000,54:463-98.
34 Kumamoto CA. Candida biofilms. Curr Opin Microbiol, 2002,5:608-11.
35 Mukherjee PK, Chandra J. Candida biofilm resistance. Drug Resist Updat,2004,7:301-9.
36 AAF1. http://www.candidagenome.org/cgi-bin/locus.pl?locus=aaf1.Candida Genome Database.
37 PGAPZA-map. http://tools.invitrogen.com/content/sfs/vectors/pgapz_map.pdf.Invitrogen Corporation.
38 URA3. http://www.candidagenome.org/cgi-bin/locus.pl?locus=URA3.Candida Genome Database.
39 卢圣栋.现代分子生物学实验技术(第二版).北京:中国协和医科大学出版社.1999.
40 PGAPZA-seq. http://tools.invitrogen.com/content/sfs/vectors/pgapza_seq.txt.Invitrogen Corporation.
41 Walther A, Wendland J. An improved transformation protocol for the human fungal pathogen Candida albicans. Curr Genet, 2003,42:339-43.
42 Silar P, Thiele DJ. New shuttle vectors for direct cloning in Saccharomyces cerevisiae. Gene, 1991,104:99-102.
43 J.萨姆布鲁克,E.F.费里奇,T.曼尼阿蒂斯,等著.金冬雁等译.分子克隆实验指南(第二版).北京:科学技术出版社.1999.
44 汪家政,范明主编.蛋白质技术手册.北京:科学出版社.2000.
45 Dan Burke,Dean Dawson, Tim Stearns. Methods in Yeast Genetics : A Cold Spring Harbor Laboratory Course Manual (2000 Edition). the Cold Spring Harbor Laboratory Press.
46 Douglas LJ. Candida biofilms and their role in infection. Trends Microbiol,2003,11:30-6.
47 Ramage G, Wickes BL, Lopez-Ribot JL. Biofilms of Candida albicans and their associated resistance to antifungal agents. Am Clin Lab, 2001,20:42-4.
48 Pla J, Gil C, Monteoliva L, et al. Understanding Candida albicans at the molecular level. Yeast, 1996,12:1677-702.
49 Goshorn AK, Scherer S. Genetic analysis of prototrophic natural variants of Candida albicans. Genetics, 1989,123:667-73.
50 Wilson RB, Davis D, Mitchell AP. Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol,1999,181:1868-74.
51 Lott TJ, Magee PT, Barton R, et al. The molecular genetics of Candida albicans. J Med Vet Mycol, 1992,30 Suppl 1:77-85.
52 Magee PT, Rikkerink EH, Magee BB. Methods for the genetics and molecular biology of Candida albicans. Anal Biochem, 1988,175:361-72.
53 Alani E, Kleckner N. A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast. Genetics,1987,117:5-12.
54 Brown AJ, Cormack BP, Gow NA, et al. Advances in molecular genetics of Candida albicans and Candida glabrata. Med Mycol, 1998,36 Suppl 1:230-7.
55 Fonzi WA, Irwin MY. Isogenic strain construction and gene mapping in Candida albicans. Genetics, 1993,134:717-28.
56 Wilson RB, Davis D, Enloe BM, et ai. A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast, 2000,16:65-70.
57 Du W, Coaker M, Sobel JD, et al. Shuttle vectors for Candida albicans: control of plasmid copy number and elevated expression of cloned genes. Curr Genet,2004,45:390-8.
58 Kurtz MB, Cortelyou MW, Miller SM, et al. Development of autonomously replicating plasmids for Candida albicans. Mol Cell Biol, 1987,7:209-17.
59 pGAPZA, pichia expression vectors for constitutive expression and purification of recombinant proteins.http://tools.invitrogen.com/content/sfs/manuals/pgapz_man.pdf.Invitrogen Corporation.
60 Waterham HR, Digan ME, Koutz PJ, et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene, 1997,186:37-44.
61 Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res, 1987,15:8125-48.
62 Kozak M. Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci U S A, 1990,87:8301-5.
63 Baillie GS, Douglas LJ. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol, 1999,310:644-56.
64 孙秋宁,Gordon Ramage,Jose L,Lopez-Ribot.念珠菌生物膜体内外抗真菌药物敏感性研究.中华皮肤科杂志,2002,35:409-410.
65 Kojic EM, Darouiche RO. Candida infections of medical devices. Clin Microbiol Rev, 2004,17:255-67.
66 Bertagnolio S, de Gaetano Donati K, Tacconelli E, et al. Hospital-acquired candidemia in HIV-infected patients. Incidence, risk factors and predictors of outcome. J Chemother, 2004,16:172-8.
67 Everaert EP, Mahieu HF, Wong Chung RP, et al. A new method for in vivo evaluation of biofilms on surface-modified silicone rubber voice prostheses.Eur Arch Otorhinolaryngol, 1997,254:261-3.
68 Chandra J, McCormick TS, Imamura Y, et al. Interaction of Candida albicans with adherent human peripheral blood mononuclear cells increases C. albicans biofilm formation and results in differential expression of pro- and anti-inflammatory cytokines. Infect Immun, 2007,75:2612-20.
69 Nikawa H, Mikihira S, Egusa H, et al. [Candida adherence and biofilm formation on oral surfaces]. Nippon Ishinkin Gakkai Zasshi, 2005,46:233-42.
70 徐柏森,杨静.实用电镜技术.南京:东南大学出版社,2008.
71 ACT1.http://www.candidagenome.org/cgi-bin/locus.pl?locus=act1. Candida Genome Database.
72 ALS3. http://www.candidagenome.org/cgi-bin/locus.pl?dbid=CAL0001605.Candida Genome Database.
73 HWP1. http://www.candidagenome.org/cgi-bin/locus.pl?locus=HWP1.Candida Genome Database.
74 Huggett J, Dheda K, Bustin S, et al. Real-time RT-PCR normalisation;strategies and considerations. Genes Immun, 2005,6:279-84.
75 Bernd Kochanowski, Udo Reischl. Quantitative PCR Protocols. Humana Press Totowa, New Jersey, 1999.
76 Nailis H, Coenye T, Van Nieuwerburgh F, et al. Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR. BMC Mol Biol, 2006,7:25.
77 Ramage G, Saville SP, Thomas DP, et al. Candida biofilms: an update.Eukaryot Cell, 2005,4:633-8.
78 Nobile C J, Mitchell AP. Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcrlp. Curr Biol,2005,15:1150-5.
79 Holmes AR, Cannon RD, Jenkinson HF. Interactions of Candida albicans with bacteria and salivary molecules in oral biofilms. J Ind Microbiol,1995,15:208-13.
80 Argimon S, Wishart JA, Leng R, et al. Developmental regulation of an adhesin gene during cellular morphogenesis in the fungal pathogen Candida albicans.Eukaryot Cell, 2007,6:682-92. Bassetti M, Righi E, Costa A, et al. Epidemiological trends in nosocomial candidemia in intensive care. BMC Infect Dis, 2006,6:21.
2 Hajjeh RA, Sofair AN, Harrison LH, et al. Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol, 2004,42(4): 1519-27.
3 Procianoy RS, Silveira RC. Epidemiology of candidemia. J Clin Microbiol, 2008,46(5): 1894; author reply 1894.
4 Anunnatsiri S, Chetchotisakd P, Mootsikapun P. Fungemia in non-HIV-infected patients: a five-year review. Int J Infect Dis, 2009,13(1):90-6.
5 Sbarbati A, Fanos V, Bernardi P, et al. Rapid diagnosis of fungal infection of intravascular catheters in newborns by scanning electron microscopy. Scanning, 2001,23(6):376-8.
6 Kumar CP, Menon T. Biofilm production by clinical isolates of Candida species. Med Mycol, 2006,44(1):99-l01.
7 Chandra J, Mukherjee PK, Leidich SD, et al. Antifungal resistance of Candidal bio films formed on denture acrylic in vitro. J Dent Res, 2001,80(3):903-8.
8 Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev, 2002,15(2): 167-93.
9 Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol, 2001,183( 18):5385-94.
10 Andes D, Nett J, Oschel P, et al. Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect Immun, 2004,72(10):6023-31.
11 Schinabeck MK, Long LA, Hossain MA, et al. Rabbit model of Candida albicans biofilm infection: liposomal amphotericin B antifungal lock therapy. Antimicrob Agents Chemother, 2004,48(5): 1727-32.
12 Ramage G, Wickes BL, Lopez-Ribot JL. A seed and feed model for the formation of Candida albicans biofilms under flow conditions using an improved modified Robbins device. Rev Iberoam Micol, 2008,25(1):37-40.
13 Hawser SP, Baillie GS, Douglas LJ. Production of extracellular matrix by Candida albicans biofilms. J Med Microbiol, 1998,47(3):253-6.
14 Baillie GS, Douglas LJ. Role of dimorphism in the development of Candida albicans biofilms. J Med Microbiol, 1999,48(7):671-9.
15 Yeater KM, Chandra J, Cheng G, et al. Temporal analysis of Candida albicans gene expression during biofilm development. Microbiology, 2007,153(Pt 8):2373-85.
16 Thomas DP, Bachmann SP, Lopez-Ribot JL. Proteomics for the analysis of the Candida albicans biofilm lifestyle. Proteomics, 2006,6(21):5795-804.
17 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):536-45.
18 Hawser SP, Douglas LJ. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect Immun, 1994,62(3):915-21.
19 Nobile CJ, Schneider HA, Nett JE, et al. Complementary adhesin fiinction in C. albicans biofilm formation. Curr Biol, 2008,18(14): 1017-24.
20 de Groot PW, de Boer AD, Cunningham J, et al. Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot Cell, 2004,3(4):955-65.
21 Granger BL, Flenniken ML, Davis DA, et al. Yeast wall protein 1 of Candida albicans. Microbiology, 2005,151(Pt 5):1631-44.
22 Zhao X, Daniels KJ, Oh SH, et al. Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces. Microbiology, 2006,152(Pt 8):2287-99.
23 Nobile CJ, Nett JE, Andes DR, et al. Function of Candida albicans adhesin Hwpl in biofilm formation. Eukaryot Cell, 2006,5(10):1604-10.
24 Li F, Palecek SP. EAP1, a Candida albicans gene involved in binding human epithelial cells. Eukaryot Cell, 2003,2(6):1266-73.
25 Singleton DR, Masuoka J, Hazen KC. Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity. J Bacteriol, 2001,183(12):3582-8.
26 Cao YY, Cao YB, Xu Z, et al. cDNA microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol. Antimicrob Agents Chemother, 2005,49(2):584-9.
27 Lamarre C, Deslauriers N, Bourbonnais Y. Expression cloning of the Candida albicans CSA1 gene encoding a mycelial surface antigen by sorting of Saccharomyces cerevisiae transformants with monoclonal antibody-coated magnetic beads. Mol Microbiol, 2000,35(2):444-53.
28 Hogan DA. Talking to themselves: autoregulation and quorum sensing in fungi. Eukaryot Cell, 2006,5(4):613-9.
29 Alem MA, Oteef MD, Flowers TH, et al. Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development. Eukaryot Cell, 2006,5(10): 1770-9.
30 Kruppa M, Krom BP, Chauhan N, et al. The two-component signal transduction protein Chklp regulates quorum sensing in Candida albicans. Eukaryot Cell, 2004,3(4): 1062-5.
31 Monge RA, Roman E, Nombela C, et al. The MAP kinase signal transduction network in Candida albicans. Microbiology, 2006,152(Pt 4):905-12.
32 Kumamoto CA. A contact-activated kinase signals Candida albicans invasive growth and biofilm development. Proc Natl Acad Sci USA,2005,102( 15):5576-81.
33 Lo HJ, Kohler JR, DiDomenico B, et al. Nonfilamentous C. albicans mutants are avirulent. Cell, 1997,90(5):939-49.
34 Nobile CJ, Mitchell AP. Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcrlp. Curr Biol, 2005,15(12):1 150-5.
35 Fu Y, Filler SG, Spellberg BJ, et al. Cloning and characterization of CAD1/AAF1, a gene from Candida albicans that induces adherence to endothelial cells after expression in Saccharomyces cerevisiae. Infect Immun, 1998,66(5):2078-84.
36 Barki M, Koltin Y, Yanko M, et al. Isolation of a Candida albicans DNA sequence conferring adhesion and aggregation on Saccharomyces cerevisiae. J Bacteriol, 1993,175(17):5683-9.
37 Richard ML, Nobile CJ, Bruno VM, et al. Candida albicans biofilm-defective mutants. Eukaryot Cell, 2005,4(8): 1493-502.
38 Alem MA, Douglas LJ. Prostaglandin production during growth of Candida albicans biofilms. J Med Microbiol, 2005,54(Pt 11):1001-5.
39 Alem MA, Douglas LJ. Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans. Antimicrob Agents Chemother, 2004,48(1):41-7.
40 Dhillon NK, Sharma S, Khuller GK. Signaling through protein kinases and transcriptional regulators in Candida albicans. Crit Rev Microbiol, 2003,29(3):259-75.
41 Mateus C, Crow SA Jr, Ahearn DG. Adherence of Candida albicans to silicone induces immediate enhanced tolerance to fluconazole. Antimicrob Agents Chemother, 2004,48(9):3358-66.
42 Mukherjee PK, Chandra J, Kuhn DM, et al. Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols. Infect Immun, 2003,71(8):4333-40.
43 Baillie GS, Douglas LJ. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemother, 2000,46(3):397-403.
44 Al-Fattani MA, Douglas LJ. Penetration of Candida biofilms by antifungal agents. Antimicrob Agents Chemother, 2004,48(9):3291-7.
45 Ramage G, Bachmann S, Patterson TF, et al. Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother, 2002,49(6):973-80.
46 Baillie GS, Douglas LJ. Effect of growth rate on resistance of Candida albicans biofilms to antifungal agents. Antimicrob Agents Chemother, 1998,42(8): 1900-5.
47 Ramage G, VandeWalle K, Lopez-Ribot JL, et al. The filamentation pathway controlled by the Efgl regulator protein is required for normal biofilm formation and development in Candida albicans. FEMS Microbiol Lett, 2002,214(1):95-100.
48 Lupetti A, Danesi R, Campa M, et al. Molecular basis of resistance to azole antifungals. Trends Mol Med, 2002,8(2):76-81.
49 Nolte FS, Parkinson T, Falconer DJ, et al. Isolation and characterization of fluconazole- and amphotericin B-resistant Candida albicans from blood of two patients with leukemia. Antimicrob Agents Chemother, 1997,41 (1): 196-9.
50 Miyazaki Y, Geber A, Miyazaki H, et al. Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans. Gene, 1999,236(1):43-51.
51 Kelly SL, Lamb DC, Taylor M, et al. Resistance to amphotericin B associated with defective sterol delta 8-->7 isomerase in a Cryptococcus neoformans strain from an AIDS patient. FEMS Microbiol Lett, 1994,122(1-2):39-42.
52 Khot PD, Suci PA, Miller RL, et al. A small subpopulation of blastospores in Candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and beta-1,6-glucan pathway genes. Antimicrob Agents Chemother, 2006,50(11):3708-16.
53 Shuford JA, Rouse MS, Piper KE, et al. Evaluation of caspofungin and amphotericin B deoxycholate against Candida albicans biofilms in an experimental intravascular catheter infection model. J Infect Dis, 2006,194(5):710-3.
54 Ramage G, VandeWalle K, Bachmann SP, et al. In vitro pharmacodynamic properties of three antifungal agents against preformed Candida albicans biofilms determined by time-kill studies. Antimicrob Agents Chemother, 2002,46(11):3634-6.
55 Soustre J, Rodier MH, Imbert-Bouyer S, et al. Caspofungin modulates in vitro adherence of Candida albicans to plastic coated with extracellular matrix proteins. J Antimicrob Chemother, 2004,53(3):522-5.
56 Bachmann SP, VandeWalle K, Ramage G, et al. In vitro activity of caspofungin against Candida albicans biofilms. Antimicrob Agents Chemother, 2002,46(11):3591-6.
57 Kuhn DM, George T, Chandra J, et al. Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother, 2002,46(6): 1773-80.
58 Mukherjee PK, Long L, Kim HG, et al. Amphotericin B lipid complex is efficacious in the treatment of Candida albicans biofilms using a model of catheter-associated Candida biofilms. Int J Antimicrob Agents, 2009,33(2): 149-53.
59 Enjalbert B, Whiteway M. Release from quorum-sensing molecules triggers hyphal formation during Candida albicans resumption of growth. Eukaryot Cell, 2005,4(7):1203-10.
60 Tsukamoto T, Matsukawa M, Sano M, et al. Biofilm in complicated urinary tract infection. Int J Antimicrob Agents, 1999,11(3-4):233-6; discussion 237-9.