【作者】 胡辉；

【导师】 张永信； 卢洪洲； 章强强；

【作者基本信息】 复旦大学 ， 内科学， 2009， 博士

【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。更多还原

【Abstract】 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.更多还原