Green Clayton B, Cheng Georgina, Chandra Jyotsna, Mukherjee Pranab, Ghannoum Mahmoud A, Hoyer Lois L
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106, USA.
Microbiology (Reading). 2004 Feb;150(Pt 2):267-275. doi: 10.1099/mic.0.26699-0.
An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.
开发了一种逆转录聚合酶链反应（RT-PCR）检测方法，用于分析白色念珠菌ALS（凝集素样序列）家族中基因的表达模式。用菌株SC5314接种皮肤黏膜念珠菌病的重组人颊黏膜上皮（RHE）模型，结果显示白色念珠菌破坏了上皮层，还形成了一个具有生物膜特征的上层真菌层。对从接种白色念珠菌的颊黏膜RHE中提取的总RNA样本进行RT-PCR分析表明，随着RHE破坏的进展，ALS1、ALS2、ALS3、ALS4、ALS5和ALS9在不同时间均能持续检测到。ALS7转录本的检测，尤其是ALS6转录本的检测，更具随机性，且与严格的时间模式无关。用于接种RHE的白色念珠菌培养物中ALS基因的表达模式与在RHE模型中观察到的相似，这表明白色念珠菌与颊黏膜RHE的接触对ALS基因表达的影响很小。对从义齿模型和导管生物膜中提取的RNA样本进行RT-PCR分析，结果显示其基因表达模式与颊黏膜RHE标本相似。在生物膜形成过程中，不同白色念珠菌菌株的生物膜RNA标本的RT-PCR分析结果一致，且与浮游细胞中的基因表达模式相当。本文所述的RT-PCR检测方法将有助于分析人类临床标本以及来自其他疾病模型的样本。该方法将进一步深入了解ALS基因及其编码蛋白在白色念珠菌与其宿主之间各种相互作用中的作用。
