title: RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms.
abstract: 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.
authors.affiliation: Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
authors.forename: Clayton B
authors.initials: CB
authors.lastname: Green
authors.affiliation: Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
authors.forename: Georgina
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Jyotsna
authors.initials: J
authors.lastname: Chandra
authors.affiliation: Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Pranab
authors.initials: P
authors.lastname: Mukherjee
authors.affiliation: Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Mahmoud A
authors.initials: MA
authors.lastname: Ghannoum
authors.affiliation: Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
authors.forename: Lois L
authors.initials: LL
authors.lastname: Hoyer
mesh_terms: D000373:Agglutinins / Q000235:genetics*; D001483:Base Sequence; D018441:Biofilms; D002176:Candida albicans / Q000235:genetics*; D002180:Candidiasis, Oral / Q000175:diagnosis*; D017931:DNA Primers; D004847:Epithelial Cells / Q000382:microbiology*; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D000373:Agglutinins; D017931:DNA Primers; D005656:Fungal Proteins