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Chapter 5 : The Conversion from Classical Studies in Fungal Pathogenesis to the Molecular Era
Category: Fungi and Fungal Pathogenesis
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This chapter focuses on pioneering works that have laid the foundation for the conversion from the classical study of fungal pathobiology to the molecular era. The most widely used and important molecular biology-based technologies that are now available and in use for the identification of pathogenic fungi and for study of fungal pathobiology are also discussed. The transition from the classical era to the molecular era necessitated the development of models, techniques, and strategies which would facilitate the definition of the genetic and molecular basis of pathogenicity according to the working hypothesis in the molecular Koch’s postulates formulated by Falkow. The chapter talks about classical versus molecular approaches applied to the identification of fungal pathogens. Nucleic acid-based molecular assays can be divided into two classes: PCR based and non-PCR. The major PCR-based assays for fungal diagnosis consist of three different strategies, each of which has a standard Taq polymerase amplification of template DNA as a central component. The discovery of effective drugs and the development of vaccines based on knowledge of molecular biology will indeed contribute to the prevention of and therapy for these infections and better management of patients. Education in medical mycology must be viewed on par with that of other infectious diseases in order to realize the full benefit of the evolution from the classical to molecular era of studies on pathogenic fungi.
Key Concept Ranking
- Restriction Fragment Length Polymorphism
Mycology milestones. The transition from classical mycology to molecular mycology was made possible through a number of biological and technological milestones that were achieved over the last 25 years.
ATMT. Transformation is mediated by selection on hygromycin. The hygromycin B phosphotransferase gene is transferred by A. tumafaciens to the C. neoformans host cell, where it integrates into the genome. L.B, left border; hph, hygromycin B phosphotransferase gene; R.B., right border, T-DNA, transfer DNA present in Ti plasmid; Vir, virulence proteins that lead to production of the single-stranded T-DNA molecule as well as formation of the channel; C, channel through which T-DNA is exported into host cells (C. neoformans).
rDNA gene cluster. Ribosomal subunits are shown as a single cluster within brackets. The four subunits are identified (18S, 5.8S, 26S, and 5.8S) as boxes. The intervening regions are ITS1 and ITS2 (internal transcribed spacer) and IGS1 and IGS2 (intergenic region). Primers for amplifying the ITS1-ITS2 region are shown by arrows (ITS1 and ITS4), with the general size of the amplicon being ~700 bp.
RFLP. Genomic DNA from five isolates (lanes 1 to 5) of C. albicans digested with EcoRI is shown. DNA appears as a smear except when the restriction enzyme yields fragments identical in size. These fragments appear as more intensely staining bands on the gel against the background of sheared DNA because they migrate to the same place on the gel. The number and pattern can be used to discriminate strains, depending on the enzyme used for digestion. L, size marker (1-kb ladder).
RAPD analysis. Four strains of C. lusitaniae were analyzed by RAPD analysis. Strains 1 and 2 are clinical isolates, and strains 3 and 4 are unrelated controls. RAPD primers are listed under each panel and are from references 69 and 163 . RAPD patterns suggest that strains 1 and 2 are related.
Pioneering transformation protocols used in different pathogenic fungi
Genome sequence status of human fungal pathogens