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Chapter 10 : Genetic and Genomic Approaches to Cryptococcus Environmental and Host Responses
Category: Clinical Microbiology; Fungi and Fungal Pathogenesis
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Infection with Cryptococcus species is often considered an AIDS-defining illness, the outbreak of Cryptococcus gattii on Vancouver Island underscores the broad potential of the Cryptococcus neoformans species complex to evade host immune defenses and cause disease. There are several simple developments that need to be made to maximize the use of C. neoformans genomes. One development is the publication of the genomes of the serotype A and C. gattii strains and a finalized nomenclature for each gene in each strain sequenced, preferably as similar to other designations as possible. Examples of larger-scale screens include a series on melanin, morphology, suppressor screens of light sensitivity of mating , and growth under hypoxic conditions. In the first, "alternative" hosts have been used, with the most success obtained thus far in the nematode Caenorhabditis elegans. Importantly, both genes were found to be required for maximal virulence in murine inhalation models. Protein-protein interactions are important in a number of regulatory events involved in cryptococcal virulence and have been detected in C. neoformans by using a variety of methods. Single gene interactions have been detected by coimmunoprecipitation studies of the nuclear heat shock transcription factor, Hsf1, with a coactivating heat shock homolog, Ssa1, during laccase induction. A major experimental advantage of C. neoformans, uncommon among the human pathogenic fungi, is the established complete sexual cycle that enables classical Mendelian crosses and genetics.
Overlap PCR for generating gene disruption constructs. The overlap PCR process first generates three products (A): a 5’ (primers 1 and 2) and 3’ (primers 3 and 4) region of 1.0–1.5 kb flanking the gene to be mutated (your favorite gene 1 [YFG1]) and one for the marker (primers 5 and 6) used to select for transformed strains. All primers are standard 18 nt in length, with the exception of numbers 2 and 3, which are 36 nt long and composed of a chimera of the YFG1 gene and the selectable marker. (B) The three pieces are mixed in equimolar amounts and amplified a second time using primers 1 and 4. (C) This product is transformed into C. neoformans using a biolistic apparatus, where the construct in many cases undergoes a double homologous recombination event at the YFG1 locus. (D) The specific targeting events are screened using primers ( 7 and 8 ; 9 and 10 ) that only amplify when the marker is inserted, and gene disruption and the presence of only one copy of the construct in the genome is subsequently confirmed by Southern blot analysis. Primers 7 and 10 are distal to 1 and 4. Primers 5, 6, 8, and 9 can be a common set used for multiple gene disruption events.
Example of a forward genetic screen, using T-DNA insertions delivered by Agrobacterium and temperature sensitivity as the phenotype. (A) C. neoformans strains are replicated on yeast extract peptone dextrose medium and incubated overnight at 30°C or 37°C. One strain (circled) shows impaired growth at 37°C. (B) The insertional mutant strain (IMα) is crossed to the wild-type strain of opposite mating type (WTa), and progeny are isolated. Here a subset of eight progeny from a genetic analysis show no growth at 37°C if they are also nourseothricin resistant, or show wild-type growth at 37°C and nourseothricin sensitivity, indicating linkage between the T-DNA insertion event and the temperature-sensitive phenotype. Mating type (a or α adjacent to the progeny number) can be used as an independent marker to demonstrate genetic recombination in the population of the progeny. (C) Genomic DNA is extracted, digested with restriction enzyme, and selfligated, and the regions flanking the T-DNA insertion are obtained by inverse PCR using the ligation as a template. (D) The inverse PCR product is sequenced, defining the junctions between T-DNA (gray) and C. neoformans DNA. (E) Sequence comparison against the genome database reveals the nature of the gene affected. In this case the T-DNA insertion is 321 bp upstream of the start codon of a gene previously characterized with a role in growth at 37°C [AGS1, which encodes an α(1–3)glucan synthase] ( 55 , 56 ).
Overview of three alternative methods to create insertional mutants of C. neoformans for use in forward genetic screens. Tagged DNA constructs containing the transformation markers such as the cryptococcal URA5 gene or the synthetic antibiotic-resistant cassettes Natr, Hygr, and Neor, conveying resistance to nourseothricin, hygromycin, and neomycin, respectively, are inserted within the cryptococcal genome using at least three methods: electroporation, biolistics, and Agro-bacterium. Electroporation uses a high-voltage pulse delivered within a metal-coated cuvette. Biolistic transformation uses gold beads coated with transforming DNA that is delivered by a helium gas pulse directly on a thin layer of fungal cells inoculated on agar plates. Agrobacterium methods use the transforming bacterium A. tumefaciens to incorporate a specialized Ti plasmid containing the selected cryptococcal transformation marker and tagged elements into the cryptococcal DNA chromosome. Transformants are then isolated using the selective media appropriate for the transformation marker.
Genes required for melanin biosynthesis in serotype A C. neoformans. Those that have emerged from forward genetic screens are underlined. Laccase is the central enzyme for catalyzing the polymerization of phenolic substrates into melanin. Laccase is a copper-dependent enzyme; copper metabolism is therefore required for melanization. Other genes play regulatory roles for laccase directly or through copper metaliation. Laccase localizes to the cell wall, and thus mutations affecting cell wall integrity may have altered melanin profiles, such as chitin synthase (Chs3) and its regulator (Csr2). Additional genes that are known to regulate melanization ( 34 ), but through unknown mechanisms, have been omitted.