Applying genetics and molecular biology to the study of the human pathogen Cryptococcus neoformans

Abstract

The basidiomycete yeast Crytococcus neoformans is a prominent human pathogen. It primarily infects immunocompromised individuals producing a meningoencephalitis that is lethal if untreated. Recent advances in its genetics and molecular biology have made it a model system for understanding both the Basidiomycota phylum and mechanisms of fungal pathogenesis. The relative ease of experimental manipulation coupled with the development of murine models for human disease allow for powerful studies in the mechanisms of virulence and host responses. This chapter introduces the organism and its life cycle and then provides detailed step-by-step protocols for culture, manipulation of the genome, analysis of nucleic acids and proteins, and assessment of virulence and expression of virulence factors.

Figure 33.1

Life cycle of C. neoformans.

Figure 33.2

(A) Targeted gene deletion: The construct contains an antibiotic resistance cassette flanked on the 5′- and 3′-ends with 1 kb regions of homology upstream and downstream of the targeted gene. (B) Epitope-tagging: The construct contains the epitope tag and an antibiotic resistance cassette flanked on the 5′-end with a 1-kb region of homology to the 3′-end of the targeted gene, and on the 3′-end with a 1-kb region of homology to the region immediately downstream of the targeted gene. (C) Promoter replacement: The construct contains an antibiotic resistance cassette and the desired promoter region flanked on the 5′-end with a 1-kb region homologous to the sequence upstream of the promoter to be replaced, and on the 3′-end with a 1-kb region homologous to the 5′-end of the targeted gene. NAT^R, nourseothricin resistance cassette; YFG, targeted gene.

Figure 33.3

(A) Example PDS-1000/He biolistic particle delivery system (Bio-Rad): (a) “VENT/HOLD/VAC” toggle switch, (b) “FIRE” toggle switch, (c) gas acceleration tube/retaining cap, (d) microcarrier launch assembly, and (e) plate holder. (B) Close-up of microcarrier launch assembly for biolistic particle delivery system: (d) microcarrier launch assembly, (f) Top to microcarrier launch assembly, (g) Macrocarrier holder. (C) Example of a YPAD plate immediately following biolistic transformation. Note the scattering of microcarriers in the center of the patch of C. neoformans cells.

Figure 33.4

(A) Intranasal infection. A silk thread is tied across two supports (such as ring stands). The anesthetized mouse is suspended from its front incisors on the thread. The inoculum of yeast cells is pipetted down one nare. (B) Intravenous infection. The mouse is anesthetized with isofluorane administered by face mask (top view), while the tail vein is dilated through a combination of a sodium acetate heating pad from below and a heating lamp from above (side view). When the mouse is laid on its side, the lateral tail vein of the mouse will be at the top of the tail (top view).

Figure 33.5

(A) Example of a survival curve. Mice were inoculated via tail-vein injection with 2×10⁵ cells/mouse of either WT (H99) or sre1Δ strains of C. neoformans. On average, mice infected with sre1Δ survived 30 days longer, indicating sre1Δ that is a hypovirulent strain. (B) Example of STM score data. Forty-eight signature-tagged strains were grown individually in liquid YPAD medium in a 96-well deep pocket plate, then pooled together to generate the inoculum. Three mice were inoculated with 5×10⁵ cells/mouse via intranasal infection. The mice were monitored to the disease endpoint, at which point they were sacrificed. Shown are a subset of the data from the lungs, following qPCR and calculation of the STM score for each signature tag in each mouse.

Figure 33.6

(A) Melanin assay: The kinetics of melanization varies depending on the incubation temperature. lac1Δ is deficient in the primary laccase enzyme responsible for melanization in C. neoformans. (B) Capsule formation assay: WT (H99) cell grown under capsule inducting conditions (DMEM, 37 °C, 5% CO₂) and visualized with India ink. Bar denotes 10 μm.