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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.
Although members of the Ascomycota phylum, particularly Sacchromyces cerevisiae, are the most studied fungi, there are 80,000 known species of the Fungi kingdom. There is a great deal of diversity in the kingdom, ranging from small harmless unicellular yeast such as S. cerevisiae to the great plant pathogen Armillaria ostoyae, one of the largest organisms in the world. This latter species is a member of the Basidiomycota phylum, a phylum less well understood than Ascomycota.
While no basidiomycete species has been studied in as much detail as S. cerevisiae, it is a fascinating and diverse group of organisms. Basidiomycetes produce many interesting secondary metabolites used in medicine, industry, and research. Members of the phylum account for about 10% (40 species) of known human fungal pathogens (Morrow and Fraser, 2009). With the onset of the AIDS epidemic, one basidiomycete in particular, Cryptococcus neoformans, has risen from a little-known pathogen to one of the top fungal killers of immunocompromised patients.
C. neoformans is primarily found as a haploid yeast, and is widely present in the environment worldwide, including in avian excreta, soil, and tree bark. Studies have shown that humans come into frequent contact with C. neoformans: individuals with no history of cryptococcosis possess antibodies against the yeast (Chen et al., 1999), and most children appear to have been exposed by the age of five (Goldman et al., 2001). This suggests that the majority of individuals encounter C. neoformans in the environment, most likely through inhalation into the lungs. Immunocompetent individuals are usually able to control and contain the infection, often leading to an asymptomatic latent state of infection. If the patient’s immune system becomes compromised at a later date, the latent infection can reactivate. In the case of the immunocompromised individual, pulmonary infection can lead to pneumonia followed by dissemination via the bloodstream to other organs. C. neoformans is one of only a few fungal species known to cross the blood-brain barrier and infect the brain (Kim, 2006), leading to meningitis that is fatal if left untreated. When the AIDS epidemic began in the 1980s, there was a concomitant surge in cryptococcosis cases worldwide. In recent years, the increased usage of antiretroviral therapy and antifungals has reduced the overall incidences of fatal cryptococcal meningitis. Yet in areas where access to treatment is limited, C. neoformans remains an important concern in the care of the immunocompromised, including AIDS, cancer, and organ transplant patients. In addition, recent outbreaks of cryptococcosis in immunocompetent individuals in the Pacific Northwest raise concerns about the risk of cryptococcal infection even in otherwise healthy individuals (Bartlett et al., 2008; Hoang et al., 2004).
As a haploid yeast cell, C. neoformans is amenable to many of the extensive protocols that have been developed for S. cerevisiae, requiring in most cases only a few adjustments. However, having diverged from the ascomycete lineage some 400 million years ago (mya) (Taylor and Berbee, 2006), there are significant differences in its cellular machinery and life cycle (see below). Comparative genomics promises to yield rich information about the evolution of shared and diverged genes, proteins, and pathways, as well as offering insight into the differences between species that allow one yeast to exist as a benign saprophyte and another to cause lethal infection in a mammalian host.
C. neoformans is classified into four different serotypes based on its reactivity with monoclonal antibodies to surface capsular polysaccharide (Kabasawa et al., 1991). These serotypes have historically been further classified into three different varieties: var. neoformans (serotype D), var. grubii (serotype A), and var. gattii (serotypes B and C). However, in recent years, var. gattii has been proposed to comprise its own species as Cryptococcus gattii, based on morphological and biochemical evidence (Kwon-Chung and Varma, 2006). C. neoformans var. neoformans and var. grubii primarily infect immunocompromised individuals, with var. grubii causing ~99% of cryptococcal infections in HIV-infected patients (Mitchell and Perfect, 1995). C. gattii has the ability to infect immunocompetent individuals, as evidenced by an emergent outbreak in the Pacific Northwest that has resulted in hundreds of human and veterinary infections. Based on analysis of mutation frequency in conserved genes, it is thought that C. neoformans and C. gattii diverged about 37 mya, while C. neoformans var. neoformans and var. grubii split 18.5 mya, and within C. gattii, serotypes B and C diverged 9.6 mya (Xu et al., 2000). To date, the genomes of five strains of C. neoformans and C. gattii have been sequenced to at least 6× coverage: JEC21 (serotype D), B-3501 (serotype D), H99 (serotype A), WM276 (serotype B), and R265 (serotype B). H99 and R265 are clinical isolates, while WM276 was isolated from the environment. JEC21 and B-3501 are laboratory-derived strains, where JEC21 was derived from B-3501 through a series of crosses and backcrosses (Heitman et al., 1999), and their genomes are 99.5% identical (Loftus et al., 2005).
Online resources for C. neoformans genome sequences
The genomes of C. neoformans and C. gattii contain about 19 Mb of DNA spread over 14 chromosomes with about 7000 predicted protein-coding genes. The genomic sequence is relatively GC-rich (48% GC content) when compared to the genome of S. cerevisiae (38% GC content). Nucleic acid enzymatic protocols from S. cerevisiae laboratories that have been adapted for use with C. neoformans take this into account with the addition of DMSO (5% final concentration) or betaine (1.3 M final concentration) to resolve secondary structures resulting from the higher GC content.
Unless otherwise noted, the use of “C. neoformans” in the text of this chapter refers to C. neoformans var. neoformans and var. grubii. Although many of the same techniques are applicable to C. gattii, their usage is less well documented and may require additional adaptations.
C. neoformans and C. gattii primarily exist as haploid yeast cells that reproduce asexually through budding. They also possess a bipolar mating system, with mating types a and α. The mating (MAT) locus regulates the sexual cycle and encodes for more than 20 genes, including genes for cell type identity and the production and sensing of pheromone. Similar to S. cerevisiae, MATa cells produce MFa pheromone that is sensed by MATα cells. In response to pheromone, MATα cells produce a conjugation tube (Fig. 33.1A). Likewise, MATa cells respond to the MFα pheromone produced by MATα cells, although the response of MATa cells is to form large swollen cells that can then fuse to the conjugation tubes of the MATα cells. The MATa and MATα nuclei divide, and the MATα nuclei travel through the conjugation tube into the MATa cell. MATa and MATα nuclei move into the hypha formed by the MATa cell, and a septum forms between the hypha and the MATa cell. The hypha may then elongate through cell growth and division. During hyphal elongation, the nuclei divide mitotically (Fig. 33.1B). One nucleus divides in such an orientation as to enter into a bulge in the cell wall that will later form a clamp connection (i). Septa form between the posterior cell wall, the tip of the hypha, and the clamp connection, leaving one nucleus in the posterior cell, two nuclei in the tip of the hypha, and one nucleus in the clamp (ii). The clamp fuses back to merge with the posterior cell (iii), allowing the nucleus present in the clamp to join the nucleus in the posterior cell (iv). During sporulation (Fig. 33.1A), a basidium forms at the tip of the hypha. In the basidium, the MATa and MATα nuclei fuse and undergo meiosis. The new MATa and MATα nuclei then undergo repetitive rounds of mitosis, eventually forming four chains of spores that emerge from the basidium. The spores are then dispersed, and germinate into haploid yeast cells (Bovers et al., 2008; McClelland et al., 2004).
In the laboratory, mating is achieved through nitrogen starvation on V8 medium, consisting of 5% (v/v) V8 juice, 3 mM KH2PO4, 4% (w/v) agar, pH 5.0. For technical details, several excellent studies have been performed examining mating conditions; we refer you to these (Escandon et al., 2007; Nielsen et al., 2003; Xue et al., 2007).
Haploid fruiting has also been observed, where cells of one mating type become diploid and form hyphae. These monokaryotic hyphae are characterized by unfused clamp connections. Similar to mating, monokaryotic hyphae also form basidia, undergo meiosis, and sporulate (Lin et al., 2005; Tscharke et al., 2003; Wickes and Edman, 1995).
C. neoformans is classified as a Biosafety Level 2 (BSL-2) organism, and as such does not require elaborate biohazard safety facilities. Current precaution recommendations include the use of a Class I or Class II biological safety cabinet for manipulation of environmental samples or spore forms. Incidences of infection in laboratory personnel are rare, limited in the literature to skin puncture accidents with needles heavily contaminated with C. neoformans (Casadevall et al., 1994).
C. neoformans may be cultured using similar medium to that used in S. cerevisiae cultivation. Common media used include YPAD and YNB. For some assays (e.g., see Sections 6.1.6 and 6.3.2), Sabouraud dextrose medium is used for culturing for historic reasons and its promotion of yeast growth over bacterial growth.
|YPAD (yeast peptone adenine dextrose)|
|1% bacto yeast extract (Becton Dickinson, Cat. No.|
|2% bacto peptone (Beckton Dickinson, Cat. No.|
|2% glucose||20 g|
|0.73 mM L-tryptophan (Sigma, Cat. No. T8941)||0.15 g|
|0.27 mM adenine (Sigma, Cat. No. A2786)||0.037 g|
|Water||to 1 l|
|YNB (yeast nitrogen base)|
|0.15% YNB w/o amino acids, w/o dextrose, w/o|
ammonium sulfate (BIO 101, Cat. No. 4027-032)
|75 mM ammonium sulfate||10 g|
|2% glucose||20 g|
|Water||to 1 l|
|3% Sabouraud dextrose broth (Becton Dickinson,|
Cat. No. 238210)
|Water||to 1 l|
Standard growth is performed in YPAD medium, typically at 30 °C, with the alternative use of the defined medium YNB. During logarithmic growth in YPAD medium at 30 °C, the doubling time of wild-type C. neoformans is approximately 110 min. Consistent with its role as a human pathogen, C. neoformans also grows robustly at 37 °C. Unlike S. cerevisiae, C. neoformans does not perform fermentation, and therefore requires a minimal amount of oxygen for growth. C. neoformans is sensitive to alkaline pH, growing poorly at pH 9. However, it is insensitive to acidic pH, exhibiting normal doubling times in conditions as low as pH 3.
Frozen stocks of C. neoformans can be maintained in 15% glycerol solution at −80 °C. These stocks may be revived following transfer by sterile applicator stick to a YPAD plate.
Our laboratory and others have used resistance to nourseothricin (NAT), G418, and hygromycin for selection in C. neoformans.
In the plasmids pHL001-STM-# and pJAF1, the genes encoding for proteins conferring resistance to NAT and G418 have been inserted in between the promoter element of C. neoformans ACT1 and the terminator element of C. neoformans TRP1 (both of these sequences were derived from the H99 strain) (Table 33.1). In the plasmid pHYG7-KB1, the gene encoding for resistance to hygromycin was inserted between the promoter element of C. neoformans ACT1 and the untranslated region (UTR) of C. neoformans GAL7 (where the ACT1 sequence was derived from H99 and the GAL7 sequence was derived from JEC21).
For selection of yeast containing the appropriate drug resistance cassette, we use YPAD agar plates made with 0.1 mg/ml nouseothricin (clonNAT, Werner BioAgents), 0.2 mg/ml G418 (VWR, Cat. No. 45000-626), and/or 0.3 mg/ml hygromycin (Sigma, Cat. No. H7772).
Manipulation of the genomic sequence of a species is a powerful tool for analyzing the importance of specific genes in the function of the organism. Homologous recombination, or the integration of an exogenous DNA construct into the genome, is a crucial step in the site-directed mutagenesis of a target gene. C. neoformans performs homologous recombination at relatively low frequencies when compared with other fungi (1–4% as compared to nearly 100% in S. cerevisiae) but transformation with linear constructs flanked by a significant amount (0.3–1 kb) of sequence homologous to the genome creates stable integrants reproducibly (Davidson et al., 2000; Nelson et al., 2003). For example, a linear construct to target a gene for deletion might contain an antibiotic resistance cassette flanked on the 5′- and 3′-ends with 1 kb sequences homologous to the 5′- and 3′-ends of the targeted gene.
Construction of linear constructs for homologous recombination uses a procedure known as fusion PCR or PCR overlap (Davidson et al., 2002). In this process, two or more DNA fragments are joined together during the polymerase chain reaction (PCR) by virtue of a shared region of homology. This region of homology is engineered during previous PCR steps using primers containing linker sequences that are then shared between the two fragments to be fused together (Fig. 33.2).
As mentioned previously, a linear construct for targeted gene deletion is designed to contain an antibiotic resistance cassette flanked by 1 kb sequences homologous to the targeted sequence. To create this construct, an antibiotic resistance cassette, such as resistance to NAT, is first amplified with primers containing 22 bp of homology to the 5′ and 3′ ends of the cassette, and 21 bp of linker sequence which is different for the 5′ and 3′ primers (these primers are designated primers 3 and 4 in Table 33.2 and Fig. 33.2A). Then, from genomic DNA we amplify 1 kb of sequence upstream and downstream of the ORF targeted for deletion. We term these sequences the 5′ and 3′ flanks for the targeted gene deletion construct. For the 5′ flank, the forward primer (1) is 22 bp of exact homology to the genomic sequence. The reverse primer (2) is 21 bp of linker sequence that is antiparallel to the linker sequence in the forward primer (3) for amplifying the antibiotic resistance cassette, followed by 22 bp of homology to the genomic sequence. For the 3′ flank, the forward primer (5) is 21 bp of linker sequence that is antiparallel to the linker sequence in the reverse primer (6) for amplifying the antibiotic resistance cassette, followed by 22 bp of homology to the genomic sequence. The reverse primer is 22 bp of exact homology to the genomic sequence. Table 33.2 contains the linker sequences we use to design these primers.
Primers 1–6 are used first for amplification of the 5′- and 3′-flanks and the antibiotic resistance cassette. Then primers 1 and 6 are used in the fusion PCR to amplify the full-length linear construct.
(50 μl final volume): 400 nM each primer, 0.25 mM dNTPs, 20 mM Tris–HCl (pH 8.8), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% (v/v) Triton X-100, 0.01% (w/v) BSA (98% electrophoresis grade, Sigma, Cat. No. A7906), 5% (v/v) DMSO, 2.5 U Pfu polymerase, 0.5 μl of template DNA (genomic DNA at 1 μg/μl or plasmid bearing antibiotic resistance cassette at 30 ng/μl).
We maintain at 4 °C a 10× stock of PCR buffer that contains 200 mM Tris–HCl (pH 8.8), 20 mM MgSO4, 100 mM KCl, 100 mM (NH4)2SO4, 1% (v/v) Triton X-100, 0.1% (w/v) BSA. We then add the primers, DMSO, dNTPs, Pfu, and template DNA separately.
PCR conditions, performed on a PTC-200 Peltier Thermal Cycler (MJ Research): 93 °C for 3 min, followed by 35 cycles of (93 °C for 30 s, 45 °C for 30 s, 72 °C for 3.5 min or appropriate amount of time for the length of your antibiotic resistance cassette), followed by 72 °C for 5 min.
Purify the PCR products by running the PCR out on a 0.8% agarose gel. Cut out the appropriate size band in the gel, and purify using a QIAquick Gel Extraction kit (Qiagen, Cat. No. 28704), following the manufacturer’s instructions. In the final step, elute from the column with 30 μl of elution buffer (EB), letting the column stand for 1 min then centrifuging for 1 min at 13,000 rpm.
(50 μl final volume): 400 nM each primer (primers 1 and 6), 0.25 mM dNTPs, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 1.3 M betaine, 1 U Taq polymerase, 0.25 U Pfu polymerase, 50 nmol each of 5′ flank, 3′ flank, and antibiotic resistance cassette (roughly equal to 2 μl of the eluted volume from the QIAquick Gel Extraction).
We maintain at 4 °C a 10× stock of PCR buffer that contains 100 mM Tris–HCl (pH 8.3), 500 mM KCl, 20 mM MgCl2. We then add the betaine, dNTPs, Pfu, and Taq polymerases separately. Betaine is maintained as a 5 M stock at 4 °C.
PCR conditions, performed on a PTC-200 Peltier Thermal Cycler (MJ Research): 72 °C for 10 min, 92.5 °C for 3.5 min, followed by 35 cycles of (92.5 °C for 12 s, 52 °C for 12 s, 72 °C for 7 min or appropriate amount of time for the length of the full targeted gene deletion construct), followed by 72 °C for 5 min.
Purify the PCR product by running the PCR out on a 0.8% agarose gel. Cut out the appropriate size band in the gel, and purify the DNA in the gel slice using a QIAquick Gel Extraction kit, following the manufacturer’s instructions. In the final step, elute from the column with 50 μl of EB, letting the column stand for 1 min, then centrifuging the column for 1 min at 13,000 rpm. Add another 30 μl of EB to the column, let stand for 1 min, then centrifuge for 1 min at 13,000 rpm.
With modifications, fusion PCR may be utilized for a variety of genetic manipulations. By selecting different sequences for amplification, we have successfully used fusion PCR to introduce epitope tags into the 5′- and 3′-ends of genes (Fig. 33.2B), and to replace the promoters of genes (e.g., for overexpression of genes or placing genes under the control of inducible promoters) (Fig. 33.2C).
The preferred method for transformation into C. neoformans is biolistic delivery. While studies have shown that C. neoformans is transformable by electroporation, these transformations have been low efficiency, resulting in some stable ectopic transformants but also many unstable transformants harboring extrachromosomal DNA material (Edman, 1992; Edman and Kwon-Chung, 1990). In addition, electroporation of different C. neoformans strains has varying degrees of success; strain H99 (serotype A) is much less tractable to transformation in this way than strain B-3501 (serotype D). In contrast, both serotypes A and D are readily transformed with relatively high efficiency by biolistic delivery (Davidson et al., 2000; Toffaletti et al., 1993).
In biolistic delivery, DNA is introduced into the yeast cell using a biolistic particle delivery system (PDS-1000/He, Bio-Rad) hooked up to a vacuum pump (Maxima C Plus M6C, Fisher Scientific) and compressed helium tank (Fig. 33.3). The targeted gene deletion constructs generated by fusion PCR (see above) are deposited onto gold bead microcarriers that are then positioned on a macrocarrier disk in the main chamber of the biolistic PDS. The air in the biolistic PDS is removed by a vacuum pump, and helium is pumped into a small chamber (termed the gas acceleration tube) positioned above the macrocarrier disk, separated from the main chamber by a pressure-calibrated rupture disk. At a high enough pressure, the rupture disk breaks and the helium blasts into the main chamber of the biolistic PDS, propelling the macrocarrier disk downward against a metal stopping screen. The force of the impact against the stopping screen propels the DNA-coated microcarriers off the macrocarrier disk at high velocity and into C. neoformans cells that have been plated onto an agar plate and positioned below.
Preparation of constructs (may be done anytime prior to the day of transformation)
|0.9% YNB w/o ammonium sulfate w/o dextrose w/o|
ammonium sulfate (BIO 101, Cat. No. 4027-032)
|1 M sorbitol||182 g|
|1 M mannitol||182 g|
|2.6% glucose||26 g|
|0.267% bacto yeast extract (Becton Dickinson, Cat.|
|0.054% bacto peptone (Beckton Dickinson, Cat. No.|
|0.133% Gelatin (Sigma, Cat. No. G-8150)||1.33 g|
|Water||to 1 l|
The genotype of the transformed strain is verified through PCR-based detection of the expected 5′- and 3′-junctions of the resistance marker with the genomic DNA. While verifying the genotypes of many transformations, it is easiest and fastest to perform colony PCR.
(50 μl final volume): 400 nM each primer, 0.25 mM dNTPs, 20 mM Tris–HCl (pH 8.8), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% (v/v) Triton X-100, 0.01% (w/v) BSA (98% electrophoresis grade, Sigma, Cat. No. A7906), 5% (v/v) DMSO, 0.15 U Pfu polymerase, 0.5 U Taq polymerase.
We maintain at 4 °C a 10× stock of PCR buffer that contains 200 mM Tris–HCl (pH 8.8), 20 mM MgSO4, 100 mM KCl, 100 mM (NH4)2SO4, 1% (v/v) Triton X-100, 0.1% (w/v) BSA. We then add the primers, DMSO, dNTPs, Pfu, and Taq polymerases separately.
PCR conditions, performed on a PTC-200 Peltier Thermal Cycler (MJ Research): 92.5 °C for 3 min, followed by 35 cycles of 92.5 °C for 15 s, 45 °C for 15 s, 72 °C for 1 min 45 s or appropriate amount of time for the length of targeted amplicon), followed by 72 °C for 5 min.
Lyophilization greatly enhances recovery of nucleic acid from C. neoformans. Dessication may weaken the structure of the polysaccharide capsule and cell wall, allowing greater disruption in later steps.
Mice are relatively susceptible to C. neoformans infection, when compared with other mammalian hosts such as rats and rabbits. Immunocompetent murine strains will succumb to pulmonary infection, and will experience dissemination to other organs including the brain, similar to human cryptococcosis. A murine model of cryptococcal infection offers several advantages over other species, including the consistency of susceptibility within a given strain, the availability of genetically modified strains (useful for examining host factors that may be involved in infection), as well as their small size and low cost. Our laboratory utilizes two routes of infection for introducing C. neoformans into a murine model of infection: intranasal and intravenous.
Inbred mouse strains may vary in their susceptibility to C. neoformans infection. For example, in some studies, BALB/c mice have been demonstrated to be more resistant to C. neoformans infection than C57BL/6 mice, as evidenced by both fungal load in the lungs following infection (Chen et al., 2008; Huffnagle et al., 1998) and degree of dissemination to other organs (Chen et al., 2008), although in some survival curve analyses, C57BL/6 mice survive slightly longer than BALB/c mice following infection with C. neoformans (Nielsen et al., 2005). There are varying theories as to the source of the differences in susceptibility in mouse strains to C. neoformans: studies have linked relative resistance of a mouse strain to the production of Th1-type cytokines, where their production is associated with pulmonary clearance (Huffnagle et al., 1998), or the presence of complement protein C5 (Rhodes et al., 1980). Our laboratory performs murine infections with 5- to 6-week-old A/J mice, which are C5-deficient and therefore slightly more susceptible to C. neoformans infection than C5-sufficient mouse strains (e.g., BALB/c and C57BL/6 mice) (Nielsen et al., 2005; Wormley et al., 2007). It bears noting that the inocula listed below have been determined by our laboratory for use with our strain of H99 C. neoformans in A/J mice. Use of other mouse strains may necessitate adjustment of the inocula to a higher or lower dosage of C. neoformans cells. Additionally, derivatives of the H99 strain (i.e., H99 stocks maintained by different laboratories) appear to have varying levels of virulence.
Studies have also shown that the age of the mice may affect their relative susceptibility to infection. Older C57BL/6 mice (e.g., 17-week-old) are better able to clear an intratracheal infection from their lungs, brains, and spleens than younger (e.g., 5-week-old) mice (Blackstock and Murphy, 2004). We have found it best to infect mice of a consistent age to reduce variability in our data.
An intranasal infection is thought to more closely mimic the natural course of infection, beginning with the inhalation of C. neoformans cells leading to pulmonary disease followed by dissemination to other organs. The mice are first anesthetized with a mixture of ketamine hydrochloride (Orion Pharma Animal Health) and medetomidine hydrochloride (Domitor®, Orion Pharma Animal Health) via intraperitoneal injection. For all murine injections, we use ½ cc insulin syringes with 28G½ needles (Becton Dickinson, 329461). The anesthetic is mixed to contain 18.75 mg/ml ketamine hydrochloride and 0.625 mg/ml medetomidine hydrochloride. We administer 30–50 μl of this formulation to each mouse (10–15 g), leading to doses of 50–60 mg/kg ketamine hydrochloride and 1.5–2.0 mg/kg medetomidine hydrochloride. The mice usually succumb to the anesthesia after 5–10 min, at which point they are weighed, their ears notched for later identification, and ointment (Artifical Tears, Webster Veterinary, Cat. No. 07-841-4071) applied to their eyes to prevent them from drying out. A silk thread (50 Denier Weight, obtainable from a sewing supply store) is strung between two supports—we use ring stands for this purpose. The mice are then suspended by their incisors upon this silk thread (Fig. 33.4A). The inoculumof C. neoformans cells (5×105 cells/mouse in 50 μl) is slowly pipetted directly into one nare using a pipette fitted with a filter tip. Take care to allow for complete dispension of the inoculum into the nare; if signs of struggling are seen in the mouse, pipetting should be suspended until the mouse no longer shows signs of discomfort. We typically anesthetize and inoculate batches of five mice at a time. Following completion of inoculation, the mice remain suspended for 10 min, to allow for complete aspiration into the lungs, before being lowered and the anesthesia reversed via intraperitoneal injection of atipamezole hydrochloride (Antisedan®, Orion Pharma Animal Health). We administer 40–50 μl of 1 mg/ml atipamezole hyrochloride per mouse, leading to a dose of 2.5–3.5 mg/kg. It typically takes 10–15 min following injection with atipamezole hydrochloride to see signs of stirring in the mice, and another 15–20 min before the mice begin to walk around again.
Inocula are prepared by growing C. neoformans in liquid YPAD overnight at 30 °C. Cells are counted by hemocytometer and, for an intranasal infection, 1×107 cells are washed twice with PBS and resuspended in 1 ml of PBS. Fifty microliters of this inoculum are used per mouse (5×105 cells). For an intravenous infection, 2×107 cells are washed in PBS and resuspended in 1 ml of PBS. One hundred microliters of this inoculum is used per mouse (2×106 cells). Inocula concentrations are confirmed by plating appropriate dilutions onto YPAD plates and counting the colony forming units (CFU) after 2 days growth at 30 °C.
An intravenous infection, via the lateral tail vein, leads to more uniform dissemination to the organs. The mice are weighed prior to infection and marked by ear notching for later identification. They are anesthetized via inhalation of 3% isofluorane in oxygen, administered by face mask, then remain on a sodium acetate rechargeable heating pad (Heat Solution, Prism Enterprises) beneath a heating lamp during the procedure (see Fig. 33.4B) in order to dilate the vein so that it is more visible for easier injection. The inoculum (2×106 cells in 100 μl PBS) is injected into the lateral tail vein. Following successful inoculation, the mice are immediately removed to their cage where they will rapidly recover from the anesthesia.
Mice are weighed prior to infection, and then monitored every 2–3 days postinfection. Signs of disease progression include hunched posture, abnormal gait, weight loss, and decreased grooming as indicated by ruffled fur. Our laboratory uses two endpoints for assessing time of survival: the point at which the mouse has lost 15% of its initial weight, or 25% of its peak weight. We find the latter to be more consistent when the mice were infected at a younger age (e.g., close to 4 weeks in age) and are hence smaller at the initial time point.
“Time-to-endpoint” survival curve analysis monitors the infection of 8–10 mice with a single strain of C. neoformans, until their endpoints (as defined above). In this manner, mice infected with less virulent strains of C. neoformans survive longer than mice infected with more virulent strains (Fig. 33.5A).
This analysis gives a gross determination of the virulence of a single strain on the entire host system. A more specific analysis might address questions such as the initial rate of colonization to a specific organ, the rate of proliferation and/or rate of killing by the host immune cells, or the rate of dissemination to other organs. This additional analysis may be performed by assessing fungal load in the organs at various time points following infection. We typically examine fungal loads in the lungs, brain and spleen, although we have also examined the liver and kidneys. To measure organ loads after the animal is euthanized, the selected organs are removed by dissection, and placed on ice in 17×100 mM polypropylene sterile tubes (Evergreen Scientific, Cat. No. 222-2393-080), one tube per organ per mouse. Take care to wash the dissecting tools in water and ethanol between organs to eliminate carryover of yeast from organ to organ. Each organ is homogenized in 5 ml sterile PBS (we use a PRO200 tissue homogenizer, PRO Scientific, Oxford, CT), then serial dilutions in PBS are plated on Sabouraud dextrose agar plates (made with Sabouraud dextrose agar, Becton Dickinson, Cat. No. 211661) containing 40 μg/ml gentamycin and 50 μg/ml carbenicillin to discourage bacterial growth. CFU are assessed, and comparisons can be made for a single strain in different organs, rate of growth in different organs over time, or between multiple strains for relative fitness.
Evaluation of infectivity and virulence for a large number of C. neoformans strains through single-strain infections as described above can quickly add up in terms of both time and cost, as many mice must be used for each strain. Pooling mutant strains into a single infection allows rapid assessment of multiple strains in a single mouse. This can be easily and effectively performed using a technique known as signature-tagged mutagenesis (STM) screening. Each mutant contains a signature tag, or a unique sequence similar to a barcode, in its DNA. When pooled together in a group, individual strains can still be identified through qPCR of pooled genomic DNA using signature-tag-specific primers. By identifying relative representation in the pool of genomic DNA before and after infection, relative rates of infectivity can be assessed rapidly and reproducibly for multiple mutants in a single infection. Using 48 unique signature tag sequences, this technique has been employed by our laboratory for the production and quantitative analysis of a library containing ~1200 targeted gene deletion strains (available without restriction from the Fungal Genetic Stock Center or the American Type Culture Collection (ATCC)) (Liu et al., 2008).
In detail, to analyze a group of 48 signature-tagged strains, the group is first grown up in liquid YPAD in 96-well deep-pocket plates (Grenier Bio-One, Cat. No 780270), one strain per well, at 30 °C without shaking for 3 days. Two hundred microliters of each culture is pooled together, and the number of cells assessed by hemocytometer. 2×107 cells (for tail vein injection) or 1×107 cells (for intravenous infection) are washed twice in sterile PBS and resuspended in 1 ml sterile PBS. This pool is used as the inoculum to infect three mice, either by intranasal infection (5×105 cells/mouse) or tail vein injection (2×106 cells/mouse). Fifty microliters (5×105 cells) of this pool is also plated in triplicate on Sabouraud dextrose agar plates containing 40 μg/ml gentamycin and 50 μg/ml carbenicillin, which are then incubated at 30 °C for 2 days. The resulting colonies are scraped off each plate, resuspended in water, flash frozen in liquid nitrogen and then lyophilized. Genomic DNA is prepared from these samples as described above. This DNA constitutes the “input DNA” for later analysis.
After monitoring and sacrifice of the animals, the organs of interest are removed and homogenized in 5 ml sterile PBS. Serial dilutions in triplicate are made in sterile PBS and plated on Sabouraud dextrose agar plates containing 40 μg/ml gentamycin and 50 μg/ml carbenicillin. These plates are incubated at 30 °C for 2 days. The resulting colonies are scraped off each plate, resuspended in water, flash frozen in liquid nitrogen, and then lyophilized. The genomic DNA that is prepared from these samples constitutes the “output DNA” of the experiment.
The input and output DNA are analyzed using qPCR using a common primer targeted to the drug resistance marker that has replaced the targeted gene, coupled with signature tag-specific primers.
(50 μl final volume): 400 nM each primer, 0.25 mM dNTPs, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 1.3 M betaine, 1 U Taq polymerase, 0.25 U Pfu polymerase, 1–4 μg genomic DNA, 2 μl 2× Sybr Green I (Molecular Probes, Cat. No. S-7563).
We maintain at 4 °C a 10× stock of PCR buffer that contains 100 mM Tris–HCl (pH 8.3), 500 mM KCl, 20 mM MgCl2. We then add the betaine, dNTPs, Sybr Green I, Pfu, and Taq polymerases separately. Betaine is maintained as a 5 M stock at 4 °C. Sybr Green I is kept at −20 °C as a 100× stock in DMSO, and diluted 1:50 in TE buffer to 2× stock immediately prior to addition to the PCR mix.
PCR conditions, performed on a DNA Engine Opticon (MJ Research): 93 °C for 4 min, followed by 40 cycles of (93 °C for 45 s, 52 °C for 25 s, 72 °C for 1 min, then a plate read by the machine), followed by 72 °C for 5 min.
The threshold cycle (CT), or cycle number where the amplified target reaches a fixed threshold, of each primer pair is used to calculate an STM score, using a variation of the 2−ΔΔCT method for quantitation analysis (Livak and Schmittgen, 2001). For each signature tag, a ΔCT is calculated by subtracting the CT of the specific primer pair from the median CT for all 48 pooled strains to (ΔCT = CT-median − CT-tag). The ΔCT values for each of the three independent input DNA samples are averaged to calculate the ΔCT-input value. The ΔCT values for each of the three independent output DNA samples is similarly calculated by subtracting the median CT for all 48 pooled strains to the CT of the specific primer pair. However, ΔCT values (ΔCT-output) for the three independent output DNA samples are not averaged. The value ΔΔCT is then calculated, where (ΔΔCT = ΔCT-output − ΔCT-input). The STM score is then equal to ΔΔCT. The STM scores from the three mice (i.e., each of the three output DNA samples) are then averaged to determine a final STM score for each mutant. Strains with reduced levels of persistence in the organ have STM scores less than 0, while strains with increased levels of persistence have STM scores greater than 0 (Fig. 33.5B). The STM score correlates with the relative fold change in persistence of a strain with respect to wild type.
This method of analysis makes the basic assumption in its normalization of the data that most of the signature-tagged strains in the pooled infection will have phenotypes similar to wild type. If you desire to assay a significant number of strains that you believe to have different survival rates than wild-type C. neoformans in the mouse, you may need to also seed the inoculum pool with signature-tagged strains that are known to have a wild-type phenotype to prevent skewing during the normalization process. We frequently use knockouts in the gene SXI1 (CNAG_06814 in the H99 sequence database of the Broad Institute) in this manner, as SXI1 is required for mating but dispensable for virulence (Hull et al., 2004).
It is important to note that this screen assays for relative persistence of a strain within a particular organ. It is not a true test of virulence per se, as it is conceivable that a strain may persist in large numbers in a tissue but fail to cause disease in the host. However, in our experience (Liu et al., 2008), the STM screen, when used to assay persistence of mutant strains in the lungs of 5-week-old A/J mice following intranasal infection, has resulted in STM scores that are both reproducible from mouse-to-mouse and pool-to-pool, but are also to a certain degree quantitative, by which we mean that the relative value of the STM score correlates with relative hypo- and hypervirulent phenotypes of the strains when assayed by survival curve analysis.
STM screens also cannot avoid in trans effects from mixing of strains; theoretically a wild-type strain may complement the phenotype of a mutant strain, allowing for a false negative result. Single-strain infections bypass this limitation of the STM screen approach.
Although analysis of virulence in the host organismal level offers obvious correlations between a particular genotype and its efficacy at disease development, it is often problematic to determine the specific host-pathogen interactions responsible for a certain virulence phenotype. It is therefore useful to examine in closer detail the interaction of C. neoformans with a particular host tissue or cell type.
Many studies of the virulence of C. neoformans have focused on its interactions with the immune system, and, in particular, its interactions with macrophages. Alveolar macrophages are thought to be the first line of defense against pulmonary cryptococcal infection. Macrophages and macrophage-derived cells have been observed in the periphery of cryptococcal-containing granuloma formations in the lungs during latent infection of immunocompetent hosts. Additionally, depletion of macrophages from the murine host through the administration of silica has proven to be detrimental to fungal clearance (Monga, 1981). C. neoformans mutants that are more susceptible to killing by macrophages are hypovirulent in “time-to-endpoint” survival curves (e.g., FHB1 which encodes for flavohemoglobin; de Jesus-Berrios et al., 2003). For these and other reasons, it is of interest to examine the interaction of macrophages and macrophage-like cells with C. neoformans yeast.
Unopsonized C. neoformans cells are rarely taken up by macrophages in the absence of activation by cytokines such as IFN-γ or potent antigens such as lipopolysaccharide (LPS). Therefore, phagocytosis assays and assessments of killing by macrophages are commonly done in the presence of both opsonins (such as anti-C. neoformans antibodies or murine or human sera) and activating agents. We most commonly use the murine macrophage-like cell line RAW264.7 (American Type Culture Collection, No. TIB-71), and have had better success using anti-C. neoformans antibody than sera as an opsonizing agent.
C. neoformans has a number of characteristics previously shown to be involved in its virulence. These include (1) ability to grow at 37 °C (2) melanization, thought to aid in resistance to host killing (Nosanchuk and Casadevall, 2003), and (3) polysaccharide capsule formation, thought to be involved in host immune system evasion (Del Poeta, 2004), (Monari et al., 2006). Below are methods to test the relative efficiency of a strain for production of the virulence factors melanin and capsule.
Melanization, or the ability for the yeast to form dark pigment compounds from catecholamine substances such as l-DOPA (3,4-dihydroxy-l-phenylalanine) by the enzyme laccase (Lac1), has long been associated with C. neoformans virulence. It has been hypothesized that melanin protects the yeast from oxidative or nitrosative damage originating from the host cells. To test strains for melanization, we utilize plates containing 100 ng/ml l-DOPA.
|2% Difco Bacto Agar (Becton-Dickinson, Cat.|
|7.6 mM l-asparagine monohydrate||1 g|
|5.6 mM glucose||1 g|
|22 mM KH2PO4||3 g|
|1 mM MgSO4·7H2O||250 mg|
|0.5 mM l-DOPA (Sigma, Cat. No. D9628)||100 mg|
|0.3 mM thiamine–HCl||1 mg|
|20 nM biotin||5 μg|
|Water||to 1 l|
To make 1 l of l-DOPA plate medium, autoclave 20 g of Difco Bacto Agar in 900 ml water so that it dissolves. In 100 ml water, add l-asparagine, glucose, KH2PO4, MgSO4·7H2O, and l-DOPA in the amounts indicated in the above recipe. Add phosphoric acid to the medium to pH 5.6, then add thiamine–HCl and biotin. Mix with the dissolved agar, and pour into plates.
Secretion of a polysaccharide capsule is one of the major virulence factors of C. neoformans. When C. neoformans is mixed with India ink, the particles of the ink are excluded by a network of capsule fibers, thereby producing a characteristic halo around the yeast cell (Fig. 33.6B). Capsule is produced at low levels in typical YPAD culture—for best visualization, capsule production must be induced. Capsule can be induced through two different methods, described as follows.
Capsule induction via low nutrient conditions:
Capsule induction via carbon dioxide exposure:
Visualization of capsule by India ink staining:
While the methods described here are by no means all inclusive for what can be accomplished with C. neoformans, we hope they provide a guide for working with this basidiomycete, as well as a starting point for adapting other techniques.
We thank Joseph Heitman, Jennifer Lodge, Gary Cox, Tamara Doering, John Perfect, Peter Williamson, Christina Hull, Andrew Alspaugh, James Kronstad, Thomas Kozel, Arturo Casadevall, June Kwon-Chung, and Suzanne Noble for sharing strains, protocols, reagents, and general expertise. We thank Jessica Brown and Oliver Liu for critical reading of this manuscript. The work was supported by an Opportunity Grant from the Herb and Marion Sandler Foundation and a grant from the NIAID (R01AI065519). C. D. C. was supported by a National Science Foundation Predoctoral Fellowship.