Figure 1.
Pseudohyphal strains derived from three origins share similar characteristics.
The tao3Δ strain is in the KN99α background. Strains C, D and E were isolated after exposure to amoeba, probably in the strain G background. All three strains derived from exposure to amoeba exhibited identical phenotypes, so just D is illustrated. F7 is a phenotypic switching isolate in the ATCC 24067A background. WT = wild type. A. Light microscopy of cells (bar = 50 µm). B. Growth under different conditions. Cells were 10-fold serially diluted and spotted onto YPD medium with or without FK506 (1 µg/ml), and grown for two days at 30°C or 37°C.
Figure 2.
Pseudohyphal strains of C. neoformans derived from independent sources bear RAM pathway mutations.
The TAO3 gene is most commonly mutated in pseudohyphal strains. Mutations in “historical” isolates are in black, spontaneous mutants in blue, T-DNA insertional mutants in brown, and from amoeba in red. A. C. neoformans var. grubii, in the strain G or KN99α backgrounds. The strains C, D and E were isolated from exposure to amoeba in the 1970s. B. C. neoformans var. neoformans, all in the ATCC 24067A strain background. Strain F7 is from a phenotypic switching study [27]. Sequence information for these mutations is provided in dataset S1.
Figure 3.
A tao3 point mutation can be reconstituted by homologous recombination with a wild type fragment of TAO3.
Strain D was transformed with a modified fragment of TAO3 by biolistic bombardment. Strains were plated on FK506 to select for wild type growth. A. and B. To ensure gene replacement rather than a reversion event, a BglII site (grey, a-g mutation in bold) was engineered near the affected codon. C. Morphology of strain D and two FK506R strains (bar = 50 µm). D. Amplification by PCR and restriction digestions with BglII. Size markers = Invitrogen 1 kb+ ladder. Strain AI210 has undergone homologous recombination, while strain AI221 is a spontaneous revertant.
Figure 4.
C. neoformans RAM pathway mutants are resistant to Acanthamoeba castellanii.
A. C. neoformans var. grubii wild type (KN99α) and tao3Δ deletion (AI235) strains grown on 5% V8 juice agar on 10 cm diameter Petri dishes. Amoeba were dropped at the intersection of the cross and plates incubated for 14 days at room temperature. B. Interactions between a mixture of wild type and tao3 mutant with amoeba, illustrating the size difference between amoeba and pseudohyphal cells (left panel) and internalization of yeast or occasional pseudohyphal cells into amoeba (right panel). Scale bar = 50 µm.
Figure 5.
Multiple mechanisms revert mutations in RAM pathway genes to wild type morphology.
Mutant strains were plated on FK506 to select for revertants. Regions surrounding the mutations were amplified and sequenced. A. Reversion of the stop codon in tao3 mutant F7. B. Reversion of a 16 bp tandem duplication in SOG2. The duplicated region is colored blue. Changes between strains that are responsible for the mutation or reversion back to wild type are colored red.
Figure 6.
The ATCC 24067A strain used in phenotypic switching is a hypermutator strain.
A. Profile of ura5 mutations in strains ATCC 24067 and ATCC 24067A. Orange lines are positions of transversions, green are positions of transitions, and black indicates indels. The black line under ATCC 24067 represents three ura5 mutants with rearrangements. B. Ten-fold serial dilutions of ATCC 24067 and ATCC 24067A plated on different media types, and grown for two days. BHP is t-butyl hydroperoxide and EtBr is ethidium bromide.
Figure 7.
A RAM suppressor phenotype is meiotically stable, and segregates in a Mendelian manner.
A. Phenotypes of wild type, tao3::NAT deletion strain, and suppressor (sup.). Cells in the suppressor strain are often lemon-shaped and have cell separation defects. Bar = 50 µm. B. Growth of parental strains and 20 progeny from a cross between suppressor (strain AI235ya) and wild type strain KN99a. Mating type was tested by crossing to a and α strains as an independent genetic locus segregating in the progeny. Cells were plated onto YPD medium, supplemented with nourseothricin or FK506.
Figure 8.
Phenotypes of C. neoformans var. grubii RAM pathway mutants related to fitness or strain isolation from the environment.
A. Equivalent growth between two strains (KN99α and AI236) on 10% pigeon guano medium, three days at 30°C. B. Growth and melanization of 10-fold serial dilutions on bird seed agar; six days at 22°C. C. Colony adherence is reduced in RAM mutants. 10-fold serial dilutions were plated on YPD agar and grown six days at 22°C (left). The plate was washed under running water and photographed (right). D. RAM pathway mutants are infertile. Wild type (KN99a×KN99α) or tao3Δ (AI236×AI257) crosses were mixed on Murashige-Skoog medium, incubated in darkness at room temperature, and the edge of the mix photographed ten days later. Wild type crosses produce a mass of filaments, terminating in chains of basidiospores whereas the tao3×tao3 cross does not. Bar = 500 µm.
Figure 9.
RAM pathway mutants are attenuated for virulence in wax moth larvae.
Survival of wax moth larvae infected with wild type strain G (n = 11), mob2 mutant DM09 at two concentrations (n = 11 of 1×105 cfus, n = 10 of 1×104 cfus), complemented strain AI255 (n = 12), and phosphate buffered saline control (n = 11).
Figure 10.
RAM pathway mutants can establish an infection within mice.
A. Cell counts 24 and 96 h post-inoculation with wild type strain G or mob2 mutant strain DM09. Three mice were used for each time point and strain, expect mob2 24 h in which two mice were used. Each error bar indicates the standard error of the mean. B. and C. H&E stained lung tissue from mice sacrificed 96 h after inoculation with wild type or the mob2 mutant strains. Bar = 50 µm.
Figure 11.
RAM pathway mutants are attenuated for virulence, and can only cause disease if they revert to wild type in a mouse model.
A. Survival of mice infection with wild type strain G (n = 9), mob2 mutant DM09 (n = 10) and complemented strain AI255 (n = 9). B. Colony forming units measured from brain and lung tissue of mice when sacrificed. For the wild type and complemented strain, three mice were used. For the mob2 mutant, all ten mice were examined. Four (#1–#4) that caused disease symptoms were sacrificed. The other six were sacrificed at day 70. Three had cleared the infection (not on graph), while three had the fungal burdens indicated. C. Cell morphology and MOB2 chromatograms and sequences from strains isolated from mice infected with the mob2 mutant strain (bar = 50 µm). The mob2 mutant carries the g-a mutation that impairs splicing. Strains from mice #4 and 5 have a reversion mutation, while strains from mice #7 and #10 are pseudohyphal and maintain the original mutation. Sequence data from additional strains is provided as dataset S3.