Tao3 mediates a phenotypic switch between amoeba-adapted and mammalian-adapted forms of Cryptococcus neoformans

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionMany microbes are capable of changing phenotypes more frequently than due to basal mutation rates alone, and this ability is coupled to pathogenesis. The human pathogenic yeast Cryptococcus neoformans is found in the environment in soil, pigeon guano and tree species, locations in which the organism is exposed to microbial predators. Previous research showed that co-incubation of C. neoformans with amoeba causes a switch from a yeast to a pseudohyphal form, enabling fungal survival in amoeba yet conversely reducing virulence in mammalian models of cryptococcosis. We identify the basis for pseudohyphal development in phenotypic-switched and amoeba-derived strains, to show that genes encoding proteins of the RAM (Regulation of Ace2p activity and cellular Morphogenesis) pathway bear mutations. Reversion to wild type yeast morphology can occur through multiple different mechanisms to suggest that underlying rates of spontaneous mutation control this process and thereby influence the pathogenic potential of an organism

RAM pathway mutants are attenuated for virulence, and can only cause disease if they revert to wild type in a mouse model.

<p><b>A.</b> Survival of mice infection with wild type strain G (<i>n</i> = 9), <i>mob2</i> mutant DM09 (<i>n</i> = 10) and complemented strain AI255 (<i>n</i> = 9). <b>B.</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 <i>mob2</i> 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. <b>C.</b> Cell morphology and <i>MOB2</i> chromatograms and sequences from strains isolated from mice infected with the <i>mob2</i> mutant strain (bar = 50 µm). The <i>mob2</i> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002936#ppat.1002936.s003" target="_blank">dataset S3</a>.</p

The ATCC 24067A strain used in phenotypic switching is a hypermutator strain.

<p><b>A.</b> Profile of <i>ura5</i> 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 <i>ura5</i> mutants with rearrangements. <b>B.</b> Ten-fold serial dilutions of ATCC 24067 and ATCC 24067A plated on different media types, and grown for two days. BHP is <i>t</i>-butyl hydroperoxide and EtBr is ethidium bromide.</p

A RAM suppressor phenotype is meiotically stable, and segregates in a Mendelian manner.

<p><b>A.</b> Phenotypes of wild type, <i>tao3::NAT</i> deletion strain, and suppressor (sup.). Cells in the suppressor strain are often lemon-shaped and have cell separation defects. Bar = 50 µm. <b>B.</b> Growth of parental strains and 20 progeny from a cross between suppressor (strain AI235ya) and wild type strain KN99<b>a</b>. Mating type was tested by crossing to <b>a</b> and α strains as an independent genetic locus segregating in the progeny. Cells were plated onto YPD medium, supplemented with nourseothricin or FK506.</p

DNA Mutations Mediate Microevolution between Host-Adapted Forms of the Pathogenic Fungus <em>Cryptococcus neoformans</em>

<div><p>The disease cryptococcosis, caused by the fungus <em>Cryptococcus neoformans</em>, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of <em>C. neoformans</em> with amoeba can cause a “switch” from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, <em>C. neoformans</em> is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under <em>in vitro</em> conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology <em>in vitro</em> or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.</p> </div

RAM pathway mutants can establish an infection within mice.

<p><b>A.</b> Cell counts 24 and 96 h post-inoculation with wild type strain G or <i>mob2</i> mutant strain DM09. Three mice were used for each time point and strain, expect <i>mob2</i> 24 h in which two mice were used. Each error bar indicates the standard error of the mean. <b>B.</b> and <b>C.</b> H&E stained lung tissue from mice sacrificed 96 h after inoculation with wild type or the <i>mob2</i> mutant strains. Bar = 50 µm.</p

Pseudohyphal strains of <i>C. neoformans</i> derived from independent sources bear RAM pathway mutations.

<p>The <i>TAO3</i> 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. <b>A.. </b><i>C. neoformans</i> var. <i>grubii</i>, in the strain G or KN99α backgrounds. The strains C, D and E were isolated from exposure to amoeba in the 1970s. <b>B.. </b><i>C. neoformans</i> var. <i>neoformans</i>, all in the ATCC 24067A strain background. Strain F7 is from a phenotypic switching study <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002936#ppat.1002936-Fries2" target="_blank">[27]</a>. Sequence information for these mutations is provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002936#ppat.1002936.s001" target="_blank">dataset S1</a>.</p

Pseudohyphal strains derived from three origins share similar characteristics.

<p>The <i>tao3</i>Δ 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. <b>A.</b> Light microscopy of cells (bar = 50 µm). <b>B.</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.</p

Phenotypes of <i>C. neoformans</i> var. <i>grubii</i> RAM pathway mutants related to fitness or strain isolation from the environment.

<p><b>A.</b> Equivalent growth between two strains (KN99α and AI236) on 10% pigeon guano medium, three days at 30°C. <b>B.</b> Growth and melanization of 10-fold serial dilutions on bird seed agar; six days at 22°C. <b>C.</b> 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). <b>D.</b> RAM pathway mutants are infertile. Wild type (KN99<b>a</b>×KN99α) or <i>tao3</i>Δ (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 <i>tao3</i>×<i>tao3</i> cross does not. Bar = 500 µm.</p