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Supplementary information
VG molecular types of C. gattii isolates from Vancouver Island. Analysis of the 8
MLST loci for strains originating on Vancouver Island identified 2 outliers. The vast
majority (>95%) of Vancouver Island outbreak specimens were represented by only two
genotypes of the VGII class that were both present in clinical, veterinary, and
environmental samples. Two exceptional VGI isolates were identified (one clinical and
one veterinary) that may be attributable to infections acquired elsewhere and thus were
not studied further.
Isolation of a C. gattii diploid isolate supports cryptic same-sex mating. During
monokaryotic fruiting of C. neoformans diploidisation of haploid  strains occurs,
leading to the production of / diploid intermediates6. Thus, if monokaryotic fruiting
also occurs in C. gattii, one prediction is that diploid isolates should occur naturally in
the population. To test this hypothesis, 95 C. gattii isolates from the outbreak were
subjected to FACS analysis (29 clinical, 7 veterinary, 59 environmental), and one
diploid environmental isolate from the Vancouver Island outbreak was identified
(RB59). MLST analysis indicates that this strain represents a homozygous diploid
composed of only the major outbreak genotype. Hence, it appears to be the product of a
major genotype isolate that underwent same-sex mating with itself, as might be
predicted to occur most commonly given that this genotype represents the vast majority
of outbreak isolates. Importantly, this is an environmental isolate and was not involved
in either a human or animal case. In the same-sex mating model, monokaryotic fruiting
occurs in the environment, leading first to diploid isolates and then to the production of
filaments and basidia, meiosis, and sporulation to produce haploid spores as the
infectious propagules. In this model, diploid isolates are predicted to be present in the
environment, but are expected to be underrepresented or absent in clinical cases in
which haploid spores are hypothesized to represent the infectious particle.
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An extension of MLST analysis to include 30 loci. Based on similarities in MLST
profiles, nine representative VGII strains were selected for more in-depth analysis using
a further 22 loci. These fell into three distinct groups. The first group included strains
that showed an identical or high level of similarity to the Vancouver Island major
MLST genotype (including Vancouver Island major genotype clinical isolate R265,
Vancouver Island environmental diploid RB59, the type strain NIH444, San Francisco
environmental isolate CBS7750, and Brazil clinical isolate ICB107). The second
included those with a similarity to the Vancouver Island minor genotype (including
Vancouver Island minor genotype clinical isolate R272, Australian clinical isolate NT13 from a fertile, recombining population, and Caribbean Islands clinical isolate
99/473). And finally a divergent VGII genotype strain, NT-8, which shares two
identical alleles with the outbreak major genotype in the 8-locus analysis and also
belongs to the fertile, sexually recombining Australian population2, 3.
When these new data were combined with the previous MLST information, the only
isolates identical to the Vancouver Island major genotype strain R265 across all 30 loci
were the Seattle clinical isolate NIH444 and San Francisco environmetal isolate
CBS7750, indicating the major genotype has been present in the Pacific Northwest for
at least thirty years. The only isolate identical to the Vancouver Island minor genotype
strain R272 was the fertile Australian isolate NT-13, indicating the minor outbreak
genotype may have originated in Australia. Comparison of the major and minor
genotypes revealed 14 loci were identical, and 16 were divergent with 1 to 13 changes
between the alleles in an average MLST length of 585 bp. However, in each case a
single isolate from patients with unknown travel history from Central or South America
was identified that differed at only one locus from the major (ICB107, a Brazilian
clinical isolate) or minor (99/473, a Caribbean Islands clinical isolate) genotype,
suggesting these genotypes may have also been introduced with eucalyptus trees in
these areas also, and subsequently undergone genetic drift. In contrast, the Australian
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isolate NT-8 shared only 7 out of 30 identical alleles with strain R265, and is therefore
more distantly related.
On the geographic origin of the outbreak strains. Extended MLST analysis revealed
two intriguing clinical isolates, one from Brazil (ICB107) and the other from the
Carribbean Islands (99/473). Examination of the 30 marker MLST panel reveals that
ICB107 shares a near identical genotype (only one single nucleotide polymorphism in
30 loci) with the Vancouver Island outbreak major genotype isolates (R265, NIH444,
and CBS7750), whereas the Caribbean Island isolate 99/473 shares a near identical
genotype (again, only one single nucleotide polymorphism in 30 loci) with the
Vancouver Island outbreak minor genotype isolates (R272 and NT-13). This analysis
raises two possible models as to the origin of the Vancouver Island outbreak genotypes.
In the first model, mating occurred in Australia and strains were subsequently exported
in association with eucalyptus trees. In this model, some transit also occurred to other
locales in the Americas in addition to the Pacific Northwest, and the isolates from Brazil
and the Caribbean are related to those from the outbreak by genetic drift. In the second
model, the original mating event occurred in South America, the isolates both then
underwent genetic drift, and were subsequently transferred to both Australia and the
Pacific Northwest. We favor the first hypothesis given that strains with identical MLST
profiles as the minor genotype exist in multiple isolates in Australia, and they are part of
a fertile actively recombining population.
A related hypothesis posits that the recombinant outbreak genotype should have
originated in a geographic locale in which the VGII molecular type exhibits the highest
level of genetic diversity. Our analysis reveals that the highest level of diversity exists
in South America (12 genotypes amongst 16 VGII isolates), with more modest diversity
observed in Australia (7 genotypes amongst 24 VGII isolates), and the lowest level of
diversity on Vancouver Island (only 2 genotypes). In this scenario, a genetic cross could
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have occurred in South America with the minor genotype then transferred to both
Australia and Vancouver Island (R272, NT-13) and the major genotype only to
Vancouver Island and the Pacific Northwest (R265, NIH444, CBS7750). However,
despite the higher level of diversity in South America strains with exact MLST profiles
that match the major nor the minor genotype have not in fact as yet been isolated there.
We therefore favor the hypothesis that the genetic cross occurred in Australia. That only
two isolated South/Central American strains have been identified that are related to but
not identical with either the major and minor genotypes suggests that they are related by
genetic drift.
Relationship of virulence of R265 and NIH444. The Vancouver Island major
genotype isolate R265 was cultured from a bronchial wash, and the NIH444 isolate
(which shares the same genotype) was derived from a sputum sample from a patient in
Seattle in the 1970s. Thus, both isolates are from the pulmonary tract of human patients.
Both are highly virulent, but the R265 isolate was more virulent than NIH444 in the
murine model, and this difference was statistically significant (P < 0.001). We
considered two hypotheses to account for this difference. In the first, R265 is more
virulent because of recent passage in humans. In the second, NIH44 is less virulent
because of laboratory passage. Because both isolates are derived from bronchial or
sputum samples, and R265 was not derived for example from the CNS or CSF fluid, we
consider it unlikely that virulence of the R265 isolate is attributable to human passage
through tissues that would select for higher virulence. Rather, we favor the hypothesis
that NIH444 has been modestly attenuated by laboratory passage, storage, or both in the
30 years ensuing since its original isolation.
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Supplemental References
1.
Fraser, J. A. et al. Convergent evolution of chromosomal sex-determining
regions in the animal and fungal kingdoms. PLoS Biol. 2, 2243-2255 (2004).
2.
Campbell, L. T. et al. Clonality and recombination in genetically differentiated
subgroups of Cryptococcus gattii. Eukaryot. Cell 4, 1403-1409 (2005).
3.
Campbell, L. T. et al. Clinical and environmental isolates of Cryptococcus gattii
from Australia that retain sexual fecundity. Eukaryot. Cell 4, 1410-1419 (2005).
4.
Wang, P., Cutler, J., King, J. & Palmer, D. Mutation of the regulator of G
protein signaling Crg1 increases virulence in Cryptococcus neoformans. Eukaryot. Cell
3, 1028-1035 (2004).
5.
Fraser, J. A., Subaran, R. L., Nichols, C. B. & Heitman, J. Recapitulation of the
sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii:
implications for an outbreak on Vancouver Island, Canada. Eukaryot. Cell 2, 1036-1045
(2003).
6.
Lin, X., Hull, C. M. & Heitman, J. Sexual reproduction between partners of the
same mating-type in Cryptococcus neoformans. Nature 434, 1017-1021 (2005).
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Supplementary figure legends
Supplementary Figure 1 Congruence of gene phylogeny in C. gattii. Maximum
parsimony analyses of multiple loci from C. gattii supports the presence of four
discrete molecular types (See also Figure 1, Supplementary Table 1). Each
allele shown is present in one or more isolate from the analysed population. The
Vancouver Island major and minor genotypes are depicted with red arrows.
1000 bootstrap replicates were sampled for the calculation of statistical support.
Thickened lines indicate branches with a bootstrap value ≥ 70%. The C.
neoformans clinical strain H99 (MAT) or 125.91 (MATa) are included as an
outgroup.