Article

Activation of Meiotic Genes Mediates Ploidy

Reduction during Cryptococcal Infection
Graphical Abstract Authors
Youbao Zhao, Yina Wang,
Srijana Upadhyay, Chaoyang Xue,
Xiaorong Lin

Correspondence
xuech@njms.rutgers.edu (C.X.),
xiaorong.lin@uga.edu (X.L.)

In Brief
Zhao et al. show that meiosis genes
facilitate ploidy reduction in the global
fungal pathogen Cryptococcus
neoformans, and they are activated
during infection. Progeny isolated from
the host that have meiotic genes
activated show increased resistance to
specific genotoxic stress, indicating the
occurrence of meiosis may empower
adaptative mutations.

Highlights
d Meiosis-specific genes are activated in C. neoformans cells
during infection

d Deletion of meiotic genes increases the proportion of large

cells during infection

d Deletion of DMC1 delays the ploidy reduction of

C. neoformans cells

d Progeny with meiotic genes activated show increased

resistance to genotoxic stress

Zhao et al., 2020, Current Biology 30, 1387–1396

April 20, 2020 ª 2020 Elsevier Ltd.
https://doi.org/10.1016/j.cub.2020.01.081
Current Biology

Article

Activation of Meiotic Genes Mediates Ploidy

Reduction during Cryptococcal Infection
Youbao Zhao,1 Yina Wang,2 Srijana Upadhyay,1,4 Chaoyang Xue,2,* and Xiaorong Lin1,3,5,*
1Department of Microbiology, University of Georgia, Athens, GA 30602, USA
2Public Health Research Institute Center, New Jersey Medical School - Rutgers, The State University of New Jersey, Newark, NJ 07103, USA
3Department of Plant Biology, University of Georgia, Athens, GA 30602, USA
4Present address: Department of Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA
5Lead Contact

*Correspondence: xuech@njms.rutgers.edu (C.X.), xiaorong.lin@uga.edu (X.L.)

https://doi.org/10.1016/j.cub.2020.01.081

SUMMARY This fungus is responsible for 15% of deaths in AIDS patients

Cryptococcus neoformans is a global human fungal
pathogen that causes fatal meningoencephalitis in
mostly immunocompromised individuals. During pul-
monary infection, cryptococcal cells form large poly-
ploid cells that exhibit increased resistance to host im-
mune attack and are proposed to contribute to the
latency of cryptococcal infection. These polyploid
titan cells can generate haploid and aneuploid prog-
eny that may result in systemic infection. What trig-
gers cryptococcal polyploidization and how ploidy
reduction is achieved remain open questions. Here,
we discovered that Cryptococcus cells polyploidize
in response to genotoxic stresses that cause DNA
double-strand breaks. Intriguingly, meiosis-specific
genes are activated in C. neoformans and contribute
to ploidy reduction, both in vitro and during infection
in mice. Cryptococcal cells that activated their meiotic
genes in mice were resistant to specific genotoxic
stress compared to sister cells recovered from the
same host tissue but without activation of meiotic
genes. Our findings support the idea that meiotic
genes, in addition to their conventional roles in classic
sexual reproduction, contribute to adaptation of eu-
karyotic cells that undergo dramatic genome changes
in response to genotoxic stress. The discovery has
additional implications for evolution of sexual repro-
duction and the paradox of the presence of meiotic
machinery in asexual species. Finally, our findings
in this eukaryotic microbe mirror the revolutionary dis-
coveries of the polyploidization and meiosis-like
ploidy reduction process in cancer cells, suggesting
that the reversible ploidy change itself could provide
a general mechanism for rejuvenation to promote indi-
vidual survival in response to stress.
INTRODUCTION
Cryptococcus neoformans is a global pathogen causing menin-
goencephalitis in mostly immunocompromised individuals [1].
[2]. Its bisexual cycle involving partners of a and a mating types
has been defined for decades [3]. However, the a mating type
predominates among natural cryptococcal populations, particu-
larly in the serotype A clade, which is responsible for the vast ma-
jority of cryptococcosis (MATa > 99%). Our previous discoveries
that a single C. neoformans strain can complete a sexual cycle in
the absence of a partner of the opposite mating type (unisexual
reproduction) [4] and that MATa alleles enhance unisexual repro-
duction in vitro [5] offer a plausible explanation for its dominance
in nature. Both unisexual and bisexual reproductions naturally
entail changes in nuclear DNA content (1C-2C-4C-1C), due to
nuclear fusion or endoreplication, premeiotic replication, and
meiosis. Although unisexual reproduction differs from bisexual
reproduction in that the non-self-recognition system for cell
fusion is dispensable [6], both sexual reproduction modes
require meiosis to achieve ploidy reduction and have only been
observed in vitro.
Meiosis is a specialized reductive cell division. A single round
of DNA replication followed by two consecutive DNA divisions
enables reduction in the nuclear DNA content. During the first
meiotic division (meiosis I), extensive DNA recombination takes
place between two parental homologous chromosomes, which
are then segregated from one another. The second round of
meiotic DNA division (meiosis II) more closely resembles mitotic
DNA division, in which sister chromatids are segregated from
each other. Diversification of genotypes during meiosis is pri-
marily attributed to re-shuffling the pre-existing genetic se-
quences. Generation of novel genetic information, however, is
the less known but critical feature of meiosis due to a much
higher rate of de novo mutations that are dependent on DNA
double-strand breaks (DSBs) [7–10].
C. neoformans mostly exists in the haploid yeast state when
cultured under standard laboratory conditions. During pulmo-
nary infection, a portion of C. neoformans cells becomes poly-
ploid in DNA content (typically tetraploid and sometimes higher)
and larger in cell size (>10 mm) than standard haploid yeast cells
(4–7 mm) [11, 12]. These large titan cells can evade host immune
surveillance and are more resistant to various stresses,
enhancing C. neoformans survival in the hostile pulmonary envi-
ronment [11, 13, 14]. Cell gigantism can be potentially promoted
by many different stimuli, such as serum, CO2, phospholipids,
or nutrient starvation [15–18]. Isolated polyploid cells can
generate haploid progeny under permissive conditions in vitro

Current Biology 30, 1387–1396, April 20, 2020 ª 2020 Elsevier Ltd. 1387
[14]. Whether meiotic machinery is involved in this ploidy reduc-
tion process remains an open question. Here, we observed the
activation of meiotic genes in cryptococcal cells during infection
with a single isolate. We showed that genotoxic stresses that
cause DSBs induce cryptococcal polyploidization and that
meiotic genes contribute to the subsequent ploidy reduction
process. Remarkably, cryptococcal cells that activated their
meiotic genes during infection are more resistant to specific gen-
otoxic stress that causes DSBs compared to sister cells recov-
ered from the same animal tissue.
RESULTS
Meiosis-Specific Genes in C. neoformans Are Activated
during Infection
Bisexual and unisexual reproductions in Cryptococcus are asso-
ciated with yeast-to-hypha transition under in vitro conditions.
Ploidy increase is achieved either through cell fusion followed
by nuclear fusion (bisex) or mostly through endoreplication (uni-
sex; Figure 1A). Meiosis occurs in the basidial heads formed
at the apexes of aerial hyphae to halve the DNA content prior
to sporulation. Meiosis-specific factors, such as the meiosis-
specific recombinase Dmc1 and the meiosis-specific compo-
nent of the cohesin complex Rec8, are highly conserved from
yeasts to humans and they are critical for ploidy reduction in
a sexual cycle [19–21]. Deletion of DMC1 (CNAG_ 07909) or
REC8 (CNAG_04404) abolishes sporulation without affecting
cryptococcal hyphal and basidia development [4, 22] (Figure 1B),
consistent with their specific roles in meiosis.
Given that a portion of Cryptococcus cells become polyploid
in vivo (Figure 1C) and these cells can then undergo ploidy reduc-
tion to produce haploid progeny when cultured in vitro [14], we
wondered whether meiotic machinery contributes to the ploidy
reduction during infection. It is, however, challenging to monitor
the occurrence of meiosis or the activation of meiotic genes
in vivo for the following reasons: (1) only a small portion of the
cryptococcal population in mouse lungs become polyploid and
only a subset of these polyploid cells will likely undergo depoly-
ploidization in vivo [13, 14]; (2) among this subset of cells, ploidy
reduction could be achieved through mitotic cycles where chro-
mosomes are randomly lost (parasexual cycle); (3) even if the
meiotic machinery is activated, the expression of meiosis-spe-
cific genes are likely transient; and (4) the clinical reference strain
H99 amplifies rapidly in mice [23–26], and mitotic divisions from
highly proliferative haploid cells likely dominate in this host.
In order to detect potential transient expression of meiotic
genes occurring at a very low frequency, we employed a flip
reporter (FR) using a modified recombination-based in vivo
expression technology [27, 28]. The FR approach links the acti-
vation of meiosis-specific genes to the production of a site-spe-
cific recombinase Cre by driving the expression of CRE with the
promoter of the meiosis genes (Figure 1D). There are two pairs of
Cre recognition sites bordering a drug selection marker that
is inversely placed after a promoter of a housekeeping gene
TEF1 (PTEF1). Consequently, the drug selection marker is not
expressed in the original strain. If meiosis genes are activated,
the CRE will be expressed, and the resulting Cre enzyme causes
inversion at its recognition loci and correct orientation of the drug
marker gene with the PTEF1 promoter, resulting in the expression
1388 Current Biology 30, 1387–1396, April 20, 2020
of a dominant drug resistance selection marker. Thus, even tran-
sient activation of a meiosis-specific gene will yield a heritable
genotypic change that leads to a permanent phenotypic change
conferring nourseothricin (NAT) resistance.
We chose to monitor the activation of meiosis-specific genes,
DMC1 and REC8. We next constructed FR strains in which CRE
is driven by the promoter of the DMC1 or the REC8 gene. The
FRDMC1 and FRREC8 strains showed no differences from the
wild-type (WT) in terms of growth, thermo-tolerance, capsule
production, or melanization (Figures S1A and S1B). To examine
whether the appearance of NAT resistance in FRDMC1 and
FRREC8 is coupled to meiosis in vitro, we first tested the FRDMC1
a and FRREC8 a strains cultured alone in media that favor yeast
mitotic growth (e.g., YPD; left upper panel, Figure 1E). No
NAT-resistant colonies were recovered from either FRDMC1 a or
FRREC8 a under this culture condition (middle and right panels,
Figure 1E). Likewise, no NAT resistance was detected when
FRDMC1 a or FRREC8 a was co-cultured with a WT a strain on
media that favor yeast mitotic growth (Figure 1E). By contrast,
NAT-resistant progeny were recovered from a cross between
the FRDMC1 a or the FRREC8 a strain and a WT a mating partner
on the mating-inducing V8 agar medium (Figure 1E). The recov-
ery of relatively small number of cells with DMC1 and REC8 acti-
vated from the cross on V8 agar medium is not unexpected
because a cryptococcal mating colony is highly heterogeneous,
with the vast majority of cells dividing mitotically and only a small
fraction of cells at the periphery of the mating colony engaging in
sexual reproduction (especially so for strains of the H99 back-
ground). Additionally, the FRDMC1 and FRREC8 systems likely
report strong, but not all biologically relevant, activation of these
meiotic genes occurring during meiosis. This postulation is
based on the observation that the number of meiotic progeny
yielded by a control cross was 50–100 times higher than the de-
tected flip events. For negative controls, we generated a promo-
terless flip reporter and a flip reporter without Cre (Figure S1C).
We found extremely low levels of false-positive activation of FR
caused by leaky expression of the promoterless Cre or sponta-
neous flip without Cre. Furthermore, their activation was not
associated with sexual reproduction (Figure S1D). Collectively,
these findings indicate that the activation of DMC1 and REC8 de-
tected in these FRDMC1 a and FRREC8 a strains is coupled with
meiosis in vitro and is likely an underestimation of meiotic events.
To determine whether DMC1 and REC8 are activated during
infection, we inoculated mice with the FRDMC1 a or the FRREC8
a strain using the intranasal infection model. The FRDMC1 a and
FRREC8 a reporter strains were as virulent as the unmarked WT
a strain based on lung fungal burden (Figure S1E). NAT-resistant
colonies were recovered from the lungs of every mouse infected
with the FRDMC1 a strain or the FRREC8 a strain (Figures 1E and
S1F), although the frequency of NAT-resistant isolates recovered
was low and varied among individual mice and the batch of the
animal experiment (Figures 1E and S1F). Collectively, our find-
ings show that the meiosis-specific genes DMC1 and REC8
are activated during infection in the murine model.
Deletion of Meiosis-Specific Genes Increases the
Proportion of Large Cryptococcal Cells during Infection
Given the conserved role of meiosis in ploidy reduction, the acti-
vation of meiosis-specific genes during cryptococcal infection
Figure 1. Meiosis-Specific Genes Are Activated in C. neoformans Cells during Infection
(A) Diagram of cryptococcal unisexual reproduction and concomitant ploidy changes.
(B) Deletion of DMC1 or REC8 blocks cryptococcal sporulation. Bilateral crosses of WT H99 (axa), dmc1D (axa), and rec8D (axa) were cultured on V8 agar
medium and imaged under a light (upper) or scanning electron microscope (bottom), respectively. White arrows point at spore chains, and red arrows point at
basidium heads.
(C) Diagram of the distribution of cell size and ploidy during cryptococcal infection.
(D) Diagram of the double-floxed Cre-mediated flip reporter to detect activation of meiosis-specific genes. The double-floxed (loxP and lox2722) dominant drug
marker NAT is placed inverted relative to the constitutively active promoter of TEF1 (PTEF1). The expression of the site-specific recombinase Cre is driven by the
promoter of a meiosis-specific gene (DMC1 or REC8). Activation of meiosis-specific genes induces the expression and production of Cre. Cre recognizes the two
pairs of lox sites, resulting in inversion of the NAT drug marker and excision of one loxP site and one lox2722 site. The incompatibility of remaining unpaired loxP
and lox2722 sites impedes further Cre-mediated inversion. This ensures correct orientation between PTEF1 and NAT and concomitant transcription of NAT and
consequent resistance to nourseothricin.
(E) Diagram of testing the flip reporter strains in vitro and in vivo (left panel). The two tables to the right show the number of flip events detected with the FRDMC1 or
the FRREC8 reporter system when the cells were cultured in vitro (upper panel) or in vivo (infected mice were euthanized at DPI 13 to measure the flip events;
bottom panel). The in vitro experiments were repeated at least three times, and all showed a consistent pattern. Results from one batch of experiments were
shown here. The in vivo flip events were detected independently from the experiments conducted in Lin lab at the University of Georgia and Xue lab at Rutgers
University.
See also Figure S1.

raises the possibility that meiotic machinery is activated in vivo,
and this could contribute to depolyploidization of titan cells. To
test the effects of deletion of these meiosis-specific genes on
cryptococcal gigantism in vivo, we inoculated mice intranasally
with WT H99, dmc1D, or rec8D cells. We then collected crypto-
coccal cells from bronchoalveolar lavage (BAL) fluid at day 3
post infection (DPI 3) to analyze their cell size. We observed an
increased proportion of titan cells from the BAL fluid infected
with the dmc1D mutant and the rec8D mutant compared to
WT (Figures 2A and 2B). An increased proportion of titan cells
in the lungs in the absence of DMC1 or REC8 was observed in
the a as well as the a mating type background (Figures 2A and
2B). Histopathological examination of fungal cells by Grocott’s
methenamine silver (GMS) staining revealed an increase in large

Current Biology 30, 1387–1396, April 20, 2020 1389

Figure 2. Deletion of Meiosis-Specific Genes Increases the Proportion of Large Cells during Cryptococcal Infection
(A) BAL fluid and histopathological examination of WT (H99a or KN99a strain alone), dmc1D (a or a strain alone), and rec8D (a or a strain alone) during lung
infection. 5 3 106 cells of each of these six strains were intranasally inoculated into mice. Cryptococcal cells were recovered from BAL fluid at DPI 3. These fungal
cells were examined under microscope with or without India ink negative staining (the white halo surrounding yeast cells reflects the capsule). For histological
examination, the lungs were fixed and stained with GMS. Fungal cells appear dark brown or black with GMS staining.
(B) Quantification of the percentage of cells larger than 10 mm in WT (a or a), dmc1D (a or a), and rec8D (a or a) during lung infection. For each of the six strains,
diameters of 400 cells were quantified based on the microscopic images. Five independent cell size quantifications for each strain were conducted for statistical
analysis. Each error bar indicates the standard deviation among the five independent quantifications. **p < 0.01 (t test); ***p < 0.001 (t test).
(C) Lung fungal burden of mice infected with WT H99 and dmc1D. 1 3 104 of haploid cryptococcal cells were intranasally inoculated into mice, and lung fungal
burden was analyzed at DPI 1, 3, and 7. Five mice were used for each group, and the error bars represent the standard deviations. N.S., p = 0.21 (t test); *p < 0.05
(t test); **p < 0.01 (t test).

cryptococcal cells in the lungs of mice infected with the dmc1D
or the rec8D mutant (brown-black staining in Figures 2A and 2B).
As the ploidy of titan cells is known to be proportional to their cell
size [16], these observations suggest that these core meiosis-
specific genes contribute to changes in cryptococcal cell size
and ploidy during cryptococcal infection.
An increase in the polyploid subpopulation could lead to
reduced proliferation due to decreased haploid subpopulation,
resulting in delayed progression of disease or reduced fungal
burden. To test this hypothesis, we inoculated mice with haploid
WT and dmc1D mutant strains and compared their lung fungal
burden at DPI 1, 3, and 7. No difference was observed at DPI
1, indicating consistent original inoculum (Figure 2C). At both
DPI 3 and DPI 7, however, the dmc1D mutant showed a subtle
but significant reduction in the lung fungal burden relative to
WT in the murine model (Figure 2C). The modest reduction in
fungal burden of the dmc1D mutant in this animal model is ex-
pected: mice are hyper-susceptible to this highly aggressive
C. neoformans reference strain H99, and most of the haploid
cells that do not become titan cells likely dominate in this model
host, consequently diminishing the virulence attenuation effects
from the deletion of the meiosis-specific gene. Nonetheless, the
result indicates that DMC1 contributes to cryptococcal disease
progression.
Deletion of DMC1 Retards Ploidy Reduction without
Affecting Polyploidization Induced by Zeocin In Vitro
Titan cell formation can be potentially promoted by many stimuli
present in the host, such as serum, CO2, phospholipids, and
nutrient starvation [15–18]. However, the nature of the stress
that ultimately triggers Cryptococcus polyploidization remains
unknown. In some eukaryotes, polyploidization is known to serve
as a response to genotoxic stress [29–33]. Here, we found that
g-irradiation, which caused DNA DSBs and high levels of cell
death at the tested doses (Figure S2A), indeed induced poly-
ploidization of C. neoformans based on propidium iodide (PI)
staining (Figure S2B). The result supports the hypothesis that
cryptococcal polyploidization is a response to genotoxic stress
that causes DSBs. To further test this hypothesis, we treated
Cryptococcus cells with different genotoxic stresses, including
different doses of ultraviolet radiation (UV), methyl methanesul-
fonate (MMS), and zeocin (Figure S2C). Zeocin is a radiomimetic
DNA-damaging chemical known to cause DNA DSBs in different
organisms [33–36]. In C. neoformans, treatment with zeocin at a
high concentration resulted in cell death (Figures S2C and S3A)
and DNA DSBs, which were detected by the TUNEL assay (Fig-
ure S3B). After treating cryptococcal cells with various genotoxic
stresses, we observed cell size enlargement only in response to
g-irradiation and zeocin, which cause DSBs, but not to UV or
MMS (Figures S2D and S2F), even though all these treatments
at the tested high doses resulted in a poor survival rate (Fig-
ure S2C). Consistent with the enlargement of cell size,
C. neoformans populations increased in ploidy following zeocin
treatment (Figures S2E and S3C), further supporting the idea
that genotoxic stresses that cause DSBs induce polyploidiza-
tion. In this process, we noticed that PI staining of zeocin-treated
cells gave some signal in the cytosol, suggesting that PI staining
may not be the best approach to monitor DNA content in zeocin-
treated cells. Therefore, we decided to use the GFP-labeled his-
tone H2B subunit, which is widely used in various eukaryotes to
reflect nuclear DNA content [33, 37] (Figure S3D). We then
treated the H2B-GFP-labeled strain with zeocin and assayed
changes in cell size and ploidy. As shown in Figures 3A–3D, zeo-
cin treatment yielded enlarged cells with an approximately 4C
nuclear DNA content, consistent with our observation with PI
staining in the non-labeled strain (Figure S3C). Unlike two-peak

1390 Current Biology 30, 1387–1396, April 20, 2020

Figure 3. Deletion of DMC1 Does Not Affect the Increase in Cell Size and Ploidy Induced by Zeocin, but It Delays the Size and Ploidy Reduc-
tion Process
(A) Microscopic images of cryptococcal cells with or without zeocin treatment. Overnight cultures of H2B-GFP-tagged H99 cells in liquid YPD medium were
treated with zeocin (80 mg/mL) for 5 h at 30
C with shaking. An aliquot of cells was fixed for microscopy (A) and flow cytometry (B) analyses. The remaining cells
were washed twice with PBS buffer and re-cultured in liquid YPD at 30
C with shaking for the time course assay (C–F).
(B) Flow cytometry analysis using the forward scatter signal (proxy for cell size) and the H2B-GFP signal (proxy for nuclear DNA content) of cryptococcal cells with
or without zeocin treatment.
(C) Diameters of untreated cells (n = 50), zeocin-treated cells (n = 52), and released cells (n = 51) of WT H99 were measured based on microscopic images. The
zeocin-released cell populations shown here in (C)–(F) were collected at 24 h after being transferred to the zeocin-free medium. Violin plots in (C) and (E) were
made with the R package of ggplot2 [38].
(D) Flow cytometry analysis of H2B-GFP-labeled WT cells in response to zeocin treatment and 24 h after released from zeocin. The total numbers of events
analyzed by flow cytometry are shown in the panel.
(E) Diameters of untreated, zeocin-treated, and released dmc1D cells (n = 50 for all) were measured based on microscopic images.
(F) Flow cytometry analysis of H2B-GFP-labeled dmc1D cells in response to zeocin treatment and 24 h after released from zeocin. The total numbers of events
analyzed by flow cytometry are shown. Experiments were independently repeated three times.
(legend continued on next page)

Current Biology 30, 1387–1396, April 20, 2020 1391

fluorescence-activated cell sorting (FACS) profiles observed in
normal cryptococcal populations, it is notable that only one
peak was observed in the FACS profile of cells treated with zeo-
cin. Similar flow cytometry profiles were observed when
analyzing Cryptococcus populations induced to undergo cell
enlargement in vitro in other studies [16–18]. This could be attrib-
utable to zeocin treatment itself, a population composed of cells
in different stages of the cell cycle or ploidy, and/or the use of
H2B-GFP, which cannot accurately reflect cell cycle compared
to direct measurement of DNA content. Nonetheless, the upshift
in ploidy and cell size was obvious upon zeocin treatment or
g-irradiation. Given that C. neoformans in the host is subject to
genotoxic insults (e.g., external ROS from host immune attack
or internal ROS induced by starvation or hypoxia), it is conceiv-
able that cryptococcal polyploidization is a response to geno-
toxic stress that causes DNA DSBs during infection.
The majority of the dmc1D population became polyploid (4C)
and large in cell size after zeocin treatment to a level comparable
to the zeocin-treated WT population (Figures 3E and 3F), indi-
cating that Dmc1 does not affect the cryptococcal polyploidiza-
tion process. After transferring the zeocin-treated WT cells to
zeocin-free YPD media, we observed a reversal of cell size and
ploidy in the population based on flow cytometry assays (Figures
3C, 3D, and 3G). Deletion of DMC1 obviously delayed the reduc-
tion of both cell size and ploidy after the enlarged cells were
released into zeocin-free YPD medium (Figures 3E–3G). The
dmc1D mutant cells eventually reversed back to the haploid sta-
tus after prolonged incubation in zeocin-free media (Figure 3G).
These results indicate that meiosis-specific factor Dmc1 contrib-
utes to depolyploidization of these large polyploid cryptococcal
cells.
Cryptococcal Cells that Activated Their Meiosis-
Specific Genes during Infection Show Resistance
Specific to Zeocin
The evidence presented above demonstrates that meiosis-spe-
cific genes are activated during infection and they contribute to
cryptococcal pathogenesis. Because we infected mice with a
single isolate of only one mating type, we expect that cells with
activated meiosis-specific genes would appear clonal to the
parental strain both genotypically and phenotypically, even if
meiosis occurred in these cells during infection. Here, we
decided to compare cryptococcal cells with or without these
meiosis genes being activated during infection. As activation of
the meiosis genes is rare in vivo, we resorted to our flip reporter
system to identify these progeny. We refer to cells that activated
their meiosis-specific genes as ‘‘flipped’’ strains. The flipped
strains are NAT resistant although the original strain and non-flip-
ped progeny are NAT sensitive (Figures 1D and 1E). We picked
68 flipped progeny recovered from the lungs and brains of
mice infected with either the FRDMC1 a strain or the FRREC8 a
strain. For comparison, we picked 24 non-flipped sister strains
(G) A time course flow cytometry analysis of H2B-GFP-labeled WT and dmc1D cells in response to zeocin (80 mg/mL) treatment and after released from zeocin for
8, 24, 30, and 36 h. Zeocin-treated cells were released to zeocin-free YPD media, and released cells were collected at the indicated time points for FACS analysis
of H2B-GFP intensity. The vertical lines in each panel indicate the H2B-GFP intensity representing the 2C DNA content. The percentages of the population that
have less than 2C DNA content (DNA < 2C) or greater than 2C DNA content (DNA > 2C) were presented in each panel above the horizontal indicator lines. More
than 10,000 cells were analyzed for each sample.
See also Figures S2 and S3.
1392 Current Biology 30, 1387–1396, April 20, 2020
recovered from the same host tissues. We then examined these
strains for their stress tolerance, including resistance to the anti-
fungal fluconazole, H2O2, UV, zeocin, and MMS. Overall, the
non-flipped strains behaved similarly as the original WT H99 in
all the stress tests, although there were subtle variations (Figures
4A and S4C). Surprisingly, the flipped strains showed a signifi-
cantly wider range of resistance level to zeocin compared to
the non-flipped sister strains or the original H99 strain (Figure 4A),
but not to the DNA-damaging stress caused by H2O2 (Fig-
ure S4C), UV irradiation, or MMS (Figures 4A and 4B). To confirm
the variation in zeocin resistance among H99, the non-flipped
strains, and the flipped strains, we conducted a time course
growth assay. As shown in Figure 4C, flipped progeny showed
a wider range of resistance to zeocin although H99 and all
the non-flipped progeny were sensitive to zeocin (Figure 4C).
Collectively, these results reveal phenotypic diversity among
the flipped progeny and implicate specific DNA-damaging
stress in activating meiosis-specific genes during cryptococcal
infection.
DISCUSSION
Cryptococcosis is one of the leading causes of death among
patients with HIV in sub-Saharan Africa [2]. Epidemiological
evidence, however, indicates a high prevalence of non-symptom-
atic cryptococcal infections in the general immunocompetent
population. C. neoformans can respond to the hostile environ-
ment in the lungs of an immunocompetent host by polyploidiza-
tion. This fungus can return to normal ploidy under permissive
conditions in vitro. Here, we discovered that C. neoformans poly-
ploidizes in response to specific genotoxic stresses that cause
DSBs, a type of stress that this fungus likely experiences in the
host, based on other studies [40]. DMC1 and REC8, two highly
conserved meiosis-specific genes, are activated during crypto-
coccal disease progression in mice, and they contribute to ploidy
reduction of polyploid cells when growth conditions become
permissive. Disruption of DMC1 reduces cryptococcal virulence
in the murine model. As immunocompetent humans are much
more resistant to Cryptococcus than mice, we postulate that
polyploid cells likely represent a much larger proportion of pulmo-
nary cryptococcal populations in humans than in mice. We spec-
ulate that, in patients with polyploid cryptococcal cells, ploidy
reduction occurs when the host immunity is impaired due to
HIV infection or immunosuppressive therapies (e.g., in organ
transplant recipients). Meiotic machinery is activated in polyploid
cells and contributes to their ploidy reduction. The generated
proliferative haploid progeny can cause fatal systemic infections.
The impact of meiotic genes on cryptococcosis might be much
more dramatic in humans than what is observed in mice. Our find-
ings open a new avenue to investigate the key and yet poorly
understood aspects of cryptococcal disease progression (i.e.,
latency and subsequent reactivation).
Figure 4. Phenotypic Diversity of the Flipped Progeny Recovered from Infected Mice
(A) A quantitative representation of phenotypic assays of the flipped and the non-flipped progeny recovered from animal lungs and brains after animals
were euthanized at DPI 13. Cells were diluted into optical density 600 (OD600) = 0.03 and spotted onto YPD agar media in a 96-spot format with the
indicated stress factors at the indicated concentrations. Images were taken after 4 days of incubation at 30
C and analyzed using Colonyzer [39]. The median
growth index values are shown as black bars. See Figure S4 and Tables S1 and S2 for the raw resource images, data, and the strain list. Mann-Whitney test was
used to analyze the growth difference between WT, the flipped, and the non-flipped strains under zeocin condition. The p values of the pairwise comparisons
were shown.
(B) Serial dilutions of the selected flipped and non-flipped progeny were spotted onto YPD agar media supplemented with the indicated stress factors at the
indicated doses. Cells were then incubated at 30
C for 4 days. The parental haploid H99 strain and a diploid strain (H99a/H99a) served as controls. The strain
labels correspond to their plate positions in Figure S4B.
(C) A time course growth assay of the flipped, non-flipped, and WT strains cultured in liquid YPD with or without zeocin. The overnight cultures of the strains used
in (B) were diluted into OD = 0.05 and inoculated into liquid YPD or YPD with 10 mg/mL of zeocin in a 48-well plate. The plates were cultured at 30
C with
continuous shaking, and growth was monitored by measuring OD600 every hour. The growth of the WT, the non-flipped, and the flipped strains based on OD600
measurement was plotted against the time after inoculation (growth curve assay). The colored range shows the distribution of growth curves of the WT (black), the
non-flipped (orange), and the flipped (green) strains.
See also Figure S4.

At a broader level, the reversible ploidy changes associated
with the progression of cryptococcosis are strikingly similar to
that of cancer, in which various cancer cells become polyploid
in response to genotoxic chemotherapy or radiation therapy.
These polyploid tumor cells can depolyploidize through a
meiosis-like process, giving rise to rejuvenated and proliferative
cancer cells with normal ploidy, which are often resistant to the
therapy [30, 41–43]. The same conserved meiotic genes, such
as DMC1 and REC8, are activated in TP53 mutant tumors,
enhanced by genotoxic treatments or spindle inactivation and
associated with reversible polyploidy [44–47]. Such reversible
ploidy events are rare (in the scale of 1–10 out of a million), as
most cancer cells die due to exposure to the genotoxic treat-
ment. However, few surviving polyploid cells successfully
reverse ploidy and become proliferative. The consequence of
such events, however, is dire and not rare.
One advantage of using the eukaryotic microbial pathogen
C. neoformans for such research is its genetic amenability and

Current Biology 30, 1387–1396, April 20, 2020 1393

robust growth. For instance, here, we could select the low pro- d METHOD DETAILS
portion of progeny that activated their meiotic genes during B Flip reporter systems
cryptococcal infection in mice with the Cre-loxP-mediated flip B Gene manipulation
reporter system. It facilitates phenotypically dissection of these B Murine cryptococcosis and fungal burden analysis
rare populations of cells. An intriguing observation from this B Bronchoalveolar lavage (BAL) fluid and histopathology
study is the increased resistance to zeocin of the progeny that analysis
activated their meiotic genes in mice compared to the sister B Crosses and the estimation of meiotic events
non-flipped progeny isolated from the same host tissue. These B g irradiation
cells had never been exposed to zeocin prior to the stress test. B DNA damage stresses
We do not think that the insertion of the flip reporter system B TUNEL Assay
into the cryptococcal genome itself caused zeocin resistance B Microscopy
in the flipped strains for the following reasons: (1) the non-flipped B Flow cytometry analysis
progeny are sensitive to zeocin and they carry the same con- B Phenotypic assays
structs as the flipped ones; (2) FRDMC1 was inserted randomly B Funding Statement
into the genome and FRREC8 was inserted into the safe haven re- d QUANTIFICATION AND STATISTICAL ANALYSIS
gion [48], and yet flipped strains derived from both reporters d DATA AND CODE AVAILABILITY
showed a similar phenotype; and (3) not all flipped strains are
resistant to zeocin despite carrying the same reporter system. SUPPLEMENTAL INFORMATION
Because we inoculated mice with a single haploid cryptococcal
Supplemental Information can be found online at https://doi.org/10.1016/j.
isolate H99, recombination or assortment of chromosomes, typi- cub.2020.01.081.
cally observed during classic meiosis in a heterozygous zygote
derived from mating of two genetically distinct parental strains, ACKNOWLEDGMENTS
is unlikely to account for such phenotypic diversity observed in
our study. However, meiosis is a mutagenic process that can We thank Beth Richardson and Dr. John Shields at the Georgia Electron Micro-
generate mutations de novo with much higher rates than mitosis scopy laboratory for SEM analyses, Julie Nelson at the UGA Cytometry Shared
Resource laboratory for her help with flow cytometry analysis using the CyAn
[7]. It is possible that the phenotypic diversity and specific resis-
ADP cell analyzer (NIH grant 1S10RR027814), and Dr. Wendy Watford for her
tance to zeocin observed in the flip strains might have resulted help with g-irradiation. We thank Drs. Douda Bensasson, Ence Yang, Alex-
from increased mutations due to bona fide meiosis or atypical ander Idnurm, Joseph Heitman, Bing Zhai, and the Lin lab members for their
mitosis with some involvement of the meiotic machinery. Future input on this project. This work was supported by the National Institutes of
in-depth comparative genomic analysis of the flipped and the Health (R21AI132125 and R01AI140719 to X.L. and R01AI123315 to C.X.)
non-flipped progeny may reveal the underlying genetic bases. and the University of Georgia (fund to X.L.). X.L. holds an Investigator Award
in the Pathogenesis of Infectious Disease from the Burroughs Wellcome
Our findings provide a new avenue to explore the molecular
Fund (1012445). The funders had no role in study design, data collection,
mechanisms underlying the mutagenic property of meiosis, the and interpretation, or the decision to submit the work for publication.
genetic bases for the preservation of meiotic machinery in
asexual organisms/cells, and the impact of meiotic genes on AUTHOR CONTRIBUTIONS
the evolution and adaption of species.
The findings reported here from a clinically important eukary- Conceived and designed the experiments, Y.Z. and X.L.; Performed the exper-
otic microbe together with the mounting literature on polyploid- iments, Y.Z., Y.W., S.U., and C.X.; Analyzed the data and wrote the paper,
Y.Z., C.X., and X.L.
ization and ploidy reduction via a meiosis/meiosis-like process
in cancer cells suggest a general mechanism for rejuvenation DECLARATION OF INTERESTS
in eukaryotes in response to genotoxic stress to promote individ-
ual survival. It is our conviction that eukaryotic cells possess an The authors declare no competing interests.
inherent capability of gametogenesis. Whether it is a bona fide
meiotic process or a meio-mitotic process [47] involving some Received: August 2, 2019
of the meiotic machinery warrants further investigation. This Revised: December 4, 2019
Accepted: January 28, 2020
and future investigations will yield novel insights into a defining
Published: February 27, 2020
feature of eukaryotes and will have far-reaching implications
for our understanding of a wide range of diseases. REFERENCES

1. Casadevall, A., and Perfect, J.R. (1998). Cryptococcus Neoformans (ASM

STAR+METHODS Press).
2. Rajasingham, R., Smith, R.M., Park, B.J., Jarvis, J.N., Govender, N.P.,
Detailed methods are provided in the online version of this paper Chiller, T.M., Denning, D.W., Loyse, A., and Boulware, D.R. (2017).
and include the following: Global burden of disease of HIV-associated cryptococcal meningitis: an
updated analysis. Lancet Infect. Dis. 17, 873–881.
d KEY RESOURCES TABLE 3. Kwon-Chung, K.J. (1976). A new species of Filobasidiella, the sexual state
d LEAD CONTACT AND MATERIALS AVAILABILITY of Cryptococcus neoformans B and C serotypes. Mycologia 68, 943–946.
d EXPERIMENTAL MODEL AND SUBJECT DETAILS 4. Lin, X., Hull, C.M., and Heitman, J. (2005). Sexual reproduction between
B Fungi partners of the same mating type in Cryptococcus neoformans. Nature
B Animal studies 434, 1017–1021.

1394 Current Biology 30, 1387–1396, April 20, 2020

 5. Lin, X., Huang, J.C., Mitchell, T.G., and Heitman, J. (2006). Virulence attri-
butes and hyphal growth of C. neoformans are quantitative traits and the
MATalpha allele enhances filamentation. PLoS Genet. 2, e187.
6. Gyawali, R., Zhao, Y., Lin, J., Fan, Y., Xu, X., Upadhyay, S., and Lin, X.
(2017). Pheromone independent unisexual development in Cryptococcus
neoformans. PLoS Genet. 13, e1006772.
7. Rattray, A., Santoyo, G., Shafer, B., and Strathern, J.N. (2015). Elevated
mutation rate during meiosis in Saccharomyces cerevisiae. PLoS Genet.
11, e1004910.
8. Arbeithuber, B., Betancourt, A.J., Ebner, T., and Tiemann-Boege, I. (2015).
Crossovers are associated with mutation and biased gene conversion at
recombination hotspots. Proc. Natl. Acad. Sci. USA 112, 2109–2114.
9. Halldorsson, B.V., Palsson, G., Stefansson, O.A., Jonsson, H., Hardarson,
M.T., Eggertsson, H.P., Gunnarsson, B., Oddsson, A., Halldorsson, G.H.,
Zink, F., et al. (2019). Characterizing mutagenic effects of recombination
through a sequence-level genetic map. Science 363, eaau1043.
10. Arbel-Eden, A., and Simchen, G. (2019). Elevated mutagenicity in meiosis
and its mechanism. BioEssays 41, e1800235.
11. Zaragoza, O., and Nielsen, K. (2013). Titan cells in Cryptococcus neofor-
mans: cells with a giant impact. Curr. Opin. Microbiol. 16, 409–413.
12. Zaragoza, O., Garcı́a-Rodas, R., Nosanchuk, J.D., Cuenca-Estrella, M.,
Rodrı́guez-Tudela, J.L., and Casadevall, A. (2010). Fungal cell gigantism
during mammalian infection. PLoS Pathog. 6, e1000945.
tien,
13. Okagaki, L.H., Strain, A.K., Nielsen, J.N., Charlier, C., Baltes, N.J., Chre
F., Heitman, J., Dromer, F., and Nielsen, K. (2010). Cryptococcal cell
morphology affects host cell interactions and pathogenicity. PLoS
Pathog. 6, e1000953.
14. Gerstein, A.C., Fu, M.S., Mukaremera, L., Li, Z., Ormerod, K.L., Fraser,
J.A., Berman, J., and Nielsen, K. (2015). Polyploid titan cells produce
haploid and aneuploid progeny to promote stress adaptation. MBio 6,
e01340-15.
15. Chrisman, C.J., Albuquerque, P., Guimaraes, A.J., Nieves, E., and
Casadevall, A. (2011). Phospholipids trigger Cryptococcus neoformans
capsular enlargement during interactions with amoebae and macro-
phages. PLoS Pathog. 7, e1002047.
16. Dambuza, I.M., Drake, T., Chapuis, A., Zhou, X., Correia, J., Taylor-Smith,
L., LeGrave, N., Rasmussen, T., Fisher, M.C., Bicanic, T., et al. (2018). The
Cryptococcus neoformans Titan cell is an inducible and regulated mor-
photype underlying pathogenesis. PLoS Pathog. 14, e1006978.
17. Trevijano-Contador, N., de Oliveira, H.C., Garcı́a-Rodas, R., Rossi, S.A.,
Llorente, I., Zaballos, Á., Janbon, G., Ariño, J., and Zaragoza, Ó. (2018).
Cryptococcus neoformans can form titan-like cells in vitro in response to
multiple signals. PLoS Pathog. 14, e1007007.
18. Hommel, B., Mukaremera, L., Cordero, R.J.B., Coelho, C., Desjardins,
C.A., Sturny-Leclère, A., Janbon, G., Perfect, J.R., Fraser, J.A.,
Casadevall, A., et al. (2018). Titan cells formation in Cryptococcus neofor-
mans is finely tuned by environmental conditions and modulated by pos-
itive and negative genetic regulators. PLoS Pathog. 14, e1006982.
19. Bishop, D.K., Park, D., Xu, L., and Kleckner, N. (1992). DMC1: a meiosis-
specific yeast homolog of E. coli recA required for recombination, synap-
tonemal complex formation, and cell cycle progression. Cell 69, 439–456.
20. Kagawa, W., and Kurumizaka, H. (2010). From meiosis to postmeiotic
events: uncovering the molecular roles of the meiosis-specific recombi-
nase Dmc1. FEBS J. 277, 590–598.
21. Mehta, G.D., Kumar, R., Srivastava, S., and Ghosh, S.K. (2013). Cohesin:
functions beyond sister chromatid cohesion. FEBS Lett. 587, 2299–2312.
22. Liu, L., He, G.J., Chen, L., Zheng, J., Chen, Y., Shen, L., Tian, X., Li, E.,
Yang, E., Liao, G., and Wang, L. (2018). Genetic basis for coordination
of meiosis and sexual structure maturation in Cryptococcus neoformans.
eLife 7, e38683.
23. Perfect, J.R., Lang, S.D., and Durack, D.T. (1980). Chronic cryptococcal
meningitis: a new experimental model in rabbits. Am. J. Pathol. 101,
177–194.
24. Janbon, G., Ormerod, K.L., Paulet, D., Byrnes, E.J., 3rd, Yadav, V.,
Chatterjee, G., Mullapudi, N., Hon, C.C., Billmyre, R.B., Brunel, F., et al.
(2014). Analysis of the genome and transcriptome of Cryptococcus neo-
formans var. grubii reveals complex RNA expression and microevolution
leading to virulence attenuation. PLoS Genet. 10, e1004261.
25. Whittington, A., and Wang, P. (2011). The RGS protein Crg2 is required for
establishment and progression of murine pulmonary cryptococcosis.
Med. Mycol. 49, 263–275.
26. Ngamskulrungroj, P., Chang, Y., Sionov, E., and Kwon-Chung, K.J. (2012).
The primary target organ of Cryptococcus gattii is different from that of
Cryptococcus neoformans in a murine model. MBio 3, 1–9.
27. Sohal, V.S., Zhang, F., Yizhar, O., and Deisseroth, K. (2009). Parvalbumin
neurons and gamma rhythms enhance cortical circuit performance.
Nature 459, 698–702.
28. Livet, J., Weissman, T.A., Kang, H., Draft, R.W., Lu, J., Bennis, R.A.,
Sanes, J.R., and Lichtman, J.W. (2007). Transgenic strategies for combi-
natorial expression of fluorescent proteins in the nervous system. Nature
450, 56–62.
29. Erenpreisa, J., Cragg, M.S., Salmina, K., Hausmann, M., and Scherthan,
H. (2009). The role of meiotic cohesin REC8 in chromosome segregation
in gamma irradiation-induced endopolyploid tumour cells. Exp. Cell Res.
315, 2593–2603.
30. Illidge, T.M., Cragg, M.S., Fringes, B., Olive, P., and Erenpreisa, J.A.
(2000). Polyploid giant cells provide a survival mechanism for p53 mutant
cells after DNA damage. Cell Biol. Int. 24, 621–633.
31. Chumduri, C., Gillissen, B., Richter, A., Richter, A., Milojkovic, A.,
Overkamp, T., Müller, A., Pott, C., and Daniel, P.T. (2015). Apoptosis resis-
tance, mitotic catastrophe, and loss of ploidy control in Burkitt lymphoma.
J. Mol. Med. (Berl.) 93, 559–572.
32. Fox, D.T., and Duronio, R.J. (2013). Endoreplication and polyploidy: in-
sights into development and disease. Development 140, 3–12.
33. Adachi, S., Minamisawa, K., Okushima, Y., Inagaki, S., Yoshiyama, K.,
Kondou, Y., Kaminuma, E., Kawashima, M., Toyoda, T., Matsui, M.,
et al. (2011). Programmed induction of endoreduplication by DNA dou-
ble-strand breaks in Arabidopsis. Proc. Natl. Acad. Sci. USA 108,
10004–10009.
34. Chankova, S.G., Dimova, E., Dimitrova, M., and Bryant, P.E. (2007).
Induction of DNA double-strand breaks by zeocin in Chlamydomonas
reinhardtii and the role of increased DNA double-strand breaks rejoining
in the formation of an adaptive response. Radiat. Environ. Biophys. 46,
409–416.
35. Shimada, K., Filipuzzi, I., Stahl, M., Helliwell, S.B., Studer, C., Hoepfner,
D., Seeber, A., Loewith, R., Movva, N.R., and Gasser, S.M. (2013).
TORC2 signaling pathway guarantees genome stability in the face of
DNA strand breaks. Mol. Cell 51, 829–839.
36. Seeber, A., Dion, V., and Gasser, S.M. (2013). Checkpoint kinases and the
INO80 nucleosome remodeling complex enhance global chromatin
mobility in response to DNA damage. Genes Dev. 27, 1999–2008.
37. Zhang, C., Gong, F.C., Lambert, G.M., and Galbraith, D.W. (2005). Cell
type-specific characterization of nuclear DNA contents within complex
tissues and organs. Plant Methods 1, 7.
38. Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis
(Springer).
39. Lawless, C., Wilkinson, D.J., Young, A., Addinall, S.G., and Lydall, D.A.
(2010). Colonyzer: automated quantification of micro-organism growth
characteristics on solid agar. BMC Bioinformatics 11, 287.
40. Thomson, G.J., Hernon, C., Austriaco, N., Shapiro, R.S., Belenky, P., and
Bennett, R.J. (2019). Metabolism-induced oxidative stress and DNA dam-
age selectively trigger genome instability in polyploid fungal cells. EMBO
J. 38, e101597.
41. Erenpreisa, J., Salmina, K., Huna, A., Jackson, T.R., Vazquez-Martin, A.,
and Cragg, M.S. (2014). The ‘‘virgin birth’’, polyploidy, and the origin of
cancer. Oncoscience 2, 3–14.
Current Biology 30, 1387–1396, April 20, 2020 1395
42. Erenpreisa, J., Salmina, K., Huna, A., Kosmacek, E.A., Cragg, M.S.,
Ianzini, F., and Anisimov, A.P. (2011). Polyploid tumour cells elicit paradi-
ploid progeny through depolyploidizing divisions and regulated autopha-
gic degradation. Cell Biol. Int. 35, 687–695.
43. Erenpreisa, J., and Cragg, M.S. (2013). Three steps to the immortality of
cancer cells: senescence, polyploidy and self-renewal. Cancer Cell Int.
13, 92.
44. Kalejs, M., Ivanov, A., Plakhins, G., Cragg, M.S., Emzinsh, D., Illidge, T.M.,
and Erenpreisa, J. (2006). Upregulation of meiosis-specific genes in
lymphoma cell lines following genotoxic insult and induction of mitotic
catastrophe. BMC Cancer 6, 6.
45. Ianzini, F., Kosmacek, E.A., Nelson, E.S., Napoli, E., Erenpreisa, J., Kalejs,
M., and Mackey, M.A. (2009). Activation of meiosis-specific genes is asso-
ciated with depolyploidization of human tumor cells following radiation-
induced mitotic catastrophe. Cancer Res. 69, 2296–2304.
46. Vitale, I., Senovilla, L., Jemaà, M., Michaud, M., Galluzzi, L., Kepp, O.,
Nanty, L., Criollo, A., Rello-Varona, S., Manic, G., et al. (2010). Multipolar
mitosis of tetraploid cells: inhibition by p53 and dependency on Mos.
EMBO J. 29, 1272–1284.
47. Salmina, K., Huna, A., Kalejs, M., Pjanova, D., Scherthan, H., Cragg, M.S.,
and Erenpreisa, J. (2019). The cancer aneuploidy paradox: In the light of
evolution. Genes (Basel) 10, E83.
48. Upadhya, R., Lam, W.C., Maybruck, B.T., Donlin, M.J., Chang, A.L.,
Kayode, S., Ormerod, K.L., Fraser, J.A., Doering, T.L., and Lodge, J.K.
(2017). A fluorogenic C. neoformans reporter strain with a robust expres-
sion of m-cherry expressed from a safe haven site in the genome.
Fungal Genet. Biol. 108, 13–25.
49. Patel, R.D., Lodge, J.K., and Baker, L.G. (2010). Going green in
Cryptococcus neoformans: the recycling of a selectable drug marker.
Fungal Genet. Biol. 47, 191–198.
1396 Current Biology 30, 1387–1396, April 20, 2020
50. Toffaletti, D.L., Rude, T.H., Johnston, S.A., Durack, D.T., and Perfect, J.R.
(1993). Gene transfer in Cryptococcus neoformans by use of biolistic de-
livery of DNA. J. Bacteriol. 175, 1405–1411.
51. Fan, Y., and Lin, X. (2018). Multiple applications of a transient CRISPR-
Cas9 coupled with electroporation (TRACE) system in the Cryptococcus
neoformans species complex. Genetics 208, 1357–1372.
52. O’Brien, C.R., Krockenberger, M.B., Wigney, D.I., Martin, P., and Malik, R.
(2004). Retrospective study of feline and canine cryptococcosis in
Australia from 1981 to 2001: 195 cases. Med. Mycol. 42, 449–460.
53. Krockenberger, M.B., Canfield, P.J., and Malik, R. (2003). Cryptococcus
neoformans var. gattii in the koala (Phascolarctos cinereus): a review of
43 cases of cryptococcosis. Med. Mycol. 41, 225–234.
54. Zhao, Y., Upadhyay, S., and Lin, X. (2018). PAS domain protein Pas3 inter-
acts with the chromatin modifier Bre1 in regulating cryptococcal morpho-
genesis. MBio 9, e02135-18.
55. Kolb, A.F. (2001). Selection-marker-free modification of the murine beta-
casein gene using a lox2722 site. Anal. Biochem. 290, 260–271.
56. Nielsen, K., Cox, G.M., Wang, P., Toffaletti, D.L., Perfect, J.R., and
Heitman, J. (2003). Sexual cycle of Cryptococcus neoformans var. grubii
and virulence of congenic a and alpha isolates. Infect. Immun. 71, 4831–
4841.
57. Rivera, A., Ro, G., Van Epps, H.L., Simpson, T., Leiner, I., Sant’Angelo,
D.B., and Pamer, E.G. (2006). Innate immune activation and CD4+ T cell
priming during respiratory fungal infection. Immunity 25, 665–675.
58. Tanaka, R., Taguchi, H., Takeo, K., Miyaji, M., and Nishimura, K. (1996).
Determination of ploidy in Cryptococcus neoformans by flow cytometry.
J. Med. Vet. Mycol. 34, 299–301.
59. Warchal, S. (2018). An R package for working with multi-well plates,
https://github.com/Swarchal/platetools.
STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Chemicals, Peptides, and Recombinant Proteins
Hygromycin Research Products Cat. NO.: H75000
International
G418 Research Products Cat. NO.: G64000
International
Nourseothricin Jena Bioscience Cat. NO.: AB-102-25G
Zeocin VWR Cat. NO.: AAJ67140-8EQ
Propidium iodide Sigma- Aldrich Cat. NO.: P4864-10ML
Experimental Models: Organisms/Strains
Mouse: A/Jcr The Jackson Laboratory CRL:563
Cryptococcus neoformans strain XL280a (MFa, mild type serotype D strain) [5] Strain ID: XL280a
Cryptococcus neoformans strain H99 (MFa, wild type serotype A strain) [23] Strain ID: H99
Cryptococcus neoformans strain KN99 (MFa, wild type serotype A strain) [49] Strain ID: KN99
Cryptococcus neoformans strain YZ83 (MFa, DMC1::NAT serotype A) This manuscript Strain ID: YZ83
Cryptococcus neoformans strain YZ463 (MFa, DMC1::NAT serotype A) This manuscript Strain ID: YZ463
Cryptococcus neoformans strain YZ478 (MFa, REC8::NAT serotype A) This manuscript Strain ID: YZ478
Cryptococcus neoformans strain YZ486 (MFa, REC8::NAT serotype A) This manuscript Strain ID: YZ486
Cryptococcus neoformans strain YZ175 (MFa, PH2B-H2B-GFP-HYG serotype A) This manuscript Strain ID: YZ175
Cryptococcus neoformans strain YZ187 (MFa, DMC1::NAT, PH2B-H2B- This manuscript Strain ID: YZ187
GFP-HYG serotype A)
Cryptococcus neoformans strain YZ63 (MFa, FRDMC1, Serotype A) This manuscript Strain ID: YZ63
Cryptococcus neoformans strain YZ659 (MFa, FRREC8, Serotype A) This manuscript Strain ID: YZ659
Cryptococcus neoformans strain YZ661 (MFa, FRREC8, Serotype A) This manuscript Strain ID: YZ661
Oligonucleotides
See Table S3 This manuscript N/A
Recombinant DNA
pYZ-FRDMC1 (pXL-PDMC1-CRE-PTEF1-NAT-NEO) This manuscript Plasmid ID: pYZ-FRDMC1
pYZ-FRREC8 (pXL-PREC8-CRE-PREC8-NAT-NEO) This manuscript Plasmid ID: pYZ-FRREC8
pYZ99 (pXL1-PH2B-H2B-GFP-HYG) This manuscript Plasmid ID: pYZ99
pYZ179 (pXL1-PTUB4-TUB4–tdTomato-NEO) This manuscript Plasmid ID: pYZ179
Software and Algorithms
R 3.6 The R Foundation https://www.rproject.org/
ggplot2 [47] https://ggplot2.tidyverse.org/
Colonyzer [48] https://research.ncl.ac.uk/
colonyzer/

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiaorong
Lin (xiaorong.lin@uga.edu). Plasmids used in these studies can be made available upon request following the signing of a material
transfer agreement with the University of Georgia. All materials generated in the current study are available from the lead contact
upon reasonable request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Fungi
The C. neoformans strains used in this study are listed in the Key Resources Table. Yeast cells were maintained on YPD medium
unless specified otherwise. Mating assays were conducted on V8 juice agar medium in the dark at 22
C. Transformants obtained

Current Biology 30, 1387–1396.e1–e5, April 20, 2020 e1

by biolistic transformation [50] or by TRACE [51] were selected on YPD with 100 mg/ml of nourseothricin (NAT), 100 mg/ml of neomycin
(NEO), or 200 mg/ml of hygromycin (HYG). For in vivo experiments, C. neoformans strains were cultured in YPD liquid medium over-
night at 30
C with shaking at 220 rpm. The fungal cells were washed with sterile saline three times and resuspended to 1 3 106 cell/ml
in saline.

Animal studies
Clinical observation in animals indicates no sex predisposition of developing cryptococcosis (e.g., cats, dogs, and koalas) [52, 53].
Here we used female A/Jcr mice to minimize the number of mice needed for this study and also to facilitate direct comparison of our
findings to published work that is typically based on results obtained from female mice. Six to eight weeks old A/Jcr mice were
purchased from the Jackson Laboratory. All mice were healthy before the initiation of those studies. Animals were supplied with
hardwood chips as bedding and housed in a temperature-controlled, air-conditioned room on a 12-hr light-dark cycle. The animal
experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals
of the National Institutes of Health and IACUC regulation at University of Georgia and at Rutgers University.

METHOD DETAILS

Flip reporter systems

The coding sequence of the site-specific recombinase Cre fused with the terminator sequence of GPD1 was amplified from the
template plasmid pJL519 [49] with primers Linlab3140/YZ and Linlab3141/YZ. The resulting PCR amplicon was digested with restric-
tion enzymes EcoRV and NotI, and cloned into the vector pXL1-PTEF1 [54], which was digested with the same restriction enzymes.
The promoter of the meiosis-specific gene DMC1 was amplified with primers Linlab3144/YZ and Linlab3145/YZ using the H99
genome as the template. PDMC1 was then cloned onto the upstream of the coding sequence of Cre after digestion with NotI and
XbaI to generate the plasmid pXL1-PDMC1-CRE. Two pairs of Cre-recognition sites, loxP and lox2722 [27, 28, 55], were designed
in a convergent orientation flanking the nourseothricin-resistant gene NAT, and FseI and PacI cutting sites were designed at the
end of the double-floxed inverted NAT cassette for subsequent cloning. The whole double-floxed inverted NAT cassette was syn-
thesized by GenScript (Piscataway, NJ), which was then cloned into pXL1-PDMC1-CRE with FseI and PacI digestion to generate
the flip reporter plasmid pYZ-FRDMC1. The flip reporter plasmid pYZ-FRREC8 was constructed in a similar way, except that the pro-
moter of REC8 was amplified using the primers Linlab4619/YZ and Linlab4620/YZ.

Gene manipulation
To delete the DMC1 open reading frame (ORF) in the H99 background, approximately 1 kb 50 and 30 flanking sequences were ampli-
fied with primer pairs Linlab3660/YZ-Linlab3661/YZ and Linlab3662/YZ-Linlab3663/YZ. The 50 and 30 flanking sequences were then
fused with the split dominant NAT drug resistance marker through overlap PCR with primer pairs Linlab3664/YZ-Linlab1539/Wang
and Linlab3665/YZ-Linlab1540/Wang, respectively. The split deletion constructs were introduced into the indicated recipient strain
by biolistic transformation as described previously [50]. Transformants generated were screened by two rounds of diagnostic PCR.
The first round of PCR was to detect integration of the construct into the correct genetic locus (primer pair Linlab3660/YZ and P-actin
reverse). The second round of PCR was to confirm the replacement of the DMC1 ORF by the drug-selection cassette (primer pair
Linlab3660/YZ and Linlab3663/YZ). A similar strategy was used to delete the REC8 ORF. The primers used in REC8 deletion and
mutant confirmation are listed in the Key Resources Table. The deletion mutants in the MATa background were obtained by dissect-
ing the meiotic progeny from crosses between the MATa deletion strains and the corresponding congenic WT a strain. The mating
type and the gene deletion of the dissected progeny were confirmed through PCR. All the primers used were listed in Key Resources
Table.
To introduce the flip reporters into C. neoformans cells, M13F and M13R were used to amplify the flip reporter cassette with the
drug selection marker from the plasmid pYZ-FRDMC1 or pYZ-FRREC8. The FRDMC1 cassette was introduced into recipient crypto-
coccal cells through biolistic transformation. The FRREC8 cassette was introduced into the safe haven [48] region of the H99a strain
through the recently developed TRACE method [51]. Stable transformants were confirmed by drug resistance after 5 passages on
non-selective media. To test the specificity of the flip reporter system in vitro, cryptococcal cells were cultured under the yeast-
growth-promoting rich YPD medium and mating-promoting V8 medium as shown in Figure 1E. The original inoculum was similar
either in YPD or on V8 media for the flip tests. The final numbers of cells examined under these two different conditions were also
similar. That means the numbers of cell replication were similar. The incubation time was different because cells grow much faster
in YPD medium compared to V8 medium. Stable transformants that did not yield NAT-resistance after overnight cultures either alone
or with KN99a [56] cells in liquid YPD were then crossed with KN99a cells on V8 juice agar media (pH = 5.0). Mating colonies on V8
plates were cultured for 10 days at 22
C in the dark. The edge of the mating colonies containing the mixture of yeasts, hyphae, and
spores were collected and plated onto YPD plates with NAT. Candidates that generate NAT-resistant progeny after mating with the a
partner on V8 agar medium, but not in liquid YPD medium, are considered the positive flip reporter strains. Those were stored for the
next step of the flip reporter assay.
To tag H2B with GFP, the ORF of H2B-coding gene and 578 bp of its upstream sequence were amplified and cloned into the pXL1-
based GFP vector with HYG as the fungal selection marker to generate pYZ99. To tag Tub4 with tdTomato, the ORF of TUB4 and
about 1 kb of its upstream sequence were amplified and cloned into the pXL1-based tdTomato vector with NEO as the fungal

e2 Current Biology 30, 1387–1396.e1–e5, April 20, 2020

selection marker to generate pYZ179. Both vectors were linearized with BglII digestion and introduced into Cryptococcus cells
through biolistic transformation. Positive transformants were confirmed by diagnostic PCR and fluorescent signals.

Murine cryptococcosis and fungal burden analysis

C. neoformans strains were cultured in YPD liquid medium overnight at 30
C with shaking at 220 rpm. The fungal cells were washed
with sterile saline three times and resuspended in saline. Female A/Jcr mice (The Jackson Laboratory, Bar Harbor, ME, USA) of 8 to
10 weeks old were sedated with ketamine and xylazine via intraperitoneal injection. Sedated mice were then inoculated intranasally
with 50 mL fungal cell suspension. After infection, animals were monitored daily for disease progression, including weight loss, gait
changes, labored breathing, or fur ruffling. Infected mice were euthanized at days as indicated. Lungs or brains were dissected and
homogenized in 2 mL cold PBS buffer. The tissue suspensions were serially diluted, plated onto YNB agar medium, and incubated at
30
C for two days to count colony forming units (CFUs). The remaining tissue suspensions were plated onto YPD medium with NAT
for the selection of flipped progeny. For flip event detection, the mice inoculated with 5x106 cells in 50 mL saline and the infected mice
were euthanized at DPI 13. For assessment of the virulence level of meiotic gene deletion mutants, the mice were inoculated with
1x104 cells in 50 mL saline and infected mice were euthanized at DPI 1, 3, and 7.

Bronchoalveolar lavage (BAL) fluid and histopathology analysis

Groups of 6 to 8 weeks old female A/Jcr mice (Jackson Laboratory, Bar Harbor, ME, USA) were infected intranasally with 5x106
C. neoformans cells in 50 mL PBS. At DPI 3, mice were euthanized, and BAL fluid was harvested and processed as previously
described [57]. In brief, a catheter was inserted into the trachea of each animal after euthanasia, and airway-infiltrating cells were
obtained by lavage with 1 mL of 1 3 PBS each time for three times. Cells in the lavage fluid were pelleted at 16,000 g, resuspended
in 3.7% formaldehyde, and incubated at 22
C for 30 min. Cells were then washed once with PBS, and > 300 cells per animal were
analyzed for size by light microscopy. Cells were classified as regular cells (diameter < 10 mm) or titan cells (diameter > 10 mm). Cells
were also visualized with India ink negative staining and observed with an Olympus CX41 microscope equipped with an Infinity digital
camera (Olympus).
For histopathological analysis, lungs were immersed in 10% buffered formalin, paraffin embedded, and stained with modified
Grocott’s methanamine silver (GMS) stain according to the manufacturer’s instructions (Richard-Allan Scientific, Kalamazoo, MI,
USA).
To determine the size of cells isolated from mice, we measured the pixel size of the cell and converted it into actual size according
to the scale of the images. The ratio of cells that are larger than 10 mm was calculated. For each strain background, the size of 400
cells from three microscopic images was determined and five-independent quantifications were conducted for each strain back-
ground for statistical analysis.

Crosses and the estimation of meiotic events

Mating partners (a and a) were grown separately in YPD at 30
C. Cells were collected and equal numbers of a and a cells were mixed
and co-cultured on V8 juice agar medium (pH = 5.0) in the dark at 22
C. Successful mating led to the formation of mating hyphae and
chains of basidiospores at the periphery of the mating colony. The mating process was monitored microscopically.
For crosses between NAT-resistant MATa and G418-resistant MATa strains, cells from the periphery of the mating patches
were collected and suspended in 1 3 PBS. The cell suspension was diluted and spread onto YPD plates with NAT and G418
to select double-drug-resistant colonies. After 3-4 days of incubation on the selective medium at 30
C, colony forming units
(CFUs) were counted. The total number of meiotic progeny yielded from the cross should be approximately four times of the
CFUs of the double-drug-resistant colonies. For crosses between FRDMC1 a and WT a strains, cells collected from the periphery
of the mating patches were plated onto YPD plates with NAT to select the NAT-resistant flipped progeny. The CFUs on the NAT
containing YPD plates indicate the meiotic frequency detected by the flip reporter. This frequency from the crosses between
FRDMC1 a and WTa strains was compared to the meiotic events calculated from the crosses between NAT-resistant MATa and
G418-resistant MATa strains. The comparison indicated the percentage of meiotic events detected by the FRDMC1 a reporter
system.

g irradiation
C. neoformans cells suspended in PBS (1 3 108 cells/mL) in microcentrifuge tubes were g-irradiated (J. L. Shepherd Irradiator) with
500, 1000, and 1500Gy at 22
C. Irradiated samples were then aliquoted into two tubes. Cells from one tube were diluted and plated
onto YPD plates to calculate survival rates; Cells from the other tube were fixed for PI staining and FACS analysis.

DNA damage stresses

In addition to g irradiation, Cryptococcus cells were challenged with other DNA damage stresses, including UV, MMS, and zeocin.
For UV irradiation, 1 3 107 cells were plated onto YPD agar medium, air-dried, and then exposed to UV at a dose of 150, 300, and 450
J/m2. For MMS treatment, 1 3 107 cells in liquid YPD medium were treated with MMS at a final concentration of 0.04% or 0.08% for
five hours at 30
C with shaking at 220 rpm. For zeocin treatment, 1 3 107 cells in liquid YPD medium were treated with zeocin at 10,
20, 40, or 80 mg/ml for five hours at 30
C with shaking at 220 rpm. After treatment, cells were collected and washed with PBS twice.
An aliquot of the washed cells was diluted and plated onto YPD agar medium to count CFUs. The survival rate of control cells without

Current Biology 30, 1387–1396.e1–e5, April 20, 2020 e3

any treatment was set as 100% and the relative survival rate of cells under different treatments were calculated. The remaining cells
were fixed with 3.7% formaldehyde for microscopy and flow cytometry analyses.

TUNEL Assay
An overnight culture of the wild-type H99 strain was washed twice with PBS. Cells were then inoculated into 10 mL of fresh YPD
or YPD plus zeocin (80 ml/ml) to reach OD600 = 1 and cultured for 5 hours at 30
C with shaking at 220 rpm. DNA strand breaks
induced by zeocin were examined by the TUNEL assay with the In Situ Cell Death Detection kit, fluorescein (Catalog No.
11684795910; Roche). Briefly, cryptococcal cells were collected, fixed with 3.7% (vol/vol) formaldehyde for 30 min at 21
C,
and washed three times with PBS. Cell wall of the fixed cells was digested with 20 mg/ml lysing enzymes (Lysing enzymes
from Trichoderma harzianum, Sigma-Aldrich, in a spheroplasting solution: 1M sorbitol, 10 mM EDTA, and 100mM sodium citrate
pH 5.8) at 37
C for 120 min. Digested cells were centrifuged at 800 g for 2 minutes and gently washed three times with PBS. The
digested cryptococcal cells were then incubated in the permeabilization solution [0.1% (vol/vol) Triton X-100 and 0.1% (wt/wt)
sodium citrate] on ice for 2 min and washed twice with PBS. The permeabilized spheroplasts were subsequently incubated with
50 mL of TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase and fluorescein isothiocyanate dUTP, at 37
C
for 60 min. Finally, the spheroplasts were washed three times with PBS and observed under an epi-fluorescence microscope
Zeiss Imager M2 (Carl Zeiss, Jena, Germany). Images were acquired with an AxioCam camera using Zen pro software (Carl Zeiss
Microscopy).

Microscopy
For fluorescent microscopy, the mCherry-tagged or GFP- tagged cells, or the PI-stained cells were observed under Zeiss Imager M2
microscope. Images were acquired with an AxioCam MRm camera and processed with the software Zen pro.
For scanning electron microscopy (SEM), MATa and MATa cells of WT H99, dmc1D, and rec8D were cultured in liquid YPD at
30
C with shaking overnight. Cells were collected and diluted in sterile water into a final density of OD600 = 3. The same number of
MATa and MATa cells of H99, dmc1D, or rec8D were mixed, and 3 mL of the a-a mixture were spotted onto V8 agar and incubated
at 22
C in dark for 14 days for sporulation. Areas 2 mm2 of the mating colonies were excised using a razor blade, placed in vials
of fixative containing 2.5% v/v glutaraldehyde in 0.1 M potassium phosphate buffer (pH = 7.2) and stored overnight at 4
C. Sam-
ples were washed with buffer and post-fixed for at 4
C for 2 h in similarly buffered 1% osmium tetroxide. These samples were then
rinsed in distilled water and dehydrated in a graded ethanol series (25%, 50%, 75%, 95%, and100%). The samples were then
critical point dried using a Samdri model 780-A Critical Point Dryer (Tousimis, Rockville, MD, USA). Samples were mounted on
sticky carbon tabs on top of aluminum stubs, sputter-coated with gold-palladium (Leica EM Ace 600 Sputter Coater, USA),
and viewed using a FEI FE-SEM. Teneo Scanning Electron Microscope (Thermo Fisher Scientific, Hillsboro, OR, USA) operating
at 10 kV.

Flow cytometry analysis

Flow cytometry analysis was performed as described previously [58]. Briefly, H2B-GFP-labeled cells were washed and resus-
pended in PBS buffer for flow cytometry analysis. Cells used for PI staining were first fixed in ice-cold 70% ethanol overnight
at 4
C. The fixed cells were washed, resuspended in 0.5 mL of NS buffer (10 mM Tris-HCl [pH = 7.2], 0.25 M sucrose, 1 mM
EDTA, 1 mM MgCl2, 0.1 mM ZnCl2, 0.4 mM phenylmethylsulfonyl fluoride, 7 mM b-mercaptoethanol). RNase A (0.5 mg/ml)
and propidium iodide (10 mg/ml) were added into the suspension and incubated for 2 h at 37
C or overnight at 4
C in the
dark. The cells were sonicated for 10 s before analysis with a CyAn ADP cell analyzer (Beckman Coulter, Hialeah, FL) or BD Acuri
C6 flow cytometer (BD Biosciences). We noted that different cell analyzers may yield FACS patterns with subtle differences. Pro-
files obtained from the same cell analyzer should be used for direct comparison. Data were analyzed with FlowJo software (Trees-
tar, Ashland, OR, USA).

Phenotypic assays
Strains were grown overnight in liquid YPD at 30
C with shaking. The cells were washed, adjusted to the same optical density
(OD600 = 0.03), and serially diluted. To test these strains for sensitivity to UV radiation, an equal number of cells were spotted
onto YPD agar medium, air-dried, exposed to 150 J/cm2 of UV, and then incubated at 30
C. To test the tolerance of fungal cells
to other stressors, an equal number of cells were spotted onto YPD agar or YPD agar supplemented with fluconazole (15 mg/ml),
zeocin (80 mg/ml), or MMS (0.05%). Cells were cultured at 30
C and images of the plates were taken at day 3 and day 4 of incubation.
The colony growth was quantified with Colonyzer [39] and visualized with the R package Platetools [59].

Funding Statement
This work was supported by National Institutes of Health (R21AI132125 and R01AI140719 to XL and R01AI123315 to CX) and the
University of Georgia (fund to XL). Dr. Lin holds an Investigator Award in the Pathogenesis of Infectious Disease from the Burroughs
Wellcome Fund (1012445). The funders had no role in study design, data collection, and interpretation, or the decision to submit the
work for publication.

e4 Current Biology 30, 1387–1396.e1–e5, April 20, 2020

QUANTIFICATION AND STATISTICAL ANALYSIS

All in vitro experiments were conducted at least in triplicates unless otherwise stated in the text or figure legends. Statistics were
measured by Student’s t test or Mann-Whitney test for significance using R version 3.6. Statistical information for each experiment
can be found in the corresponding figure legend.

DATA AND CODE AVAILABILITY

The authors declare that the data supporting the findings of this study are available within the manuscript and its Supplemental
Information. All materials and datasets generated and/or analyzed in the current study are available from the corresponding author
upon reasonable request.

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