Deubiquitinase Ubp5 Is Required for the Growth and Pathogenicity of Cryptococcus gattii
Deubiquitinase Ubp5 Is Required for the Growth and Pathogenicity of Cryptococcus gattii
Yunfang Meng 0 1
Chao Zhang 1
Jiu Yi 1
Zhaojing Zhou 1
Zhenzong Fa 1
Jingyu Zhao 1
Yali Yang 1
Wei Fang 1
Yan Wang 1
Wan-qing Liao 0 1
0 Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Medical Mycology, Second Military Medical University , Shanghai , China , 2 PLA Key Laboratory of Mycosis, Department of Dermatology and Venereology, Changzheng Hospital , Shanghai , China , 3 Shanghai Dermatology Hospital , Shanghai , China , 4 Department of Pharmacology, School of Pharmacy, Second Military Medical University , Shanghai , China , 5 Department of Dermatology, Shandong Provincial Hospital Affiliated to Shandong University , Jinan, Shandong , China
1 Editor: Kirsten Nielsen, University of Minnesota , UNITED STATES
Cryptococcus gattii is a resurgent fungal pathogen that primarily infects immunocompetent hosts. Thus, it poses an increasingly significant impact on global public health; however, the mechanisms underlying its pathogenesis remain largely unknown. We conducted a detailed characterization of the deubiquitinase Ubp5 in the biology and virulence of C. gattii using the hypervirulent strain R265, and defined its properties as either distinctive or shared with C. neoformans. Deletion of the C. gattii Ubp5 protein by site-directed disruption resulted in a severe growth defect under both normal and stressful conditions (such as high temperature, high salt, cell wall damaging agents, and antifungal agents), similar to the effects observed in C. neoformans. However, unlike C. neoformans, the C. gattii ubp5Δ mutant displayed a slight enhancement of capsule and melanin production, indicating the evolutionary convergence and divergence of Ubp5 between these two sibling species. Attenuated virulence of the Cg-ubp5Δ mutant was not solely due to its reduced thermotolerance at 37°C, as shown in both worm and mouse survival assays. In addition, the assessment of fungal burden in mammalian organs further indicated that Ubp5 was required for C. gattii pulmonary survival and, consequently, extrapulmonary dissemination. Taken together, our work highlights the importance of deubiquitinase Ubp5 in the virulence composite of both pathogenic cryptococcal species, and it facilitates a better understanding of C. gattii virulence mechanisms.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Cryptococcosis is one of most prominent invasive fungal diseases; it can invade both
immunocompromised and immunocompetent hosts and often manifests as life-threatening
meningoencephalitis. Among its two major pathogenic agents, Cryptococcus neoformans (Cn) is known
to mainly infect the immunocompromised population and is responsible for the vast majority
Competing Interests: The authors have declared
that no competing interests exist.
of cases of cryptococcosis globally[
]. The other agent, Cryptococcus gattii (Cg), was originally
believed to be restricted to healthy individuals in tropical and subtropical countries such as
Australia and Papua New Guinea[
]. The outbreak of C. gattii cryptococcosis in temperate
regions such as Vancouver Island, British Columbia, and the Pacific Northwest has redrawn
public attention to this resurgent fungal pathogen[
As the sibling species of C. neoformans, C. gattii is also an encapsulated budding yeast, but it
exhibits distinct morphological, biochemical, and ecological patterns. For example, C. gattii
yields both round and bacilliform cells, and it is consistently found inhabiting decaying trees
but not bird droppings like C. neoformans[
]. Although these pathogens are not routinely
discriminated in clinical practice, their interspecific differences are significant for the clinical
manifestation and management of infection. Brain infection caused by C. gattii is associated
with an increased number of cryptococcomas, more neurological complications, and a slower
response to therapy, and it usually requires additional diagnostic follow-ups and more frequent
neurosurgical intervention, as compared with infection with C. neoformans. The unique
pattern of C. gattii in terms of its epidemiological and clinical features may be largely due to its
distinctive mechanisms of pathogenesis. Previous studies have suggested that C. gattii infection
results in defective induction of host immune responses, such as the arrested migration of
neutrophils and the reduced expression of several protective pro-inflammatory cytokines[
Furthermore, C. gattii also displays some divergent virulence-regulatory mechanisms
compared with C. neoformans, such as the antioxidant superoxide dismutase (Sod1) and
trehalose6-phosphate synthase (Tps1 and Tps2)[
]. It is clear that C. gattii may rely on the
variegated expression of virulence genes or some unknown but unique virulence traits to adapt to
the host environment in vivo. A complete understanding of its unique mechanisms of
pathogenesis is essential for allowing an accurate diagnosis and more appropriate intervention
strategies in C. gattii infection, and these mechanisms remain to be further elucidated.
Ubiquitination is a critical reversible post-translational modification for regulating cell
growth and physiology in eukaryotes[
]. Ubiquitin homeostasis is mainly determined by the
processing of its precursors and its recycling from substrates via deubiquitinases (DUBs).
DUBs are a conserved superfamily of proteases that are involved in a variety of biological
processes, such as the cell cycle, signal transduction, and the stress response[
], and they have
recently emerged as attractive targets in anticancer therapy[
]. For example, the
deubiquitinase Usp7 has been linked to human hematopoietic tumors based on its ability to regulate the
degradation of the tumor suppressor p53[
]. In model fungi, DUBs have also been reported
to be essential for several cellular functions such as nutrient sensing, sexual reproduction, and
]. However, few studies have reported the roles of deubiquitinase in the
virulence of human fungal pathogens. Using a systematic genetic analysis, Liu et al. first
demonstrated that some DUBs might be involved in melanization and pathogenesis in C.
]. Thus, from the remaining DUBs, we further identified Ubp5, which is essential for
sexual reproduction, the stress response, and the virulence composite in C. neoformans[
Interestingly, the same deubiquitinase gene has also been shown to be up-regulated in several
hypervirulent C. gattii isolates from the Vancouver Island outbreak, the expression profiles of
which display a significant correlation with the cryptococcal intracellular proliferation rate
inside macrophage-like cells[
]. Hence, we hypothesize that deubiquitinase Ubp5 may possess
a divergent function in the pathogenesis of C. gattii.
In the present study, we evaluated the biological functions of Ubp5 in Cryptococcus gattii
using the hypervirulent strain R265 as a model. Deletion of Ubp5 in C. gattii revealed a severe
growth defect under both normal and stressful conditions, and it also attenuated virulence in
non-vertebrate and mammalian hosts. In contrast to the findings for C. neoformans, enhanced
capsule production and melanin synthesis were observed in the C. gattii ubp5Δ mutant,
2 / 16
indicating that the utilization of Ubp5 has evolved for distinct regulatory purposes in the
virulence composite of these sibling species. Taken together, our study demonstrates the functional
convergence and divergence of Ubp5 among pathogenic Cryptococcus species, facilitating a
better understanding of C. gattii virulence mechanisms.
Characterization of the C. gattii gene UBP5
The C. gattii gene UBP5 (CNBG_6153) displayed approximately 87% nucleotide identity to
UBP5 from C. neoformans var. grubii (CNAG_05650) or C. neoformans var. neoformans
(CNBL2960). A phylogenetic analysis of the protein alignment was performed using the
deubiquitinase Ubp5 orthologs of the C. neoformans species complex and 10 other fungal species.
This protein was classified into distinct clades of basidiomycetous yeasts, ascomycetous yeasts,
molds, and zygomycetous molds, consistent with their evolutionary relationship (S1 Fig).
Among the basidiomycetes, C. neoformans var. grubii and C. neoformans var. neoformans
belonged to the same species, which was distinct from C. gattii. Interestingly, the Ubp5
orthologs of C. gattii and C. neoformans var. grubii formed one sister clade, suggesting an
evolutionary divergence among the pathogenic cryptococcal species. Analysis of the predicted C. gattii
protein Ubp5 revealed the presence of MATH (amino acids 55 to 206), UCH (amino acids 208
to 525), and USP7 (amino acids 631 to 1103) motifs. These motifs and their arrangement were
common in Ubp5 orthologs from all of the analyzed fungi, and the three domains displayed
identities of approximately 98%-99% in the C. neoformans species complex, indicating that the
protein structure of deubiquitinase Ubp5 was evolutionarily conserved.
Ubp5 is required for cell propagation of C. gattii
To determine the biological functions of deubiquitinase Ubp5 in C. gattii, we constructed the
Cg-ubp5Δ mutant and its reconstituted strain Cg-ubp5Δ+UBP5 on the background of the R265
hypervirulent isolate. Similarly to C. neoformans[
], the lack of Ubp5 significantly delayed the
growth of C. gattii, even on rich medium at 30°C. As shown in Fig 1A, the Cg-ubp5Δ mutant
required an incubation of approximately 40 hours to achieve stationary phase, while the WT
strain rapidly entered stationary phase by 24 hours. The reconstituted strain of CgUBP5
displayed a partially restored growth rate similar to the WT strain. We also compared the colony
sizes of these three strains after a five-day incubation on YPD agar at 30°C. The remarkable
differences in colony size further confirmed the decreased growth rate exhibited by the Cg-ubp5Δ
mutant (Fig 1B), suggesting that Ubp5 was involved in the propagation of C. gattii.
C. gattii Ubp5 is involved in fungal tolerance to multiple stressors in vitro
Next, we evaluated the effect of UBP5 disruption on stress responses in C. gattii. Similarly to
the Cn-ubp5Δ mutant[
], the Cg-ubp5Δ mutant strains displayed enhanced susceptibility to
various stressors in vitro (Fig 2). The results revealed that the Cg-ubp5Δ mutant was
hypersensitive to high temperature, exhibiting a partial growth defect at 37°C and complete fungistasis
at 39°C. Similar phenotypes were also observed in the mutant strain following exposure to
osmotic shock or cell membrane/wall damaging agents. In response to oxidative and
nitrosative damage, the Cg-ubp5Δ mutant strains exhibited slight sensitivity, but it did not differ from
the effects observed on YNB medium, suggesting that Ubp5 might not be directly involved in
stress tolerance to peroxide and nitric oxide in C. gattii. Contrasting results have been obtained
in C. neoformans, in which Ubp5 was essential for cryptococcal resistance to both H2O2 and
]. In addition, we also tested the impact of the deletion of UBP5 on the susceptibility of
3 / 16
Fig 1. Growth curve assay and colony size assessment. A. The WT, mutant, and reconstituted strains
were grown in YPD broth overnight at 30°C. Next, 106 cells of each strain were transferred to 30 mL fresh
YPD broth in flasks and incubated at 30°C. The OD600 values were measured for each group at four-hour
intervals. The growth rate of the Cg-ubp5Δ mutant was significantly reduced compared with the other two
strains. B. One hundred cells from each strain were spread onto YPD agar, incubated for 5 days at 30°C and
C. gattii to several antifungal drugs. In comparison to the WT strain, the Cg-ubp5Δ mutant
strains exhibited a 2-fold reduction in the MIC for amphotericin B and at least a 4-fold
reduction in the MICs of other common antifungal agents such as flucytosine, terbinafine, and azoles
(Table 1). Interestingly, reconstitution of Cg-UBP5 failed to restore the survival of C. gattii at
39°C in spite of restoring its tolerance to the other in vitro stressors, which might be due to
damage caused by ectopic integration and/or repeated biolistic transformations. These data
indicate that deubiquitinase Ubp5 positively regulates the fungal stress response, but with a
subtle distinction, in both C. neoformans and C. gattii species.
Deletion of UBP5 enhances capsule and melanin production in C. gattii
We tested whether the deletion of UBP5 could influence other known pathogenic factors, such
as the polysaccharide capsule and melanin production in C. gattii. Unlike the Cn-ubp5Δ
], deletion of Ubp5 led to a slight enlargement in the size of the C. gattii capsule in
DMEM when grown in the presence of CO2 (Fig 3A). A minimum of 50 cells from each strain
4 / 16
Fig 2. C. gattii Ubp5 is involved in fungal responses to various stressors. The R265, Cg-ubp5Δ, and Cg-ubp5Δ+UBP5 strains were grown on YPD
broth to saturation at 30°C and then serially diluted 10-fold (1–106 dilutions). 3 μL suspension of 108 cells/mL were spotted on YPD or YNB agar (containing
different stress-inducing agents), incubated for five days and photographed.
were measured, and the average capsule size (relative volume) of the Cg-ubp5Δ strain was
96.2% compared with 91.0% and 92.3% in the WT and reconstituted strains, respectively (Fig
3B, P<0.001). Furthermore, deletion of Ubp5 had a similar effect on the pathogenic factor
laccase (Fig 4). The C. gattii ubp5Δ strain displayed slight hypermelanization and produced leaky
melanin around the colonies compared with the WT strain when incubated on L-DOPA
medium at 30°C for 5 days. A similar difference in melanin production was more evident on
caffeic acid medium, which revealed a sharp distinction in the melanin phenotype due to Ubp5
Fig 3. UBP5 mutation enhances capsule production in C. gattii. A. The WT, mutant, and reconstituted
strains were incubated on DMEM medium for capsule induction at 37°C for three days. Capsules were
assessed by India ink staining and visualization at 100X magnification (scale bar = 10 μm). B. Relative
capsule volume on DMEM medium. Relative capsule volume = (Total Volume–Packed Volume)/Total
Volume (N = 50). Wilcoxon test was performed to examine the capsule difference between R265 and
Cgubp5Δ strains. The results revealed enhanced capsule production in the Cg-ubp5Δ mutant strain (P<0.01).
deletion in C. neoformans. We also noted that the complemented strain showed less
melanization than the WT strain in both L-DOPA and caffeic acid, which might be attributed to
trancriptional alteration of UBP5 and/or secondary mutation caused by ectopic integration. Taken
together, these data establish a distinct role for Ubp5 in capsule or melanin production in
Fig 4. Ubp5 negatively regulates melanin production in C. gattii. Strains grown in YPD broth were washed twice with PBS buffer, and a 5 μL suspension
of 107 cells/mL was spotted on L-DOPA and Caffeic Acid media and incubated for 5 days at 30°C for melanin induction.
6 / 16
Fig 5. Ubp5 mediates the survival and proliferation of C. gattii in macrophages. Activated J774A.1
macrophages were infected with R265, Cg-ubp5Δ, and Cg-ubp5Δ+UBP5 strains of C. gattii. After 2 hours of
coincubation at 37°C with 5% CO2, the extracellular yeasts were removed, and the co-cultures were
incubated for 24 hours under the same conditions. The macrophages were lysed, and the samples were then
incubated on YPD agar at 30°C for 4 days to quantify the cryptococcal colonies. Each strain was assayed
four times (average ± SEM, P<0.0001).
Role of Upb5 on C. gattii parasitism inside macrophages
Cryptococcus spp. have been generally accepted as facultative intracellular pathogens, and the
hypervirulence of some C. gattii strains has been closely associated with their potent
proliferative capacity inside macrophages[
]. Thus, we assessed the ability of the Cg-ubp5Δ
mutant to parasitize macrophages by co-incubating them with activated macrophages.
Co-culture with activated J774A.1 macrophages revealed a 65.7% reduction in the intracellular
survival of the mutant strain after 24 h compared with the background strain R265 (P<0.0001)
(Fig 5). Reconstitution of CgUBP5 completely restored the intracellular proliferation of the
mutant inside macrophages. Similar results were obtained in repeated experiments, suggesting
that deubiquitinase Ubp5 is essential for C. gattii survival inside macrophages.
C. gattii Upb5 is essential for virulence in mammals
To gain insight into the overall impact of the C. gattii Ubp5 deletion on the total virulence
composite, we first performed a survival assay using the murine inhalation model.
Immunocompetent BALB/c mice were inoculated intranasally with 105 cells of R265, the Cg-ubp5Δ
strain, or the reconstituted strain Cg-ubp5Δ+UBP5. As shown in Fig 6A, mice infected with the
WT strain R265 survived for 33 days, and the average survival time was 29±3.73 days. The
group infected with the reconstituted strain displayed a similar survival pattern, in which all of
the mice were sacrificed by day 43, and the average survival time was 28±5.75 days (P = 0.157).
In contrast, mice in the Cg-ubp5Δ mutant group did not die even at 80 days after infection,
suggesting a significant attenuation of C. gattii virulence due to the deletion of Ubp5 (P<0.001).
To investigate the potential impact of Ubp5 deletion on alveolar delivery or cryptococcal
migration from the lungs, we next evaluated fungal burdens in the lung and brain in the above
three groups at 4, 7, 14, and 21 days post-infection. Total lung CFU analyses at different time
points post-infection revealed a high cryptococcal burden in the WT group (Fig 6B & 6C).
However, the C. gattii strain lacking UBP5 resulted in significantly reduced pulmonary fungal
burden at different time points after infection (P<0.001), which also displayed a gradual
downward trend with an extended duration of infection. Furthermore, no viable colonies were found
in the brain of mice infected with the Cg-ubp5Δ strain, in contrast to the other groups. The
mice infected with reconstituted strain Cg-ubp5Δ+UBP5 displayed a slight reduction in CFU in
7 / 16
Fig 6. Deletion of Ubp5 attenuates the virulence of C. gattii in a murine inhalation model. A. Survival
curve of mouse inhalational cryptococcosis with R265, Cg-ubp5Δ, and Cg-ubp5Δ+UBP5 strains over 80
days. All of the mice infected with R265 and Cg-ubp5Δ+UBP5 were sacrificed, but all of the mice in the
Cgubp5Δ group survived (P<0.001). B&C. Fungal burden in the lung and brain. Organs were removed at 4, 7,
14, and 21 days post-infection in the three groups.
both the lung and brain compared with the WT strain but a significant increase in CFU
compared with the Cg-ubp5Δ strain (P<0.001, Fig 6B & 6C), suggesting that ectopic integration of
UBP5 partially restored the virulence of the ubp5Δ mutant. These data indicate that C. gattii
requires Ubp5 to parasitize the lung and disseminate into other organs, especially the central
Impact of Upb5 on the C. elegans model
Since deletion of UBP5 enhanced the susceptibility of C. gattii to high temperature, we
wondered whether attenuated in vivo virulence of ubp5Δ mutant was only attributed to its reduced
thermotolerance. Caenorhabditis elegans provides an important model of pathogenesis at
room temperature that can be utilized to exclude the potential effect of high temperature
8 / 16
Fig 7. Survival analysis in the C. elegans model. Forty C. elegans per group were fed on lawns of the WT,
mutant, or reconstituted strain. Ubp5 deletion attenuated the virulence of C. gattii (P<0.001).
sensitivity on cryptococcal virulence[
]. In the C. elegans/C. gattii system, the Cg-ubp5Δ
mutant (LT50 = 12 days) was less virulent than the WT (LT50 = 8 days) or reconstituted (LT50
= 9 days) strains, as determined by survival analysis (P<0.001, Fig 7). This finding was
consistent with results obtained in the murine inhalation model with these strains. Our data suggest
that the lack of UBP5 may attenuate the virulence of C. gattii independently of its influence on
C. gattii is known as the major cryptococcal pathogen in immunocompetent hosts worldwide
other than in China[
]. This pathogen, which was previously considered to be endemic in
tropical and sub-tropical regions, has received global scientific interest due to its association
with fatal outbreaks in humans and mammals, as well as its expanding geographical range[
]. Experimental studies investigating the mechanisms underlying the pathogenicity of C.
gattii are scarce. In the present study, we conducted a detailed characterization of the
deubiquitinase Ubp5 in the biology and virulence of C. gattii using the hypervirulent strain R265, and we
defined its properties as either distinctive or shared with C. neoformans.
The first phenotype observed for the Cg-ubp5Δ strain was its poor growth performance
under both stressful and normal conditions. Similarly to its function in C. neoformans, the
deubiquitinase Ubp5 of C. gattii positively regulated its responses to a variety of stressors in vitro,
such as high temperature, high salt content, and antifungal drugs, among others. There may be
several explanations for the role of Ubp5 in stress responses. First, many misfolded or damaged
proteins accumulate inside the cryptococcal cell following prolonged exposure to various
stressors, and this phenomenon relies in part on the ubiquitin-mediated degradation pathway
for the maintenance of cellular homeostasis. The ubiquitin-proteasome pathway is critical for
regulating various cellular processes, especially the stress response, in various eukaryotic
species such as Saccharomyces. cerevisiae, Schizosaccharomyces. pombe, and Candida. candida[
]. In C. neoformans, several ubiquitin-system genes, such as UBI4 (polyubiquitin) and UBC6
(ubiquitin conjugating enzyme), also display significant transcriptional changes under stressful
]. As a core component of the ubiquitin-proteasome system, deubiquitinase is
essential for maintaining the dynamic balance of ubiquitin by processing ubiquitin precursors
or proofreading ubiquitin-protein conjugation, and thus, it participates in the stress responses
]. Second, ubiquitination and deubiquitination might be an important
modification mechanism in some signaling pathways associated with the stress response in fungi. For
example, the HOG pathway is negatively regulated via ubiquitin-mediated degradation of the
upstream component Ssk1 in S. cerevisiae[
]. In C. neoformans, many genes encoding
9 / 16
ubiquitin-conjugating enzymes are significantly up-regulated in some HOG pathway mutants,
while some genes encoding components (such as MAPKKK and Cpb1) of the MAPK and Ca2
+/calcineurin signaling pathways also display significant transcriptional changes in the
]. The relationship of deubiquitinase with various signaling pathways
remains to be further illuminated in Cryptococcus spp. Finally, the reduced stress tolerance of
the Cg-ubp5Δ mutant was also associated with its slower proliferation rate, even in rich media.
Yeast cell growth is a complex biological process that relies on the coordination of multiple
factors, such as cell division, cell size, nutrients and energy metabolism[
]. In S. cerevisiae,
deubiquitination is an important modification mechanism that is involved in energy uptake
and the cell cycle[
]. Cg-Ubp5 might exploit similar strategies to regulate cell growth in C.
However, deletion of the deubiquitinase Ubp5 led to subtle differences in fungal
susceptibility to some stressors between C. gattii and C. neoformans. For example, the C. gattii ubp5Δ
strain was less sensitive to oxidative and NO stress but was more susceptible to cell
wall/membrane damaging agents compared with the Cn-ubp5Δ mutant in C. neoformans[
Cryptococcus spp. rely on multiple signaling pathways and regulatory mechanisms to respond to each
], and the deubiquitinase Ubp5 might act on distinct substrates in its two major
pathogens to differentially regulate the stress response. However, it might also be due to the
functional similarities and redundancies of the DUB protein family such that selective
environmental pressure could drive the microevolution of the function of Ubp5 in Cryptococcus spp.
The functional difference in Ubp5 between the two sibling species was more significant in
terms of the expression of other pathogenic factors. In C. neoformans, CnUbp5 positively
regulated both melanin and capsule production, which might be associated with its roles in
regulating copper ion metabolism or polysaccharide attachment to the cell wall[
deletion of CgUbp5 led to opposite phenotypes (enhanced melanin and capsule production) in
C. gattii, which further confirmed the functional reconfiguring of the homologous gene in
cryptococcal evolution. Cryptococcus spp. exploit a similar mechanism for both capsule and
melanin production, in which vesicles containing the protein components are excreted into the
extracellular space and the components are then attached to the cell wall[
factors that participate in cell wall remodeling, such as chitin and chitosan, are also involved in
capsule or melanin assembly[
]. For example, lack of chitosan in C. neoformans led to a
"leaky melanin" phenotype like Cg-ubp5Δ mutant in this study. Considering the
hypersusceptibility of the Cg-ubp5Δ mutant to Congo Red, we speculate that CgUbp5 might indirectly
regulate capsule or melanin production via its role in cell wall synthesis; however, this
hypothesis requires further exploration.
The Cryptococcus neoformans species complex utilizes multiple pathogenic factors to
overcome the hostile environment in vivo and cause damage to the host[
]. Each pathogenic factor
potentially provides a different relative contribution to the overall virulence phenotype of this
]. In C. gattii, deletion of deubiquitinase CgUbp5 resulted in significantly
attenuated virulence in a mammalian host, although the Cg-ubp5Δ mutant displayed slightly
enhanced production of capsule and melanin. Excluding the effect of the reduced
thermotolerance, the survival assay in C. elegans model further confirmed the role of CgUbp5 in regulating
the virulence composite of C. gattii. The results of our study suggest that the deubiquitinase
Ubp5 is essential for the overall virulence phenotype in both C. gattii and C. neoformans but
with some common and/or specialized mechanisms[
It is believed that cryptococcal spores or desiccated yeast cells are first inhaled into the lungs
and then disseminate into extrapulmonary regions in the central nervous system when the host
]. In the present study, the fungal burden in a murine model
suggested that C. gattii lacking deubiquitinase Ubp5 was easily cleared by host pulmonary
10 / 16
defense responses. This phenomenon was closely associated with the decreased survival rate of
the Cg-ubp5Δ mutant inside macrophages. Alveolar macrophages have been demonstrated to
be the first line of host defense, a primary haven for latent infection, and also to function as a
Trojan horse for hematogenous dissemination during cryptococcal infection. Enhanced
intracellular replication within host macrophages is an important feature of the hypervirulent
C. gattii strains recovered from the Vancouver Island outbreak, which are characterized by the
upregulation of multiple genes including CgUBP5[
]. Consistent with this perspective, our
work further indicates that CgUbp5 is required for the survival of C. gattii in the pulmonary
space and, thus, its extrapulmonary dissemination.
In summary, our work supports the importance of deubiquitinase Ubp5 in the virulence
composite of C. gattii strain R265. Ubp5 was found to be involved in cellular propagation, the
stress response, capsule and melanin production, and thus pathogenicity of C. gattii in both
non-vertebrate and vertebrate models. Furthermore, our results revealed the evolutionary
convergence and divergence of Ubp5 in these two major cryptococcal pathogens to a certain
extent, suggesting that C. gattii exploit some specialized mechanisms to adapt to environments
in vivo and in vitro. However, the detailed mechanism by which Ubp5 deletion affects host
immunological responses during cryptococcal infection remains unknown. It will be important
to explore the potential of deubiquitinase as an anticryptococcal target.
Materials and Methods
Strains and media
R265, a VGIIa clinical C. gattii isolate from the Vancouver Island outbreak[
], was used as a
background strain in this study. Mutant and complemented strains of R265 were constructed
by biolistic transformation. All of the strains were maintained on non-selective yeast extract
peptone dextrose solid medium (YPD, 1% yeast extract, 2% peptone, 2% dextrose, and 2%
agar). Selective media containing geneticin (G418) or nourseothricin were used for the
screening of mutant or reconstituted strains as previously reported[
Construction of mutant and reconstituted C. gattii strains
The primers used in this study are listed in S1 Table. For gene deletion, overlap PCR was
employed to generate the knock-out cassette of Cg-UBP5, including the flanking fragments and
NEO resistance gene[
]. The purified PCR products were precipitated onto gold
microparticles and introduced into R265 cells by biolistic transformation[
]. Stable transformants were
screened using selective medium that included G418 and then confirmed via diagnostic PCR,
DNA sequencing, and Southern blot analysis. Southern blot examination was performed as
A whole DNA fragment of Cg-UBP5 containing the ORF, promoter and terminator region
was amplified from R265 genomic DNA for mutant complementation. The purified PCR
fragments were linked to the digested plasmid pCH233 by Xba I using the Infusion1EcoDryTM
Cloning System (Clontec, Mountain View, US). The linked fragments were then introduced
into the mutants by biolistic transformation. Stable colonies were screened on selective
medium containing nourseothricin, and finally confirmed by diagnostic PCR and Southern
In vitro phenotypic assays
The yeast cells were cultured to saturation in YPD broth at 30°C, washed twice with 1×PBS
buffer, and then quantified using a Countstar Automated Cell Counter. To evaluate the stress
11 / 16
response of C. gattii strains, the cells were serially diluted 10-fold (1–106 dilutions). 3 μL
suspension of 108 cells/mL were spotted on different stress media, incubated for five days and
then photographed. For the high temperature stress test, the yeast cells were incubated at 30°C,
37°C and 39°C on YPD agar. For the oxidative and NO stress test, the cells were incubated on
Yeast Nitrogen Base (YNB) agar containing 2 mM H2O2 or 0.75 mM NaNO2 (pH 4.0). To
evaluate the response to high salt and osmotic stress, 1.5 M NaCl, 1.5 M KCl or 1.5 M sorbitol was
added to the YPD agar. To assess cell wall/membrane integrity stress, 0.5% Congo Red or
0.02% SDS was added to the YPD agar.
The antifungal susceptibility test was performed as previously reported[
]. The MICs of
common antifungal agents (including amphotericin B, flucytosine, terbinafine, and azoles)
against the R265, Cg-ubp5Δ, and Cg-ubp5Δ+UBP5 strains were determined using the Clinical
and Laboratory Standards Institute broth microdilution reference method (CLSI, 2002), and
Candida parapsilosis ATCC22019 served as a quality control strain.
To measure capsule production, fungal cells were incubated on DMEM medium for three
days in the presence of 5% CO2 at 37°C[
]. The capsule was stained with India ink and
visualized by microscopy. The relative capsule volume was calculated for at least 50 cells for each
strain according to the following formula: (Total Volume–Packed Volume)/Total Volume. To
analyze melanin production, a 5 μL suspension of 107 cells/mL for each strain was spotted on
L-DOPA and Caffeic Acid medium[
] and then incubated for 5 days at 30°C.
Macrophage killing assays
J744.A1. macrophage cells were used to assay the intracellular survival of different C. gattii
strains as previously described[
]. In brief, each strain was incubated overnight at 30°C
and then opsonized with cryptococcal monoclonal antibody (C66441M, bought from Meridian
Life Science, Inc. Saco, US). A total of 106 yeast cells were co-incubated with 105 J744.A1. cells
that had been activated with interferon-gamma and lipopolysaccharide in 96-well tissue culture
plates. After a 2-hour co-culture, the extracellular yeasts were washed away with PBS buffer,
and fresh DMEM medium was added. After 24 hours of incubation, the macrophages were
lysed with 0.5% SDS, and viable fungal cells were calculated by quantitative culture on YPD
agar at 30°C for 3 days.
Virulence assays in vivo
BALB/c mice were intranasally infected according to an established protocol[
]. For the
survival assay, ten mice per group were challenged with 105 CFU of the mutant (Cg-ubp5Δ),
wildtype(WT) (R265), or complemented (Cg-ubp5Δ+UBP5) strain in 50 μL of PBS. All of the mice
were monitored daily for signs of infection and sacrificed via carbon dioxide euthanasis based
on predetermined endpoints such as weight loss ( 15%), neurological symptoms, and an
inability to access food or water. To assess the organ fungal burden, lungs and brains were
removed from the sacrificed mice (12 mice per group) after 3, 7, 14, and 21 days.
A Caenorhabditis elegans (C. elegans) model was also used to evaluate the virulence of each
strain under room temperature as previous reported[
]. Briefly, a total of 40 standard C. elegans
strain N2 Bristol in each group were incubated to the young adult developmental stage on a lawn
of Escherichia coli OP50. Subsequently, they were transferred to plates containing WT, mutant,
or complemented strains. The viability of C. elegans was determined every day by microscopy.
The data obtained for the mouse and C. elegans model survival assays were plotted as
KaplanMeier survival curves and analyzed with the log-rank test using SPSS 18.0 software. The LF50
12 / 16
(time for half of the worms to die) was also calculated to estimate survival differences in the C.
elegans model. The remaining statistical analyses were conducted with the student’s t test or
Mann-Whitney test. The results were considered statistically significant when the P value was
less than 0.05.
The animal studies were proved by the Committee on Ethics of Biomedicine Research, Second
Military Medical University, and carried out in strict accordance with the recommendations in
the Regulations for the Administration of Affairs concerning Experimental Animals of the
State Science and Technology Commission (China). Animal model was established under
isofluorane anesthesia, and all efforts were made to minimize animal suffering and distress.
S1 Fig. Phylogenetic tree analysis of Ubp5 orthologs. The alignment of predicted Ubp5
orthologs from various fungal species was performed using the DNASTAR 6.13 ClustalW
multiple-sequence alignment. The organism sources and accession numbers (NCBI database)
for the protein sequences are as follows: C. gattii R265, KGB80315; C. gattii WM276,
XP_003197136; C. neoformans var. grubii (CnVG) H99, AFR99081; C. neoformans var.
neoformans (CnVN) JEC21, XP_572460; Ustilago maydis, XP_758786; Candida albicans, KGQ89526;
Clavispora lusitaniae, XP_002617519; Candida glabrata, XP_449943; Saccharomyces cerevisiae,
EWH16885; Aspergillus fumigatus, XP_748018; Talaromyces marneffei, XP_002147746;
Colletotrichum fioriniae, XP_007599895; Fusarium graminearum, XP_009255591; Mucor
S1 Table. Primers used in this study.
We sincerely acknowledge the kind gift R265 strain from Prof. Xiaorong Lin (Texas A&M
University, College Station, Texas, USA).
Conceived and designed the experiments: WF WL. Performed the experiments: YM CZ JY ZZ.
Analyzed the data: WF ZF JZ. Contributed reagents/materials/analysis tools: YY YW. Wrote
the paper: WF YM CZ.
13 / 16
Acad Sci U S A. 2004; 101(49):17258–63. Epub 2004/12/02. doi: 10.1073/pnas.0402981101 PMID:
15572442; PubMed Central PMCID: PMCPmc535360.
14 / 16
15 / 16
1. Heitman J , Kozel TR , Kwon-Chung KJ , Perfect JR , Casadevall A . Cryptococcus: from human pathogen to model yeast . Washington, DC: ASM Press; 2011 .
2. Chen S , Sorrell T , Nimmo G , Speed B , Currie B , Ellis D , et al. Epidemiology and host- and varietydependent characteristics of infection due to Cryptococcus neoformans in Australia and New Zealand . Australasian Cryptococcal Study Group. Clin Infect Dis . 2000 ; 31 ( 2 ): 499 - 508 . Epub 2000/09/15. doi: 10 .1086/313992 PMID: 10987712 .
3. Sorrell TC . Cryptococcus neoformans variety gattii . Med Mycol . 2001 ; 39 ( 2 ): 155 - 68 . Epub 2001/05/11. PMID: 11346263 .
4. Kidd SE , Hagen F , Tscharke RL , Huynh M , Bartlett KH , Fyfe M , et al. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada) . Proc Natl
5. Springer DJ , Chaturvedi V . Projecting global occurrence of Cryptococcus gattii . Emerg Infect Dis . 2010 ; 16 ( 1 ): 14 - 20 . Epub 2009/12/25. doi: 10 .3201/eid1601.090369 PMID: 20031037; PubMed Central PMCID : PMCPmc2874352 .
6. Chaturvedi V , Chaturvedi S. Cryptococcus gattii: a resurgent fungal pathogen . Trends in microbiology. 2011 ; 19 ( 11 ): 564 - 71 . Epub 2011/09/02. doi: 10 .1016/j.tim. 2011 . 07 .010 PMID: 21880492; PubMed Central PMCID : PMCPmc3205261 .
7. Xue C , Tada Y , Dong X , Heitman J. The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants . Cell host & microbe . 2007 ; 1 ( 4 ): 263 - 73 . Epub 2007/ 11/17. doi: 10 .1016/j.chom. 2007 . 05 .005 PMID: 18005707 .
8. Perfect JR , Dismukes WE , Dromer F , Goldman DL , Graybill JR , Hamill RJ , et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america . Clin Infect Dis . 2010 ; 50 ( 3 ): 291 - 322 . Epub 2010/01/06. doi: 10 .1086/649858 PMID: 20047480 .
9. Brouwer AE , Siddiqui AA , Kester MI , Sigaloff KC , Rajanuwong A , Wannapasni S , et al. Immune dysfunction in HIV-seronegative, Cryptococcus gattii meningitis . J Infect . 2007 ; 54 ( 3 ): e165 - 8 . Epub 2006/ 11/18. doi: 10 .1016/j.jinf. 2006 . 10 .002 PMID: 17109966 .
10. Dong ZM , Murphy JW . Effects of the two varieties of Cryptococcus neoformans cells and culture filtrate antigens on neutrophil locomotion . Infect Immun . 1995 ; 63 ( 7 ): 2632 - 44 . Epub 1995/07/01. PMID: 7790079; PubMed Central PMCID : PMCPmc173353 .
11. Cox GM , Harrison TS , McDade HC , Taborda CP , Heinrich G , Casadevall A , et al. Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages . Infect Immun . 2003 ; 71 ( 1 ): 173 - 80 . Epub 2002/12/24. PMID: 12496163; PubMed Central PMCID : PMCPmc143417 .
12. Narasipura SD , Ault JG , Behr MJ , Chaturvedi V , Chaturvedi S. Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence . Mol Microbiol . 2003 ; 47 ( 6 ): 1681 - 94 . Epub 2003/03/08. PMID: 12622821 .
13. Ngamskulrungroj P , Himmelreich U , Breger JA , Wilson C , Chayakulkeeree M , Krockenberger MB , et al. The trehalose synthesis pathway is an integral part of the virulence composite for Cryptococcus gattii . Infect Immun . 2009 ; 77 ( 10 ): 4584 - 96 . Epub 2009/08/05. doi: 10 .1128/iai.00565-09 PMID: 19651856; PubMed Central PMCID : PMCPmc2747965 .
14. Petzold EW , Himmelreich U , Mylonakis E , Rude T , Toffaletti D , Cox GM , et al. Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans . Infect Immun . 2006 ; 74 ( 10 ): 5877 - 87 . Epub 2006/09/22. doi: 10 .1128/iai.00624-06 PMID: 16988267; PubMed Central PMCID : PMCPmc1594924 .
15. Clague MJ , Urbe S. Ubiquitin : same molecule, different degradation pathways . Cell . 2010 ; 143 ( 5 ): 682 - 5 . Epub 2010/11/30. doi: 10 .1016/j.cell. 2010 . 11 .012 PMID: 21111229 .
16. Komander D , Clague MJ , Urbe S. Breaking the chains: structure and function of the deubiquitinases . Nature reviews Molecular cell biology . 2009 ; 10 ( 8 ): 550 - 63 . Epub 2009/07/25. doi: 10 .1038/nrm2731 PMID: 19626045 .
17. Nicholson B , Marblestone JG , Butt TR , Mattern MR . Deubiquitinating enzymes as novel anticancer targets . Future oncology (London, England) . 2007 ; 3 ( 2 ): 191 - 9 . Epub 2007/03/27. doi: 10 .2217/ 147966188.8.131.52 PMID: 17381419; PubMed Central PMCID : PMCPmc2291548 .
18. Cheon KW , Baek KH . HAUSP as a therapeutic target for hematopoietic tumors (review) . International journal of oncology . 2006 ; 28 ( 5 ): 1209 - 15 . Epub 2006/04/06. PMID: 16596237 .
19. Auesukaree C , Damnernsawad A , Kruatrachue M , Pokethitiyook P , Boonchird C , Kaneko Y , et al. Genome-wide identification of genes involved in tolerance to various environmental stresses in Saccharomyces cerevisiae . Journal of applied genetics . 2009 ; 50 ( 3 ): 301 - 10 . Epub 2009/07/30. doi: 10 . 1007/bf03195688 PMID: 19638689 .
20. Enyenihi AH , Saunders WS . Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae . Genetics . 2003 ; 163 ( 1 ): 47 - 54 . Epub 2003/02/15. PMID: 12586695; PubMed Central PMCID : PMCPmc1462418 .
21. Kahana A. The deubiquitinating enzyme Dot4p is involved in regulating nutrient uptake . Biochem Biophys Res Commun . 2001 ; 282 ( 4 ): 916 - 20 . Epub 2001/05/16. doi: 10 .1006/bbrc. 2001 .4669 PMID: 11352638 .
22. Liu OW , Chun CD , Chow ED , Chen C , Madhani HD , Noble SM . Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans . Cell . 2008 ; 135 ( 1 ): 174 - 88 . Epub 2008/10/16. doi: 10 .1016/j.cell. 2008 . 07 .046 PMID: 18854164; PubMed Central PMCID : PMCPmc2628477 .
23. Fang W , Price MS , Toffaletti DL , Tenor J , Betancourt-Quiroz M , Price JL , et al. Pleiotropic effects of deubiquitinating enzyme Ubp5 on growth and pathogenesis of Cryptococcus neoformans . PLoS One . 2012 ; 7 ( 6 ): e38326 . Epub 2012 /06/22. doi: 10 .1371/journal.pone.0038326 PONE-D- 12-11810 [pii]. PMID: 22719877; PubMed Central PMCID : PMC3375289 .
24. Ma H , Hagen F , Stekel DJ , Johnston SA , Sionov E , Falk R , et al. The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation . Proc Natl Acad Sci U S A . 2009 ; 106 ( 31 ): 12980 - 5 . Epub 2009/08/05. doi: 10 .1073/pnas.0902963106 PMID: 19651610; PubMed Central PMCID : PMCPmc2722359 .
25. Coelho C , Bocca AL , Casadevall A . The intracellular life of Cryptococcus neoformans . Annual review of pathology . 2014 ; 9 : 219 - 38 . Epub 2013/09/21. doi: 10 .1146/annurev-pathol- 012513 -104653 PMID: 24050625 .
26. Ma H , May RC . Virulence in Cryptococcus species . Adv Appl Microbiol . 2009 ; 67 : 131 - 90 . Epub 2009/ 02/28. doi: 10 .1016/s0065- 2164 ( 08 ) 01005 - 8 PMID: 19245939 .
27. Mylonakis E , Ausubel FM , Perfect JR , Heitman J , Calderwood SB . Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis . Proc Natl Acad Sci U S A . 2002 ; 99 ( 24 ): 15675 - 80 . Epub 2002/11/20. doi: 10 .1073/pnas.232568599 PMID: 12438649; PubMed Central PMCID : PMCPmc137775 .
28. Fang W , Fa Z , Liao W. Epidemiology of Cryptococcus and cryptococcosis in China . Fungal Genet Biol . 2015 ; 78 : 7 - 15 . Epub 2014/12/03. doi: 10 .1016/j.fgb. 2014 . 10 .017 PMID: 25445309 .
29. Byrnes EJ 3rd, Bartlett KH , Perfect JR , Heitman J . Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals . Microbes and infection / Institut Pasteur . 2011 ; 13 ( 11 ): 895 - 907 . Epub 2011/06/21. doi: 10 .1016/j.micinf. 2011 . 05 .009 PMID: 21684347; PubMed Central PMCID : PMCPmc3318971 .
30. Finley D , Ozkaynak E , Varshavsky A . The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses . Cell . 1987 ; 48 ( 6 ): 1035 - 46 . Epub 1987/03/27. PMID: 3030556 .
31. Leach MD , Stead DA , Argo E , MacCallum DM , Brown AJ . Molecular and proteomic analyses highlight the importance of ubiquitination for the stress resistance, metabolic adaptation, morphogenetic regulation and virulence of Candida albicans . Mol Microbiol . 2011 ; 79 ( 6 ): 1574 - 93 . Epub 2011/01/29. doi: 10 . 1111/j.1365- 2958 . 2011 . 07542 . x PMID : 21269335 ; PubMed Central PMCID : PMCPmc3084552 .
32. Ogiso Y , Sugiura R , Kamo T , Yanagiya S , Lu Y , Okazaki K , et al. Lub1 participates in ubiquitin homeostasis and stress response via maintenance of cellular ubiquitin contents in fission yeast . Mol Cell Biol . 2004 ; 24 ( 6 ): 2324 - 31 . Epub 2004/03/03. PMID: 14993272; PubMed Central PMCID : PMCPmc355854 .
33. Ko YJ , Yu YM , Kim GB , Lee GW , Maeng PJ , Kim S , et al. Remodeling of global transcription patterns of Cryptococcus neoformans genes mediated by the stress-activated HOG signaling pathways . Eukaryot Cell . 2009 ; 8 ( 8 ): 1197 - 217 . Epub 2009/06/23. doi: 10 .1128/ec.00120-09 PMID: 19542307; PubMed Central PMCID : PMCPmc2725552 .
34. Upadhya R , Campbell LT , Donlin MJ , Aurora R , Lodge JK . Global transcriptome profile of Cryptococcus neoformans during exposure to hydrogen peroxide induced oxidative stress . PLoS One . 2013 ; 8 ( 1 ): e55110 . Epub 2013 /02/06. doi: 10 .1371/journal.pone.0055110 PMID: 23383070; PubMed Central PMCID : PMCPmc3557267 .
35. Kimura Y , Tanaka K. Regulatory mechanisms involved in the control of ubiquitin homeostasis . Journal of biochemistry . 2010 ; 147 ( 6 ): 793 - 8 . Epub 2010/04/27. doi: 10 .1093/jb/mvq044 PMID: 20418328 .
36. Komada M. Controlling receptor downregulation by ubiquitination and deubiquitination . Current drug discovery technologies . 2008 ; 5 ( 1 ): 78 - 84 . Epub 2008/06/10. PMID: 18537571 .
37. Sato N , Kawahara H , Toh-e A , Maeda T . Phosphorelay-regulated degradation of the yeast Ssk1p response regulator by the ubiquitin-proteasome system . Mol Cell Biol . 2003 ; 23 ( 18 ): 6662 - 71 . Epub 2003/08/29. PMID: 12944490; PubMed Central PMCID : PMCPmc193698 .
38. Gutteridge A , Pir P , Castrillo JI , Charles PD , Lilley KS , Oliver SG . Nutrient control of eukaryote cell growth: a systems biology study in yeast . BMC biology . 2010 ; 8 : 68 . Epub 2010/05/26. doi: 10 .1186/ 1741 -7007-8-68 PMID: 20497545; PubMed Central PMCID : PMCPmc2895586 .
39. Ramanathan A , Schreiber SL . Multilevel regulation of growth rate in yeast revealed using systems biology . Journal of biology . 2007 ; 6(2):3 . Epub 2007/05/03. doi: 10 .1186/jbiol56 PMID: 17472733; PubMed Central PMCID : PMCPmc2373900 .
40. Bozza WP , Zhuang Z. Biochemical characterization of a multidomain deubiquitinating enzyme Ubp15 and the regulatory role of its terminal domains . Biochemistry . 2011 ; 50 ( 29 ): 6423 - 32 . Epub 2011/06/30. doi: 10 .1021/bi200529z PMID: 21710968 .
41. Bahn YS , Jung KW . Stress signaling pathways for the pathogenicity of Cryptococcus . Eukaryot Cell . 2013 ; 12 ( 12 ): 1564 - 77 . Epub 2013/10/01. doi: 10 .1128/ec.00218-13 PMID: 24078305; PubMed Central PMCID : PMCPmc3889573 .
42. Eisenman HC , Casadevall A . Synthesis and assembly of fungal melanin . Appl Microbiol Biotechnol . 2012 ; 93 ( 3 ): 931 - 40 . Epub 2011/12/17. doi: 10 .1007/s00253-011-3777-2 PMID: 22173481; PubMed Central PMCID : PMCPmc4318813 .
43. Reese AJ , Doering TL . Cell wall alpha-1 ,3 -glucan is required to anchor the Cryptococcus neoformans capsule . Mol Microbiol . 2003 ; 50 ( 4 ): 1401 - 9 . Epub 2003/11/19. PMID: 14622425 .
44. Eisenman HC , Nosanchuk JD , Webber JB , Emerson RJ , Camesano TA , Casadevall A . Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans . Biochemistry . 2005 ; 44 ( 10 ): 3683 - 93 . Epub 2005/03/09. doi: 10 .1021/bi047731m PMID: 15751945 .
45. Rodrigues ML , Nakayasu ES , Oliveira DL , Nimrichter L , Nosanchuk JD , Almeida IC , et al. Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence . Eukaryot Cell . 2008 ; 7 ( 1 ): 58 - 67 . Epub 2007/11/28. doi: 10 .1128/ec.00370-07 PMID: 18039940; PubMed Central PMCID : PMCPmc2224146 .
46. Banks IR , Specht CA , Donlin MJ , Gerik KJ , Levitz SM , Lodge JK . A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans . Eukaryot Cell . 2005 ; 4 ( 11 ): 1902 - 12 . Epub 2005/11/10. doi: 10 .1128/ec.4.11. 1902 - 1912 . 2005 PMID: 16278457; PubMed Central PMCID : PMCPmc1287864 .
47. Fonseca FL , Nimrichter L , Cordero RJ , Frases S , Rodrigues J , Goldman DL , et al. Role for chitin and chitooligomers in the capsular architecture of Cryptococcus neoformans . Eukaryot Cell . 2009 ; 8 ( 10 ): 1543 - 53 . Epub 2009/07/21. doi: 10 .1128/ec.00142-09 PMID: 19617395; PubMed Central PMCID : PMCPmc2756858 .
48. Baker LG , Specht CA , Donlin MJ , Lodge JK . Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans . Eukaryot Cell . 2007 ; 6 ( 5 ): 855 - 67 . Epub 2007/04/03. doi: 10 .1128/ec.00399-06 PMID: 17400891; PubMed Central PMCID : PMCPmc1899242 .
49. Kronstad J , Saikia S , Nielson ED , Kretschmer M , Jung W , Hu G , et al. Adaptation of Cryptococcus neoformans to mammalian hosts: integrated regulation of metabolism and virulence . Eukaryot Cell . 2012 ; 11 ( 2 ): 109 - 18 . Epub 2011/12/06. doi: 10 .1128/ec.05273-11 PMID: 22140231; PubMed Central PMCID : PMCPmc3272904 .
50. McClelland EE , Bernhardt P , Casadevall A . Estimating the relative contributions of virulence factors for pathogenic microbes . Infect Immun . 2006 ; 74 ( 3 ): 1500 - 4 . Epub 2006/02/24. doi: 10 .1128/iai.74.3. 1500 - 1504 . 2006 PMID: 16495520; PubMed Central PMCID : PMCPmc1418678 .
51. Giles SS , Dagenais TR , Botts MR , Keller NP , Hull CM . Elucidating the pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans . Infect Immun . 2009 ; 77 ( 8 ): 3491 - 500 . Epub 2009/ 05/20. doi: 10 .1128/iai.00334-09 PMID: 19451235; PubMed Central PMCID : PMCPmc2715683 .
52. Kronstad JW , Attarian R , Cadieux B , Choi J , D'Souza CA , Griffiths EJ , et al. Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box . Nature reviews Microbiology . 2011 ; 9 ( 3 ): 193 - 203 . Epub 2011/02/18. doi: 10 .1038/nrmicro2522 PMID: 21326274; PubMed Central PMCID : PMCPmc4698337 .
53. Hu G , Kronstad JW . Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition . Current genetics . 2006 ; 49 ( 5 ): 341 - 50 . Epub 2006/01/07. doi: 10 .1007/s00294-005- 0054 -x PMID : 16397763 .
54. May RC , Park Y-D , Shin S , Panepinto J , Ramos J , Qiu J , et al. A Role for LHC1 in Higher Order Structure and Complement Binding of the Cryptococcus neoformans Capsule . PLoS Pathogens . 2014 ; 10 ( 5 ):e1004037. doi: 10.1371/journal.ppat.1004037 PMID: 24789368
55. Vidotto V , Aoki S , Ponton J , Quindos G , Koga-Ito CY , Pugliese A. A new caffeic acid minimal synthetic medium for the rapid identification of Cryptococcus neoformans isolates . Rev Iberoam Micol . 2004 ; 21 ( 2 ): 87 - 9 . Epub 2004/11/13. PMID: 15538835 .
56. Alby K , Bennett RJ . Interspecies pheromone signaling promotes biofilm formation and same-sex mating in Candida albicans . Proc Natl Acad Sci U S A . 2011 ; 108 ( 6 ): 2510 - 5 . Epub 2011/01/26. doi: 10 . 1073/pnas.1017234108 PMID: 21262815; PubMed Central PMCID : PMCPmc3038756 .