The Cryptococcus gattii species complex: Unique pathogenic yeasts with understudied virulence mechanisms

Abstract

Members of Cryptococcus gattii/neoformans species complex are the etiological agents of the potentially fatal human fungal infection cryptococcosis. C . gattii and its sister species cause disease in both immunocompetent and immunocompromised hosts, while the closely related species C . neoformans and C . deneoformans predominantly infect immunocompromised hosts. To date, most studies have focused on similarities in pathogenesis between these two groups, but over recent years, important differences have become apparent. In this review paper, we highlight some of the major phenotypic differences between the C . gattii and neoformans species complexes and justify the need to study the virulence and pathogenicity of the C . gattii species complex as a distinct cryptococcal group.

Full text

REVIEW
The Cryptococcus gattii species complex:
Unique pathogenic yeasts with understudied
virulence mechanisms
Lamin Saidykhan
1,2
, Chinaemerem U. Onyishi
1
, Robin C. MayID
1
*
1Institute of Microbiology & Infection and School of Biosciences, University of Birmingham, Edgbaston,
Birmingham, United Kingdom, 2Division of Physical and Natural Science, University of The Gambia,
Brikama Campus, West Coast Region, The Gambia
*r.c.may@bham.ac.uk
Abstract
Members of Cryptococcus gattii/neoformans species complex are the etiological agents of
the potentially fatal human fungal infection cryptococcosis. C.gattii and its sister species
cause disease in both immunocompetent and immunocompromised hosts, while the closely
related species C.neoformans and C.deneoformans predominantly infect immunocompro-
mised hosts. To date, most studies have focused on similarities in pathogenesis between
these two groups, but over recent years, important differences have become apparent. In
this review paper, we highlight some of the major phenotypic differences between the C.
gattii and neoformans species complexes and justify the need to study the virulence and
pathogenicity of the C.gattii species complex as a distinct cryptococcal group.
Introduction
Cryptococcus gattii (sensu stricto) was first recognized and described as a distinct cryptococcal
strain from Cryptococcus neoformans in 1970 [1]. Initially recognized as a variety of Cryptococ-
cus, the Cryptococcus neoformans var. gattii lineage was subsequently elevated to species status
as C.gattii [2]. Further genetic, biochemical, morphological, ecological, and serological charac-
terization of C.gattii environmental and clinical isolates provided more evidence for the classi-
fication of C.gattii as a unique cryptococcal species [2–10].
The C.gattii divergence from C.neoformans is estimated to have occurred 37 to 49 million
years ago [11,12]. Since then, C.gattii has maintained diversity by continuous recombination
and evolution into novel lineages with significant genetic diversity that warranted their classifi-
cation into monophyletic genotypes [12–14]. Recently, the five recognized C.gattii genotypes,
VGI, VGII, VGIII, VGIV, and VGVI were elevated to five individual species: C.gattii,C.deu-
terogattii,C.bacillisporus,C.tetragattii, and C.decagattii, respectively, while the two main line-
ages of C.neoformans were raised to species level, becoming C.neoformans and C.
deneoformans [15]. Different phylogenetic analysis based on concatenated genetic loci unani-
mously identified C.deuterogattii/VGII to be the basal lineage of the C.gattii species complex
[11,12,14]. VGI, VGIV, and VGIII diverged from VGII approximately 12.4 million years ago
[12,16]. Thereafter, C.tetragattii/VGIV diverged from C.bacillisporus/VGIII and C.gattii/VGI
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OPEN ACCESS
Citation: Saidykhan L, Onyishi CU, May RC (2022)
The Cryptococcus gattii species complex: Unique
pathogenic yeasts with understudied virulence
mechanisms. PLoS Negl Trop Dis 16(12):
e0010916. https://doi.org/10.1371/journal.
pntd.0010916
Editor: Joshua Nosanchuk, Albert Einstein College
of Medicine, UNITED STATES
Published: December 15, 2022
Copyright: ©2022 Saidykhan et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Funding: CUO has been supported by a PhD
scholarship from the Darwin Trust of Edinburgh.
LS has been supported by a PhD scholarship from
the Islamic Development Bank. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.

sister clades 11.7 million years ago, finally followed by the split between C.bacillisporus/VGIII
and C.gattii/VGI 8.5 million years ago.
All seven species of Cryptococcus are capable of causing the life-threatening disease crypto-
coccosis in humans [17]. C.neoformans (sensu stricto), which accounts for 99% of cases world-
wide [18–20], typically presents as fungal meningitis in immunocompromised patients. In
contrast, infections by the C.gattii species complex occurs more commonly in otherwise
healthy individuals and can often present as fungal pneumonia. During the 20th century, most
research considered C.gattii and C.neoformans to be interchangeable in their biology. How-
ever, the emergence of C.deuterogattii as the cause of the most devastating and unprecedented
fungal outbreak in a healthy human population [21,22] refocused attention on this species,
and, as a result, recent research has highlighted key differences between the C.gattii species
complex (CGSC) and C.neoformans species complex (CNSC). In particular, the apparently
low propensity for CGSC species to disseminate from the lung to the central nervous system,
and their ability to act as primary pathogens in healthy individuals, remain key unanswered
questions [5,23,24].
Phylogeny and speciation
To date, the majority of C.gattii studies have focused on C.deuterogattii/VGII, because of its
exceedingly high pathogenicity [21,25] and its role as the predominant etiological agent of the
devastating Pacific Northwest Outbreak (PNW) [21,22,26,27]. VGII is not only the cause of
the outbreak [27,28] but also possesses a high recombination frequency (via sexual macroevo-
lution and asexual microevolution) producing the highly clonal lineages VGIIa, VGIIb, VGIIc,
and VGIIx, which were responsible for the dissemination of the outbreak [21,29–31]. The high
clonality of the VGII subtypes has been found to emanate from VGII exhibition of nonclassical
same-sex mating, in which sexual reproduction occurs between two alpha mating-type
(MATα-MATα) parents [28], which has only been previously described in C.neoformans [32].
Hence, the PNW outbreak owes its origin and dissemination to this VGII-specific reproduc-
tive phenotype.
Morphological differences between C.gattii and C.neoformans
species complexes
Most of the virulence-related phenotypic differences between C.gattii and C.neoformans are
morphological. Within the Cryptococcus species complex as a whole, variation in morphologi-
cal traits such as cell body/capsule size, shape, budding, surface morphology, and cell wall
structure and composition are key factors employed not only for the identification of the dif-
ferent cryptococcal lineages [2,33–36], but also to support their elevation to distinct species
[15]. In an in vitro study of 70 cryptococcal clinical isolates (53 C.neoformans and 17 C.gattii),
Fernandes and colleagues documented that cellular and capsular enlargement in response to a
host-relevant environment is more common in C.gattii while capsule shedding and produc-
tion of micro cells were primarily C.neoformans traits (Table 1) [37]. In the same study, “giant
cells” (also known as titan cells; [38]) measuring >15 μm were predominantly found in CGSC
rather than in CNSC (50.0% versus 10.75%, respectively), an observation that was recapitulated
in a Drosophila model of infection [39].
Using an in vitro titan induction system, we recently showed that the capacity to produce
titan cells, an atypical morphotype that is formed when cryptococcal yeast cells transform into
extremely enlarged polyploid cells, is more abundant in CGSC than in CNSC [40]. Interest-
ingly, this correlates with a “staggered” cell cycle in C.deuterogattii, in which cell size increase
precedes DNA replication—something that is not seen in C.neoformans [40]. Whereas C.
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neoformans titan cells undergo cell division to produce daughter cells [40–43], CGSC titan
cells exhibit a growth arrest to form large unbudded cells [40] (Fig 1). It is possible that this dif-
ference may partially explain C.deuterogattii’s lower ability to disseminate outside the lungs,
since C.neoformans titan cells likely rely on their small-sized daughter cells for dissemination
[41,42].
These morphological differences also lead to differences in the host response. For example,
the presence of enlarged CGSC cells was associated with high CD4
+
T cell count, while the for-
mation of small phenotype “micro” cells by CNSC correlates with meningeal irritation and an
aggressive inflammatory response [37].
Compositional differences in capsule are also likely to play a major role in varying virulence
profiles. While exploring the interaction of C.gattii with the phagocytic amoebae Acantha-
moeba castellanii, Malliaris and colleagues [45] discovered a significantly lower phagocytosis
Table 1. Comparison of morphological attributes between 70 clinical isolates of cryptococci from HIV/AIDS patients in Botswana-Africa (adapted from [37] and
summarized).
Species/genotype (No. of isolates) Mean Cell diameter (μm) Mean Capsule thickness (μm) Giant cells (%) Micro cells (%) Shed capsule (%)
C.neoformans/ VNI (17) 7.0 5.5 12 82 94
/ VNII (2) 6.9 3.9 0 0 50
/VNBI (25) 8.3 7.3 20 52 80
/VNBII (9) 7.1 5.3 11 44 67
All C.neoformans (53) 7.32 5.5 10.75 44.5 72.75
C.gattii /VGI (1) 10.2 15.7 50 0
C.tetragattii /VGIV (16) 9.9 9.3 50 0 0
All C.gattii (17) 10.05 12.0 50 0 0
https://doi.org/10.1371/journal.pntd.0010916.t001
Fig 1. Micrograph showing the budding nature of C.deuterogattii yeast (left panel) vs. titan (right panel) cells [44]. Scale bar = 5 μm.
https://doi.org/10.1371/journal.pntd.0010916.g001
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profile and reduced virulence of an acapsular cryptococcal mutant strain, cap67, when the
mutant strain was coated with capsular extract from C.gattii (serotype B), versus extract from
C.neoformans (serotype A). Although the underlying mechanism is not known, the result sug-
gests the presence of structural difference(s) in C.gattii capsular polysaccharide, which have a
direct impact on virulence. Chemical and biophysical differences in the structure of the major
capsular polysaccharides in CGSC versus CNSC have been well documented [46]; conse-
quently, it will be interesting in the future to assess how these biochemical differences relate to
the differing host response.
After the polysaccharide capsule, the most important virulence determinant of cryptococci
[46,47] is cell wall melanisation [33,48–53]. Melanin is a negatively charged hydrophobic pig-
ment formed by the oxidative polymerization of phenolic compounds [51] and its synthesis is
catalysed by laccase. The production of melanin is not only essential for maintaining cell wall
integrity but also protects the fungi from environmental stressors, such as UV light and high
temperature, and the host immune system [54]. Interestingly, its pattern of distribution varies
between strains; for instance, being homogenous in the C.deuterogattii/VGII hypervirulent
outbreak strain CDCR265 [33] but heterogeneously distributed in C.neoformans H99. Inter-
estingly, in a Galleria mellonella infection model, melanization profiles (as determined by lac-
case activity) of the four C.gattii molecular types have been directly associated with virulence,
such that C.gattii species complex strains with higher melanin production showed higher
lethality towards Galleria larvae.
Immunomodulatory attributes of C.gattii species complex
The morphological and molecular traits of CGSC discussed above influence how the host
immune system responds to infection. The C.deuterogattii outbreak in the Pacific Northwest
(outside its regions of endemicity) that started in 1999 [55] had a mortality rate ranging from
8.7% to 50% even when treated with antifungal drugs [56–61], highlighting significant differ-
ences in the host response to this infection.
Innate immune response to C.gattii species complex
Cryptococcal infection begins with the inhalation of the fungi into the lungs. Lung-resident
macrophages are among the first host immune cells that inhaled fungi interact with; however,
there are relatively few studies that investigate the precise mechanisms by which phagocytes
respond to the presence of CGSC, as opposed to CNSC, in the host. As with C.neoformans, the
capsule of CGSC cells, which is composed of a majority glucuronoxylomannan (GXM) and a
minority galactoxylomannan (GalXM) polysaccharide, functions as a fungal virulence factor
and has antiphagocytic properties [62]. The phagocytosis of foreign particles is initiated by the
recognition of pathogen-associated molecular patterns (PAMPs) by host pattern recognition
receptors (PRRs), such as members of the Toll-like receptor (TLR) family and the C-type lectin
receptor (CLR) family [63]. GXM from C.deuterogattii was found to be recognised by Dectin-
3, a CLR, ultimately leading to NF-κB and ERK-dependent inflammatory responses (Fig 2A)
[64]. In the same study, wild-type and Dectin3
−/−
mice were infected with C.deuterogattii
intratracheally, and it was observed that Dectin3
−/−
mice had decreased survival, greater lung
and brain fungal burden, and decreased TNF-αand IL-6 production (Fig 2B). Thus, engage-
ment of Dectin-3 with C.deuterogattii GXM may activate a broader anticryptococcal immune
response. In another study, HEK293A cells transfected with TLR2/1 and TLR2/6 were able to
induce NF-κB activation after stimulation with GXM isolated from five different Cryptococcus
strains among which were C.gattii serotype B strain CN23/10.993 (C.deuterogattii/VGII) and
serotype C strain HEC40143 (VGIII/C.bacillisporus) and C.neoformans serotype A strains
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T
1
444 and HEC3393 (VNI) and serotype D strain ATCC 28938 (VNIV) [65,66]. Interestingly,
GXM from the C.deuterogattii strain resulted in the greatest activation of NF-κB, suggesting
the existence of structural and immunomodulatory differences between the strains [65] in a
way that is reminiscent of the study by Malliaris and colleagues [45] discussed above. The five
GXM samples were also used to stimulate nitric oxide (NO) production by RAW264.7 macro-
phages, and it was found that GXM samples from both CGSC strains were able to induce NO
production, while those from the three CNSC strains did not [65].
In a study that sought to define the cytokine profile produced by cryptococcal infection,
human peripheral blood mononuclear cells (PBMCs) from healthy individuals were infected with
heat-killed CGSC and CNSC clinical isolates. It was found that CGSC isolates induced a greater
expression of IL-1β, TNF-α, IL-6, IL-17, and IL-22, compared to CNSC isolates [67]. Meanwhile,
there was no difference in IL1Ra levels between the strains. Lastly, it was observed that the modu-
lation of CGSC-induced cytokine production required TLR4 and TLR9, but not TLR2.
Adaptive immune response to C.gattii species complex
The uptake of fungi into phagosomes and subsequent phagosome maturation results in the
degradation of the fungus and the presentation of fungal peptides on major histocompatibility
complex (MHC) molecules [68]. These peptides are then recognised by CD4
+
T cells, thereby
activating the adaptive immune response [69]. Additionally, the cytokine profile produced by
the activation of PRRs results in the differentiation of naïve T cells into Th1, Th2, or Th17 cells
[70]. The Th1 and Th17 responses are known to be protective against C.neoformans,Candida
albicans, and Aspergillus fumigatus [71–73]. Meanwhile, the Th2 response is anti-inflamma-
tory and promotes fungal survival and dissemination in C.neoformans [23,68]. It has been
shown that mice infected with C.deuterogattii and C.gattii had reduced Th1 and Th17 cells in
their lungs compared to those infected with C.neoformans H99 [24]. This suggests that CGSC
is able to successfully infect immunocompetent hosts by dampening the activation of the pro-
tective Th1/Th17 response and enhancing the nonprotective Th2 response [24]. The dimin-
ished Th1/Th17 response was likely driven by a decrease in the expression of MHC-II on the
surface of dendritic cells from C.deuterogattii- and C.gattii-infected mice. Huston and col-
leagues [74] have also shown that dendritic cells infected with C.deuterogattii fail to exhibit
Fig 2. The role of Dectin-3 in host response to C.deuterogattii infection in vitro and in vivo.(A) The C-type lectin receptor, Dectin-3, recognises C.
deuterogattii (Cd) capsular glucuronoxylomannan (GXM) [64]. The recognition of GXM leads to the activation of NF-κB and ERK signalling pathways to drive
proinflammatory cytokine production. (B) Dectin-3 deficient mice showed increased susceptibility to C.deuterogattii infection [64]. Figure created with
BioRender.com.
https://doi.org/10.1371/journal.pntd.0010916.g002
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increased expression of MHC-II molecules, CD86, CD83, CD80, and CCR7, which are needed
for T cell activation. Therefore, at least two CGSC strains are able to subvert dendritic cell–
mediated activation of the adaptive immune response.
Another mechanism by which CGSC is thought to subvert immune recognition is through
its capsule polysaccharide. Urai and colleagues [66] found that mice infected with C.deutero-
gattii/VGII strain JP02 showed poorer survival and decreased inflammatory cell infiltration
when compared to mice infected with C.neoformans H99. This difference in virulence was
attributed to the C.deuterogattii capsule structure because exposing dendritic cells to purified
GXM from JP02 did not induce IL6, IL12p40, and TNF-αproduction but stimulation with
H99 GXM did. This finding is reinforced by a 2021 study by Ueno and colleagues [75] that
found that an acapsular cap60ΔC.gattii (VGI) mutant was easily phagocytosed and killed by
dendritic cells; however, when capsular polysaccharide from C.gattii was deposited onto the
acapsular mutant, phagocytosis was hindered and IL6 and IL23 proinflammatory cytokine
expression was dampened [75]. Therefore, capsular polysaccharide aids immune evasion by
shielding C.gattii sensu stricto from recognition by dendritic cells, thereby preventing the
expression of proinflammatory cytokines and hindering clearance of the fungi [75]. This abil-
ity to evade immune detection also provides an explanation for why strains within the C.gattii
species complex are able to infect both immunocompetent and immunocompromised individ-
uals while C.neoformans strains predominantly infect immunocompromised people.
It is important to mention that the result by Urai and colleagues [66] and Ueno and col-
leagues [75] is in contrast with earlier findings by Fonseca and colleagues [65], which showed
that GXM from C.deuterogattii were the most potent in inducing TLR-mediated NF-κB
expression and NO production in macrophages. It also contrasts with results from Schoffelen
and colleagues [67], which found that PBMC exposed to CGSC strains produced more proin-
flammatory cytokines than those exposed to CNSC strains. This may be explained as differ-
ences in macrophage versus dendritic cell response. Alternatively, it may represent variation
within the CGSC, highlighting the need to be precise about the “C.gattii” species used. Aside
from the within CGSC variation, all these studies point to the existence of significant variation
in immune response to CGSC and CNSC infection. As more is understood about host interac-
tion with cryptococcal species, novel therapeutic agents can be developed to decrease the case–
fatality rate of infection with cryptococci.
What are the drivers of the C.deuterogattii outbreak?
In 1999, an outbreak of C.gattii (now recognised as C.deuterogattii) was identified in British
Columbia, which went on to become the largest life-threatening primary fungal outbreak in a
healthy population. This unprecedented outbreak has driven an intense research effort to dis-
cover the underlying determining factors [25,26,76,77]. Over the last decade, studies focused
on outbreak strains revealed a hypervirulent C.gattii molecular type C.deuterogattii/VGII, to
be the primary agent driving the pathogenesis [21,78,79] and spread [28,80] of the outbreak.
C.deuterogattii/VGII outbreak isolates harbour several unique cellular and genetic attri-
butes that contribute to their hypervirulence and pathogenicity. Compared to other CGSC lin-
eages, VGII outbreak strains display higher resistance to host immune defence mechanisms
[78,79] and an overall increased survival profile within the host [79,81]. Upon phagocytosis by
macrophages, C.deuterogattii/VGII responds to the host oxidative burst by triggering rapid
intracellular proliferation [78]. A follow-up study revealed that there is in fact a “division of
labour” mechanism where a subpopulation of fungal cells undergo growth arrest, thereby facil-
itating the rapid proliferation of neighbouring fungal cells [79], in a process that is mediated
through the exchange of extracellular vehicles (EVs) [81]. Interestingly, the pattern of titan cell
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formation also differs in this outbreak lineage (discussed above), and it will be interesting to
explore and potentially establish the correlation between this unique VGII-titan feature, divi-
sion of labour responses, and their combined influence on virulence.
In addition to cellular phenotypes, novel genomic traits found in PNW outbreak isolates
are thought to contribute to their hypervirulence [16,31]. Genomic analysis among the four
VG lineages reveals that, unlike other VG lineages, VGII outbreak isolates have acquired genes
encoding proteins involved with membrane trafficking such as Friend of Prmt1 (Fop), and
genes involved in heat tolerance such as heat shock protein 70 (HSP70), genes that are known
to be required for virulence in C.neoformans [82]. Perhaps the most striking genomic trait of
VGII is the lack of RNA interference (RNAi) machinery due to the absence of genes encoding
the Argonaute proteins Ago1 and Ago2 [16], PAZ, Piwi, and DUF1785 genes, which are key
regulator of the RNAi machinery in C.neoformans [31,83]. In fact, analysis from whole
genome studies discovered a total of 146 genes (including the RNAi-associated ones) missing
in VGII outbreak isolates, which is three times more than those lost in VGI-III/VGIV com-
bined [31]. Although the true significance of the absence of these genes/pathways as a whole is
not yet elucidated, it is likely that they contribute to the unique host–pathogen interaction
seen in this lineage [28,84].
Understudied virulence-associated phenotypes of C.gattii
Studies elucidating the virulence of CGSC phenotypes and their impact on pathogenesis and
disease outcomes are somewhat overshadowed by that of C.neoformans [76]. Often, the biol-
ogy, virulence factors, and pathogenicity of Cryptococcus are highlighted using C.neoformans
as a model or the primary theme of study [46,52,85,86]. Below are some examples of under-
studied CGSC virulence phenotypes.
Capsule
The cryptococcal polysaccharide capsule is essential for both cellular function and pathogene-
sis of Cryptococcus, protecting the fungus against desiccation in the environment and playing
the synergistic role of a shield and virulence mechanism in animal host [46,47]. Thus, the cap-
sule is considered the most important virulence determinant of cryptococci. Evidence has
shown that capsular size and its impact on host immune response in the C.gattii lineage differs
from that of C.neoformans [37,84]. Despite these differences in capsule properties, the biosyn-
thesis properties [18], biophysical properties [46,47,87,88], and immunogenic properties [89]
of cryptococcal capsule have primarily been characterized in C.neoformans. Although the
physical impact of capsule on fungal biology is likely to be similar for both species, the impact
of varying capsule composition on CGSC virulence remains less well understood [90].
Morphogenesis
The most dramatic host adaption phenotype exhibited by Cryptococcus is the formation of
titan cells [43,91,92], a phenomenon that has thus far been largely studied in C.neoformans
[92–96]. We and others have recently investigated titan cell formation across the broader Cryp-
tococcus species complex, which has revealed some subtle differences between lineages [44].
The impact of these differences on the CGSC–host interaction remains largely unknown [97–
99]. Similarly, while several signalling pathways, receptors, and genes have been identified as
driving titan cell formation in C.neoformans (including the cyclic AMP (cAMP)/protein
kinase A (PKA) pathway (putatively associated with C.neoformans pathogenesis), G protein-
coupled receptors (Gpr4 and Gpr5), St3a [38], and the CLN1 genes [93]), such investigations
are yet to be performed with C.gattii and its sister species.
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Extracellular vesicles
Extracellular vesicles (EVs) are rounded bilayered particles produced by prokaryotic and
eukaryotic cells to mediate intercellular communication by transferring information between
cells [100]. EVs have significant roles in the cellular and pathogenic lifestyle of cells including
stress response, intercellular competition, lateral gene transfer (via RNA or DNA), pathogenic-
ity, and detoxification [101]. The first fungal EV was described in C.neoformans, and, there-
fore, the biology of EVs and its role in C.neoformans virulence is well studied and
documented [102–105]. Cryptococcal EVs are carriers of several virulence molecules such as
capsular GXM, laccase, urease, and a repertoire of immunogenic proteins and thus have been
termed “virulence bags” [104,105].
The biological and functional features of EVs including biosynthesis, secretory pathways,
composition, structure, virulence properties and mechanism, influence in pathogen–host
interaction, and immunogenic-related attributes have been well characterized in C.neofor-
mans [102,104–107]. However, our knowledge and concepts of C.neoformans-derived EVs
cannot be directly applied to CGSC strains, since evidence has shown distinctive features of
EVs in the two species complexes [102]. For instance, the size of C.deuterogattii EVs is signifi-
cantly smaller than C.neoformans and C.deneoformans [102]. Although homologous EV-pro-
tein families, such as the putative glyoxal oxidase (Gox proteins) and Ricin-type beta-trefoil
lectin domain-containing protein (Ril), were characterized in all three species, the Sur7/Pal1
family of tetraspanin membrane proteins was exclusive to C.deuterogattii [102]. Moreover,
two of the ferroxidase Cfo proteins investigated were found to be expressed only by C.deutero-
gattii EVs but not in the two other species [108].
In terms of function, the novel discovery of C.deuterogattii EV-based long-distance patho-
gen-to-pathogen communication (which has not been reported for non-gattii strains) [81], its
role in exploiting host immune cells, and ultimate impact on virulence [79,81] provide strong
evidence that the functional mechanism of EVs differs in the two lineages. This discovery and
other findings on CGSC-specific EV traits highlight the need for probing the already-studied
concepts of C.neoformans-derived EV and novel EV-associated phenotypes in these other spe-
cies. Research focused on CGSC EVs will not only diversify our understanding of cryptococcal
EVs but also holds potential for revealing new paradigms around the biological and pathogenic
functions of fungal EVs more broadly.
We conclude this section with a list of C.neoformans phenotypic virulence traits that are
somewhat neglected and could potentially be studied in C.gattii (Table 2).
Table 2. List of virulence-related phenotypic traits whose underlying molecular, genetic, and metabolic mechanism has been studied in C.neoformans but not C.
gattii.
Virulence factor Virulence factor regulatory genes/pathways Mode of action/functional mechanism References
Capsule Cap59 gene Encodes transmembrane proteins and is involved in GXM synthesis [109],
[110,111]
Cap64 gene Complements an acapsular strain and is required for capsule synthesis [112]
Cap60 gene Encodes proteins localized to the nuclear membrane and cytoplasm;
required for both capsule formation and virulence
[113]
Uge1p gene; putative UPD-galactose
epimerase
Required for GalXM synthesis and consequent crossing of the blood–
brain barrier
[114]
Putative G1/S cyclin (Cln1) Regulates the cell cycle during capsule formation; required for virulence
at 37˚C
[115]
Rim101 transcription factor via cAMP/PKA
pathways
Required for polysaccharide attachment to the cell wall surface [116]
CLN1 gene Encodes C.neoformans cyclin Cln1; critical for balancing DNA
replication and cell division during titan cell formation; negatively
regulates in vivo titan cell formation
[93]
(Continued)
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Concluding remarks
Despite their similar appearance, it is now clear that many features of the CGSC are remark-
ably different from those in the CNSC (Fig 3). However, studies elucidating the virulence of C.
gattii phenotypes and their impact on pathogenesis and disease outcomes have been overshad-
owed by those focusing on CNSC. While the prevalence of cryptococcosis due to CNSC makes
this a logical choice, several unique features of C.gattii and its sister species warrant further
Table 2. (Continued)
Virulence factor Virulence factor regulatory genes/pathways Mode of action/functional mechanism References
Titan cell formation Rim101 transcription factor via cAMP/PKA
pathways
Required for the generation of titan cells [117]
Usv101 transcription factor, GPA1, CAC1,
Ric8, and PKA1 genes associated with
cAMP/PKA pathway
Negatively regulates titan cell formation in vivo and in vitro [118] [41]
[117]
G1/S cyclin (Cln1) Required for cell wall stability and production of melanin; protective
against oxidative damage; positively regulates the production of laccase
[119]
Cell wall Proteinase Proteolytic activity against host proteins including collagen, elastin,
fibrinogen, immunoglobulins, and complement factors
[120]
Degradative enzymes
(proteinase, phospholipases,
urease)
Phospholipase enzyme (PLB1 gene) Regulates phospholipase B (PLB), lysophospholipase hydrolase, and
lysophospholipase transacylase activities; positively regulates virulence
in vivo and is required for intracellular growth and vomocytosis
[52] [121]
Urease (Ure1 gene) Required for virulence in mice, CNS invasion; and vomocytosis [122] [121]
CNA1 gene Encodes C.neoformans calcineurin A; required for growth at
mammalian physiological temperature; required for virulence in
immunocompromised animal model
[40]
https://doi.org/10.1371/journal.pntd.0010916.t002
Fig 3. Schematic diagram illustrating CGSC distinct phenotypic virulencetraits as compared to C.neoformans.Upon inhalation from the
environment, CGSC yeast cells/spores responds to the lung extracellular niche by exhibiting phenotypic traits including larger capsule (with
less immunogenic properties), larger cell body (with higher degree of homogeneity), and thinner but more compacted cell wall with higher
chitosan content than C.neoformans. The manner in which CGSC strains exhibit these host-adaptive traits is perhaps responsible for its low
affinity to dissemination from the lungs to the brain. Within the host macrophage, the intracellular phenotypes (mitochondrial fusion and
“division of labour” proliferation mechanism mediated by extracellular vesicles) exhibited by C.deuterogattii (which drives the fatal Pacific
Northwest outbreak) are absent in C.neoformans.
https://doi.org/10.1371/journal.pntd.0010916.g003
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PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010916 December 15, 2022 9 / 17

Key Learning Points
• Although morphologically similar, members of the Cryptococcus gattii species complex
and Cryptococcus neoformans species complex exhibit important differences in biology
and pathogenesis.
• Their interaction with the human immune system is one such key difference, with evi-
dence for more profound immune-dampening mechanisms within the C.gattii species
complex, contributing towards its enhanced ability to infect healthy hosts.
• Both species complexes form titan cells, but the triggers and mechanisms by which
they do so are subtly different.
• Extracellular vesicle release occurs in both species, but only C.deuterogattii has thus
far been shown to use these vesicles to coordinate a virulence strategy.
• The recent recognition that lineages within both species are sufficiently different to
warrant elevation to species level should serve as a prompt for a more detailed exami-
nation and appreciation of the varying biology within this genus.
Top Five Papers
1. Byrnes EJ, 3rd, Li W, Lewit Y, Ma H, Voelz K, Ren P, et al. Emergence and patho-
genicity of highly virulent Cryptococcus gattii genotypes in the northwest United
States. PLoS Pathog. 2010;6(4):e1000850. Epub 2010/04/28. doi: 10.1371/journal.
ppat.1000850. PubMed PMID: 20421942; PubMed Central PMCID:
PMC2858702.
2. Hagen F, Khayhan K, Theelen B, Kolecka A, Polacheck I, Sionov E, et al. Recogni-
tion of seven species in the Cryptococcus gattii/Cryptococcus neoformans species
complex. Fungal Genet Biol. 2015;78:16–48. Epub 2015/02/28. doi: 10.1016/j.fgb.
2015.02.009. PubMed PMID: 25721988.
3. Kidd SE, Hagen F, Tscharke R, Huynh M, Bartlett KH, Fyfe M, et al. A rare geno-
type of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver
Island (British Columbia, Canada). Proceedings of the national academy of sci-
ences. 2004;101(49):17258–63.
4. Voelz K, Johnston SA, Smith LM, Hall RA, Idnurm A, May RC. ‘Division of
labour’in response to host oxidative burst drives a fatal Cryptococcus gattii out-
break. Nat Commun. 2014;5(1):1–12.
5. Cheng PY, Sham A, Kronstad JW. Cryptococcus gattii isolates from the British
Columbia cryptococcosis outbreak induce less protective inflammation in a
murine model of infection than Cryptococcus neoformans. Infect Immun.
2009;77(10):4284–94. Epub 2009/07/29. doi: 10.1128/iai.00628-09. PubMed
PMID: 19635827; PubMed Central PMCID: PMC2747943.
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investigation, including their ability to infect otherwise healthy individuals, their role in an
unprecedented fungal outbreak and several distinct phenotypic traits that differ from those in
C.neoformans. Such features are likely to underlie important differences in clinical presenta-
tion between the two pathogens, most notably in their varying patterns of dissemination from
the lungs to the brain. In the future, it will be important to revisit many paradigms of crypto-
coccal pathogenesis in CGSC strains and, indeed, other Cryptococcus species and, in doing so,
reveal the full range of diversity within this genus.
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