Connecting virulence pathways to cell-cycle progression in the fungal pathogen Cryptococcus neoformans
Connecting virulence pathways to cell-cycle progression in the fungal pathogen Cryptococcus neoformans
Proliferation and host evasion are critical processes to understand at a basic biological level for improving infectious disease treatment options. The human fungal pathogen Cryptococcus neoformans causes fungal meningitis in immunocompromised individuals by proliferating in cerebrospinal fluid. Current antifungal drugs target “virulence factors” for disease, such as components of the cell wall and polysaccharide capsule in C. neoformans. However, mechanistic links between virulence pathways and the cell cycle are not as well studied. Recently, cell-cycle synchronized C. neoformans cells were profiled over time to identify gene expression dynamics (Kelliher et al., PLoS Genet 12(12):e1006453, 2016). Almost 20% of all genes in the C. neoformans genome were periodically activated during the cell cycle in rich media, including 40 genes that have previously been implicated in virulence pathways. Here, we review important findings about cell-cycle-regulated genes in C. neoformans and provide two examples of virulence pathways-chitin synthesis and G-protein coupled receptor signaling-with their putative connections to cell division. We propose that a “comparative functional genomics” approach, leveraging gene expression timing during the cell cycle, orthology to genes in other fungal species, and previous experimental findings, can lead to mechanistic hypotheses connecting the cell cycle to fungal virulence.
Cryptococcus neoformans; Cell-cycle transcription; Virulence factors; Gene regulatory networks
Human fungal pathogens cause more than a million
lifethreatening illnesses each year (Brown et al. 2012).
Antifungal drug development focuses on targeting the pathogen
without causing significant side effects in the host. The
cell cycle is highly conserved across eukaryotic species,
because it is an essential process for growth and division.
Thus, cell-cycle machinery is not an ideal candidate for
antifungal drug design. However, connections between the
cell cycle and fungal-specific virulence factors are poorly
understood. An improved basic biological understanding
of fungal proliferation and links to virulence pathways can
increase drug treatment options.
The cell division cycle is a fundamental biological
process underlying growth and reproduction. The cell cycle
is divided into four phases (Gap 1, Synthesis, Gap 2, and
Mitosis), where cells precisely duplicate their genomic
content and then faithfully segregate cellular contents into
two new cells (Morgan 2007). These cell-cycle events, such
as DNA replication and spindle formation, are regulated by
cyclin-dependent kinases (CDKs) and their cyclin-binding
partners (Bloom and Cross 2007; Evans et al. 1983;
Hartwell et al. 1974; Nasmyth 1993; Nurse and Thuriaux 1980).
In addition to driving periodic cellular events, many genes
encoding cell-cycle regulators are themselves periodically
transcribed. Programs of periodic gene expression have
been observed in many eukaryotes including fungi, plants,
mice fibroblasts, and human cell lines (Bar-Joseph et al.
2008; Grant et al. 2013; Ishida et al. 2001; Menges et al.
2005; Oliva et al. 2005; Peng et al. 2005; Rustici et al.
2004). In the budding yeast Saccharomyces cerevisiae,
transcription of periodic genes in the proper cell-cycle
phase is controlled by transcription factors, which are also
regulated at the protein level by cyclin/CDKs and by
ubiquitin ligases such as the APC/C (Bristow et al. 2014;
Landry et al. 2014; Lee et al. 2002; Orlando et al. 2008;
Ostapenko and Solomon 2011; Simmons Kovacs et al. 2012;
Simon et al. 2001) (reviewed in: Benanti 2016; Haase and
Cryptococcus neoformans (Basidiomycota) is a distantly
related budding yeast to S. cerevisiae (Ascomycota)
(Stajich et al. 2009), but the cell cycle is not as well
characterized in C. neoformans. C. neoformans can cause a
respiratory infection with pneumonia-like symptoms in the lungs,
followed by dissemination and proliferation in the human
central nervous system. Fungal meningitis and other
infections are a leading cause of death in immune-compromised
individuals (Brown et al. 2012; Park et al. 2009). The most
effective antifungal treatments for cryptococcosis target
C. neoformans cells without affecting host cells.
Therefore, the translational realm of the C. neoformans field
studies “virulence factors” for fungal disease, such as the
yeasts’ cell wall and polysaccharide capsule (O’Meara and
Alspaugh 2012). The cell cycle has not traditionally been
considered a virulence factor, but many virulence
functions appear to be under cell-cycle control, so mechanisms
controlling cell division will impact fungal virulence. For
example, one G1 cyclin gene has been identified in C.
neoformans (CNAG_06092), and its mRNA is expressed
periodically during the cell cycle (Kelliher et al. 2016). The
CLN1 gene is not essential for viability, but the knockout
strain is defective in proliferation at 37 °C, less virulent
in an insect model, grows to abnormally large cell sizes,
lacks melanin production, and shows polysaccharide
capsule defects compared to wild-type controls (García-Rodas
et al. 2014, 2015). These genetic findings directly connect
cell-cycle machinery defects to canonical virulence
pathways. Interestingly, a recent screen for essential genes C.
neoformans identified ribosomal RNA and other metabolic
regulators but did not identify any putative cell-cycle genes
(Kuwada et al. 2015). Thus, there is still much to be learned
about the cell cycle in C. neoformans, as the only known
G1 cyclin gene and other putative key regulators of the cell
cycle do not appear to be required for viability.
In this review, we highlight the importance of
combining transcriptome dynamics with functional studies. We
investigate two virulence pathways in C. neoformans that
contain genes that are periodically expressed during the
cell cycle. We find that multiple enzymes controlling
chitin synthesis are co-expressed periodically in a specific
cell-cycle phase. On the other hand, genes involved in
mating pheromone sensing are expressed in different
cellcycle phases. After identifying the expression timing of the
virulence genes of interest, we predict roles of these genes
during the cell cycle by incorporating the previous genetic
and cell biological findings about gene function and by
comparing to sequence orthologs in S. cerevisiae. These
two virulence pathways serve as examples for the types of
mechanistic hypotheses that can be generated from
understanding the gene expression dynamics of the C.
neoformans cell cycle. We close with a discussion of the
direction of this functional genomics work—constructing gene
regulatory networks that explain how large programs of
periodic genes are controlled during the fungal cell cycle.
Periodic cell-cycle genes have been characterized
extensively in the budding yeast S. cerevisiae (Bristow et al.
2014; Cho et al. 1998; de Lichtenberg et al. 2005; Eser
et al. 2014; Granovskaia et al. 2010; Hereford et al. 1981;
Orlando et al. 2008; Pramila et al. 2006; Spellman et al.
1998). In S. cerevisiae, many cell-cycle genes peak in
mRNA expression level before their protein products are
used in cell cycle events. One canonical example is DNA
replication origin firing, where replication origin proteins
are assembled into a complex before S phase, activated,
and then degraded or changed in localization to prevent
rereplication (Bell and Dutta 2002). This “just-in-time
transcription” phenomenon can be visualized for the conserved
DNA helicase complex that acts during origin firing in both
S. cerevisiae and C. neoformans (Fig. 1a, b). Our recent
publication describes in detail how different time series
experiments for C. neoformans and S. cerevisiae cells
are aligned on a common “cell-cycle time” axis using the
CLOCCS algorithm (Orlando et al. 2007, 2009) (Kelliher
et al. 2016: S1 File). According to this common timeline,
origin-firing genes are transcribed in early G1 phase
during each cell cycle (Guo et al. 2013; Kelliher et al. 2016).
These findings suggest a common function for MCM genes
in S. cerevisiae and in C. neoformans.
Comparative genomics was also applied to groups of
coregulated cell-cycle genes (Kelliher et al. 2016). We found
that DNA replication (S phase) and mitosis (M phase)
genes in S. cerevisiae and C. neoformans were highly
conserved in periodicity and timing of expression during the
fungal cell cycle. These analyses required identification of
orthologous genes in the two species of budding yeast, and
our recent publication describes in detail how orthologous
genes were identified in C. neoformans and S. cerevisiae
(Kelliher et al. 2016: S1 File). Almost one thousand
additional periodic genes were identified in C. neoformans,
many of which have not been previously linked to the cell
cycle. We posit that identifying the phase in which these
Fig. 1 Timing of expression provides mechanistic insights for
DNA replication, chitin synthase, and GPCR subunits during the
C. neoformans and S. cerevisiae cell cycles. The MCM2-7 genes
involved in DNA replication origin firing are plotted in C.
neoformans (respectively: CNAG_03341, CNAG_00099, CNAG_06182,
CNAG_04052, CNAG_03962, and CNAG_05825) (a) and S.
cerevisiae (respectively: YBL023C, YEL032W, YPR019W, YLR274W,
YGL201C, and YBR202W) (b) to visualize activation timing before
S phase of the cell cycle. Chitin synthase genes in C. neoformans
are expressed after S phase (c), while S. cerevisiae orthologs vary in
their expression timing (d). C.n. CHS4 is orthologous to S.c. CHS3
(YBR023C, red c, d). Both CHS6 and CHS8 have orthology to CHS1
(YNL192W) and CHS2 (YBR038W) in S. cerevisiae. According to a
global sequence similarity measure (Kelliher et al. 2016: S4 Table),
C.n. CHS6 is most similar to S.c. CHS2 (green c, d), and C.n. CHS8
is more similar to S.c. CHS1 (blue c, d). GPCR subunits in C.
neoformans are expressed at different times during the cell cycle (e), and
S. cerevisiae orthologs are less periodic and vary in expression
timing (f). C.n. GPA2 is orthologous to both S.c. GPA1 (YHR005C) and
GPA2 (YER020W, red lines e, f), and GPG2 is orthologous to STE18
(YJR086W) in S. cerevisiae. In all plots, orthologous gene pairs are
shown in the same color, and ortholog identification data can be
found in the previous work (Kelliher et al. 2016: S4 Table, S1 File).
Line plots are shown on an fpkm unit scale, which were normalized
separately for each yeast experiment. All transcripts are plotted on a
common cell-cycle timeline in CLOCCS lifeline points as described
(Kelliher et al. 2016: S1 File). Periodicity rankings for each C.
neoformans gene can be found in S2 Table, and S. cerevisiae genes can
be found in S1 Table (Kelliher et al. 2016)
unknown genes are expressed can provide mechanistic
insights. For example, S. cerevisiae genes that play a role
in bud emergence peak in expression before G1/S phase.
Orthologous genes in C. neoformans were not highly
conserved in periodicity or timing of expression at G1/S phase
(Kelliher et al. 2016: Figure 4). This putative divergence in
budding gene timing is supported by data that C.
neoformans bud emergence can occur in a range of times between
G1 and G2 phases, depending on culturing conditions such
as oxygen levels and cell concentration (Ohkusu et al.
2001, 2004). Thus, gene orthology alone is not
necessarily informative regarding biological function across fungal
systems. Below, we investigate gene expression timing of
two virulence pathways to connect virulence mechanisms
to cell-cycle progression.
Chitin synthesis in C. neoformans may be directly
linked to cell-cycle progression
Genes involved in virulence pathways are of critical
importance for understanding the biology and for treating the
opportunistic fungal pathogen C. neoformans (Buchanan
and Murphy 1998; Liu et al. 2008). Our recent
publication identified 40 periodic genes that have been previously
identified by genetic screens for virulence phenotypes
(Kelliher et al. 2016: S3 Table). Here, we asked if any
metabolic pathways were enriched in this list of 40 virulence
genes using the database FungiDB (Stajich et al. 2012).
The most significant Metabolic Pathway hit (PWY-6981)
included four genes involved in chitin biosynthesis. These
four chitin synthase enzymes—CHS4 (CNAG_00546),
CHS5 (CNAG_05818), CHS6 (CNAG_06487), and CHS8
(CNAG_07499)—are periodically expressed during the C.
neoformans cell cycle (Kelliher et al. 2016).
Chitin synthesis is a ubiquitous and dynamic process
across fungal species (Langner and Göhre 2016). The
previous work has characterized the family of chitin synthase
genes and shown that chitin and chitosan levels accumulate
along with population density in C. neoformans, unlike the
budding yeasts S. cerevisiae and Candida albicans (Banks
et al. 2005). The CHS3 gene is highly expressed in
proliferating C. neoformans cells, and single chs3 mutants are
temperature sensitive at 37 °C, which is a highly relevant
virulence factor for human infection (Bloom and Panepinto
2014). In addition to the previous work on steady-state
expression levels of chitin synthase genes from
asynchronous C. neoformans cells (Banks et al. 2005: Figure 3),
the cell-cycle time series data set now provides much more
dynamical detail. We visualized the periodic chitin
synthase genes to determine their timing of peak expression
during the cell cycle.
The four periodic chitin synthase genes are co-expressed
and peak in expression after the S phase MCM genes in C.
neoformans (Fig. 1a, c). The previous work showed that
chitin/chitosan levels in the cell wall vary between S.
cerevisiae and C. neoformans (Banks et al. 2005), and thus,
it was important to compare these chitin genes to their
putative orthologous genes in S. cerevisiae. In C.
neoformans, chitin synthase genes are much more coordinately
expressed in time than their S. cerevisiae orthologs (Fig. 1c,
d). The S. cerevisiae gene CHS2 is most similar in
dynamics and expression timing to the group of C. neoformans
chitin synthases. The S. cerevisiae CHS2 gene plays a role
in cell wall remodeling during cytokinesis (Oh et al. 2012;
Sburlati and Cabib 1986), while CHS1 and CHS3 affect
chitin levels in the S. cerevisiae cell wall during
alpha-factor arrest (shmoo formation), during bud emergence, and
generally during cell growth (Shaw et al. 1991).
We hypothesize that the four periodically expressed
chitin synthase enzymes in C. neoformans are utilized
after S phase for bud growth and/or during cytokinesis for
extracellular matrix remodeling. Unlike S. cerevisiae, the
expression of CHS4, CHS5, CHS6, and CHS8 is tightly
coordinated, suggesting they act at the same time to
perform a similar function. The transcription factor(s)
controlling the coordinated activation of CHS genes is unknown.
The CRZ1 transcription factor (CNAG_00156) is known to
regulate CHS6 expression levels under various stress
conditions (Lev et al. 2012), but the CRZ1 transcript did not
score highly for cell-cycle periodicity (Kelliher et al. 2016:
S2 Table). If the transcriptional regulator(s) can be
identified, a combination drug therapy (Bahn 2015; Zhang et al.
2014) to stall the fungal cell cycle in G2 or M phase and
simultaneously inhibit chitin synthase could render cells as
poorly virulent as chs3 mutants in the laboratory (Banks
et al. 2005). Chitin synthesis represents a promising
antifungal target for further study.
A subset of G-protein coupled receptor subunits
are expressed at different times during the C.
neoformans cell cycle
Given the 40 periodic genes with previously identified
virulence phenotypes (Kelliher et al. 2016: S3 Table), we
also used FungiDB to ask if any Gene Ontology terms were
enriched (Stajich et al. 2012). G-protein coupled
receptor signaling (GO:0007186) was one of the top five most
significant GO terms. G-protein coupled receptor (GPCR)
signaling pathways have been studied extensively in C.
neoformans for their role in sensing and responding to
the cellular environment (Xue et al. 2008). The three
periodically expressed GPCR subunits have previously been
implicated in the signaling pathway that allows haploid
C. neoformans cells to sense the opposite mating type via
mating pheromones. During infection, C. neoformans cells
are typically haploid and proliferating asexually. However,
meiotic spores are thought to initiate the first steps of host
colonization in the lungs, and thus, understanding the
biology of both the asexual and sexual phases of C. neoformans
growth is essential (Kozubowski and Heitman 2012).
Three periodic GPCR subunits are expressed at different
times during the cell cycle (Fig. 1e). GPG2 (CNAG_05890)
is a Gγ subunit, which can bind to Gβ subunits in two
different signaling pathways: Gib2 (CNAG_05465),
associated with nutrient sensing, and Gpb1 (CNAG_05465),
associated with mating pheromone sensing (Palmer et al.
2006). GPA2 (CNAG_00179) and GPA3 (CNAG_02090)
are Gα subunits, and are expressed in different phases of
the cell cycle (Fig. 1e). The previous work has shown that
GPA2 activates mating, while GPA3 inhibits mating, but
both Gα genes must be deleted for a fungal sterility
phenotype (Hsueh et al. 2007). The mating pathway in S.
cerevisiae is well understood and has fewer components than C.
neoformans (Bardwell 2004; Dohlman and Thorner 2001).
In S. cerevisiae, the G-protein subunits involved in
mating pheromone signaling are GPA1 (Gα), STE18 (Gγ), and
STE4 (Gβ). The S. cerevisiae GPA1 subunit is periodically
expressed during the cell cycle, but its peak expression
timing does not precisely match its ortholog in C. neoformans
(Fig. 1e, f).
Unlike GPA1 in S. cerevisiae, the Gα subunits of the
C. neoformans mating pathway (GPA2 and GPA3) peak in
different phases of the cell cycle. Perhaps, C. neoformans
cells are capable of sensing mating pheromone throughout
the cell cycle, rather than exclusively G1 phase.
Alternatively, these GPCRs may have been repurposed for other
functions in C. neoformans. The strong peak of GPA2
expression at each G1 phase in C. neoformans does suggest
that, like S. cerevisiae, cells may be “deciding” whether or
not to mate before commitment to each cell cycle (Fig. 1e,
f). Intriguingly, mating in C. neoformans is also linked to
light–dark cycles and regulated by the circadian rhythm
transcription factor orthologs BWC1 (CNAG_05181) and
BWC2 (CNAG_02435) (Idnurm and Heitman 2005). These
two TF genes do not score as highly periodic during the C.
neoformans cell cycle (Kelliher et al. 2016: S2 Table), but
the contribution of circadian rhythms to virulence is not
well understood for many pathogenic species and warrants
further study (Hevia et al. 2016).
In this review, we highlight what can be learned about gene
function by the pattern of expression throughout the cell
cycle. By combining information about expression
dynamics with orthology and functional studies from model
systems, we demonstrate that new mechanistic hypotheses can
be rapidly generated. Here, we begin to elucidate
connections between the cell cycle and virulence pathways using
these approaches. We show that four chitin synthases in
C. neoformans are co-expressed after S phase, unlike
their putative orthologs in S. cerevisiae (Fig. 1c, d). We
also demonstrate that two Gα subunits involved in
mating pheromone signaling are expressed in different
cellcycle phases, where their putative S. cerevisiae ortholog is
expressed only prior to G1 phase (Fig. 1e, f). By
combining information from timing of expression during the cell
cycle, evolutionarily related genes, and previous functional
work in a “comparative functional genomics” approach, we
can build mechanistic, testable hypotheses about virulence
gene function in non-model organisms. Both C.
neoformans and S. cerevisiae budding yeasts provide supporting
evidence for the “just-in-time transcription” hypothesis,
where sets of genes are co-expressed at a given time to
perform a function during a specific cell-cycle phase (Kelliher
et al. 2016). In future work, the approaches described here
can be applied to the many additional periodic genes in C.
neoformans with unknown cell-cycle functions.
A long-term goal of this work is to characterize the
regulatory pathways that control periodic gene expression
during the fungal cell cycle. Identification of
transcription factors and of their binding sites in the genome will
be essential knowledge to approach this problem. These
data sets are available in S. cerevisiae, which quantitative
models have used to predict an interconnected network of
periodically expressed transcription factors that are capable
of driving cell-cycle transcription (Hillenbrand et al. 2016;
Li et al. 2004; Orlando et al. 2008; Sevim et al. 2010;
Simmons Kovacs et al. 2012). Transcription factor (TF)
deletion collections have been generated and carefully
phenotyped in both C. neoformans (Jung et al. 2015) and C.
albicans (Homann et al. 2009). One promising avenue of
future work would be to synchronize mutant cells in the
cell cycle and determine if single or double TF mutants
affect cell-cycle progression (Simmons Kovacs et al. 2012).
One historical example of a characterized gene
regulatory network that regulates periodic gene expression is
the circadian rhythm, which is present in almost all
organisms to anticipate environmental light–dark cycles (Zhang
and Kay 2010). Using Neurospora crassa as a model for
the eukaryotic circadian clock, mutations in the frequency
(FRQ) locus were identified in screens for arrhythmic
fungi (Loros and Feldman 1986; McClung et al. 1989).
The FRQ protein participates in a core negative feedback
loop that regulates the circadian period length (Hurley
et al. 2016). The screens that identified FRQ in N. crassa
and its orthologs in other eukaryotes represent the utility
of genetic approaches when one gene has a large effect on
the biological phenotype of interest (analogous to mapping
human disease genes with Mendelian inheritance).
Complex phenotypes and quantitative traits are more
challenging to solve using genetic approaches alone. For
example, the positive regulators of the N. crassa circadian
network, white collar-1 (WC-1) and white collar-2 (WC-2),
were characterized more than a decade later than FRQ due
to complex and partially redundant roles in activating
lightresponsive genes in the network (Crosthwaite et al. 1997).
Redundancy in biological processes can be addressed with
double mutant screens that are more effective at identifying
core genes controlling a dynamic process (Costanzo et al.
2013). However, robustness can come from multiple genes
with partially overlapping functions or, in response to
genetic perturbation, from “compensation” in the strength
of interactions in a network transcription factors, which has
directly been demonstrated in the mammalian circadian
clock network (Baggs et al. 2009). Intricate network
interactions provide robustness to the network, but can foil the
traditional genetic approaches, and thus network modeling
of dynamical systems will become an invaluable approach
for learning complex mechanisms.
We posit that cell-cycle networks, like the circadian
rhythm, are also ancient in origin and contain highly
redundant regulatory pathways (Simmons Kovacs et al. 2008).
In the recent C. neoformans publication, the cell-cycle
network topology at G1/S phase was highlighted as a region
of partial conservation between fungal species (Kelliher
et al. 2016: Figure 6). In fact, the transcriptional
machinery involved in the cellular “decision” to commit to the cell
cycle, enter quiescence, or select another fate is
functionally conserved in G1/S phase from S. cerevisiae to human
cells (Miles and Breeden 2016). Further work is needed to
understand interacting genes in pathogenic fungi and also
in the conservation of these fungal and animal gene
networks (Brown and Madhani 2012; Medina et al. 2016). In
addition to quantifying mRNA abundance, the localization
and protein activity of TFs and other cell-cycle regulators
will be important directions for future work in C.
neoformans (Chong et al. 2015; Kuwada et al. 2015).
Building networks of genes that control a given
process is critical for a full understanding of the dynamics
of a biological system. Understanding dynamics and
network topology also allows us to predict how gene
networks will respond to perturbation (such as drug
treatment) and combat drug resistance, which is a major
problem in infectious diseases. Preliminary networks
of interacting genes have been established in the wheat
pathogen Fusarium graminearum (Guo et al. 2016), in
nitrogen utilization in the pathogenic yeast C. albicans
(Ramachandra et al. 2014), and during ordered
capsule assembly in C. neoformans (Maier et al. 2015). We
propose that synchronizing populations of cells for the
cell cycle could build on this pioneering work and
elucidate direct connections between proliferation and
virulence factors. Another useful direction for future work
on understanding gene regulatory networks in C.
neoformans will be to profile cell-cycle synchronized cells in
non-rich media and/or at high temperature, as it is already
known that steady-state gene expression levels change in
response to poor media conditions (Janbon et al. 2014).
The opportunistic fungal pathogen C. neoformans
expresses nearly 20% of all genes periodically during the
cell cycle (Kelliher et al. 2016), and we have begun to
make connections between virulence pathways and the
cell cycle. To elucidate the network of transcription factors
and other cell-cycle regulators that control periodic
virulence pathways, future work will assay cell-cycle
phenotypes from the C. neoformans TF deletion collection (Jung
et al. 2015) in rich media and in poor media conditions that
mimic infection (Janbon et al. 2014). An improved
understanding of cell-cycle biology in fungal species will lead
to more informed, and potentially combination therapies to
treat fungal diseases.
Acknowledgements We thank members of the Haase
Laboratory for helpful discussions. We also thank J. Uehling for feedback
on the manuscript. This work is supported by the Defense Advanced
Research Projects Agency (DARPA) Grant #D12AP00025.
Open Access This article is distributed under the terms of the
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