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Molecular and Cellular Biology, January 2000, p. 352-362, Vol. 20, No. 1
Departments of
Genetics,1
Medicine,2
Microbiology,3 and Pharmacology
and Cancer Biology4 and Howard Hughes
Medical Institute,5 Duke University Medical
Center, Durham, North Carolina 27710
Received 16 June 1999/Returned for modification 27 July
1999/Accepted 4 October 1999
Cryptococcus neoformans is an opportunistic fungal
pathogen with a defined sexual cycle. The gene encoding a
heterotrimeric G-protein Cryptococcus neoformans
is an opportunistic fungal pathogen that infects the central nervous
system to cause meningoencephalitis in individuals with compromised
immune function (26, 39). Virulence is associated with
mating type (28), production of melanin (29, 30, 48,
56) and a polysaccharide capsule (4, 16, 30), and
growth at 37°C (30, 45).
The life cycle of this organism has been defined (25).
Mating occurs between MATa and MAT Heterotrimeric guanine nucleotide binding proteins interact with
G-protein-coupled receptors to sense external signals and regulate cell
growth and development (14). G-protein-mediated signals
include responses to hormones and neurotransmitters, vision and
olfaction, and pheromone-induced mating in S. cerevisiae and Schizosaccharomyces pombe. Heterotrimeric G proteins are
comprised of alpha ( In most examples, the G In C. neoformans, the G Strains and media.
C. neoformans strains used in
this study included H99 (serotype A, MAT Isolation of the C. neoformans GPB1 gene.
Primers 5'-AT(ATC)TA(TC)GC(GATC)ATGCA(TCT)TGG and
5'-AA(AG)TC(AG)TA(GATC)CC(GATC)GC encompassed conserved
residues IYAMHW and AGYDDF, respectively, of G Nucleic acid manipulations.
DNA and RNA were extracted from
cells that were lyophilized overnight and broken with glass beads (4 mm
diameter) by the use of a Vortex mixer. Total DNA for Southern blot
analysis was isolated as described in reference 46.
Total DNA for PCRs was obtained as described in reference
18. Total RNA was extracted with a buffer containing
150 mM sodium acetate, 100 mM LiCl, 4% sodium dodecyl sulfate, 10 mM
EDTA, 10 mM EGTA, and 20 mM
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The G-Protein
Subunit GPB1 Is Required for
Mating and Haploid Fruiting in Cryptococcus
neoformans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
subunit, GPB1, was cloned and disrupted.
gpb1 mutant strains are sterile, indicating a role for this
gene in mating. GPB1 plays an active role in mediating responses to
pheromones in early mating steps (conjugation tube formation and cell
fusion) and signals via a mitogen-activated protein (MAP) kinase
cascade in both MAT
and MATa cells. The
functions of GPB1 are distinct from those of the G protein GPA1,
which functions in a nutrient-sensing cyclic AMP (cAMP) pathway
required for mating, virulence factor induction, and virulence.
gpb1 mutant strains are also defective in monokaryotic
fruiting in response to nitrogen starvation. We show that
MATa cells stimulate monokaryotic fruiting of
MAT cells, possibly in response to mating pheromone,
which may serve to disperse cells and spores to locate mating partners.
In summary, the G subunit GPB1 and the G subunit GPA1 function in
distinct signaling pathways: one (GPB1) senses pheromones and regulates
mating and haploid fruiting via a MAP kinase cascade, and the other
(GPA1) senses nutrients and regulates mating, virulence factors, and pathogenicity via a cAMP cascade.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
cells and involves cell fusion, filamentation, nuclear migration and
fusion, meiosis, and sporulation. Mating type is linked to physiology
and virulence. MAT strains are more prevalent in the
environment, and most clinical isolates are MAT
(27); MAT strains are more virulent in mice than are congenic MATa strains (28). In
response to nitrogen starvation, MAT cells differentiate
to form filaments, basidia, and spores (haploid fruiting)
(62). Thus, genes linked to the MAT locus
regulate the physiology and virulence of C. neoformans. A
homolog of the Saccharomyces cerevisiae and Candida
albicans STE12 transcription factor is encoded by the C. neoformans MAT locus (61), and
ste12 mutant strains have defects in haploid fruiting
(67). Recent studies of a GTP-binding protein, GPA1, underscore the importance of signaling cascades in C. neoformans virulence (2, 54).
), beta (), and gamma (
) subunits. In
response to binding of ligand to receptors, the G complex is
recruited, leading to GDP-GTP exchange on G and release of G.
-GTP subunit actively transduces signals.
However, G subunits can also signal (5, 21-23, 32, 33, 36,
52, 64). For example, the S. cerevisiae G
complex Ste4-Ste18 is released from the G subunit Gpa1 by pheromone
(6, 32, 33, 59). The G complex recruits the Ste5
scaffold, allowing activation of the Ste5-bound kinase Ste11 by
membrane-localized Ste20 kinase (32-34, 47). The G
subunit Gpa1 plays a negative role in S. cerevisiae mating
(9, 40). In contrast, in Schizosaccharomyces pombe, the G subunit Gpa1 positively signals mating and,
together with Ras1, activates a mitogen-activated protein (MAP) kinase cascade (8, 42, 44, 58, 65). In the chestnut blight fungus
Cryphonectria parasitica, the G subunit CPGB-1 regulates sporulation and virulence, likely with the G subunit CPG-1 (13, 21).
subunit GPA1 is required for
mating and virulence (2). GPA1 regulates responses to
nutritional starvation signals required for mating and induction of the
virulence factors capsule and melanin. Cyclic AMP (cAMP) suppresses the mating and virulence defects of gpa1 mutant cells,
suggesting that GPA1 activates adenylyl cyclase similarly to Gs in
mammals and Gpa2 during S. cerevisiae pseudohyphal growth
(24, 37). In the present study, we investigated the roles of
a G-protein subunit, GPB1, in C. neoformans.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) and the
isogenic ade2 mutant M049. Strains JEC34 and JEC43 are
isogenic ura5 mutant serotype D MATa and MAT strains, respectively (41). Strain BAC20
is a gpa1::ADE2 mutant of the
MATa strain JEC20 (provided by B. Allen and A. Alspaugh). Yeast extract-peptone-dextrose (YPD) and yeast nitrogen base
(YNB) media, synthetic medium, V8 agar, filament agar, niger seed agar
for melanin production, and low-iron medium plus 56 µM
ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) for induction of capsule formation were as described in references 2 and 55.
subunits. PCR
parameters were as follows: 94°C for 40 s, 40°C for 1 min, and
72°C for 2 min (40 cycles). C. neoformans cDNA (strain
B3501; 200 ng) served as the template. PCR products were excised,
cloned, and used to clone the GPB1 gene. For size-selected
libraries, DNA was cleaved with HindIII and
electrophoresed, and 4.9-kb fragments were excised. DNA was recovered
by using a QIAEX DNA extraction kit (Qiagen), ligated in
HindIII-cleaved plasmid pUC18, and transformed into Escherichia coli, and bacterial colonies were screened with
the GPB1 PCR product as a probe (49).
-mercaptoethanol, extracted with phenol
(pH 4.0), and precipitated with LiCl.
Two-hybrid assays. For two-hybrid interactions, a GPB1 cDNA was cloned in plasmids pGBT9 and pGAD424 (Clontech) to yield plasmids pGBT9::GPB1 and pGAD424:GPB1, expressing GAL4(DB)-GPB1 and GAL4(AD)-GPB1 fusion proteins, respectively. DNA of plasmids pGAD424::GPB1 and pGBT::GPA1 (2) or of plasmids pGBT9::GPB1 and pGAD424::GPA1 (2) was used to transform the yeast strain PJ69-4A (20).
GPB1 gene disruption. pCnGPB1 is a pUC18-derived clone containing a 4.9-kb HindIII fragment spanning the GPB1 gene from strain H99. For the gpb1::ADE2 gene disruption, pCnGPB1 was digested with ApaI (for which there is a unique cleavage site in the GPB1 gene), blunt ended with T4 DNA polymerase, and dephosphorylated with calf intestinal alkaline phosphatase. Two plasmids were constructed for the GPB1 gene disruption. Either a 2.4-kb XhoI or 2.9-kb KpnI-BamHI DNA fragment containing the ADE2 gene from C. neoformans serotype D strain B3501 (51, 53) was blunt ended and inserted at the blunted ApaI site in plasmid pCnGPB1 to yield the gpb1::ADE2 disruption alleles. The ade2 serotype A strain M049 was grown for 40 h in liquid YPD and transformed with the gpb1::ADE2 disruption allele by the use of a biolistic DNA delivery apparatus (Bio-Rad) as described elsewhere (53). Transformants were selected on synthetic medium lacking adenine but containing 1 M sorbitol. Primers used for PCRs to verify the presence of gpb1::ADE2 alleles were 5'-AGAGAGCTCAGCGCACAC-3' and 5'-GTAGTCATCGTAGCCGGC-3'.
Mating assays.
Mating assays were conducted by coculturing
MAT strains with the tester MATa strain
JEC20 on V8 or filament agar medium containing 0.5% galactose
(inducing) or 0.5% glucose (repressing) (62). Plates were
incubated at 22°C, and resultant colonies were examined by using a
Nikon Eclipse E400 microscope.
Expression of GPB1, GAL7-GPB1,
GAL7-CPK1, GAL7-STE12, MF1,
and Ras1-Q67L.
A ura5 derivative of the
gpb1 mutant strain was obtained by plating cells on
5-fluoroorotic acid medium, and the ura5 mutation was then
complemented by introducing plasmid pCnTel1 (10, 31). A
2.5-kb XbaI fragment containing the wild-type
GPB1 gene was inserted into plasmid pCnTel1
(pCnTel1::GPB1) for complementation tests. The genomic clone
of the C. neoformans Fus3/Kss1 MAP kinase homolog
CPK1 (pCnTel1::CPK1) and the CPK1 cDNA
clone (R. Davidson and J. Heitman, unpublished data) were cloned
under the control of the C. neoformans GAL7
promoter (60), and the resulting plasmids (pCnTel1::GAL7-CPK1) were used for epistasis. The cDNA clone
of the C. neoformans STE12 gene expressed from
the GAL7 promoter in plasmid pCGS-1 (61) was also
used. Plasmids were transformed in circular form by biolistic
transformation into the gpb1 ura5 mutant. The
GPB1 cDNA clone was placed under the control of the GAL7 promoter (pCnTel1
::GAL7-GPB1) and used to
transform a ura5 strain of H99, the gpb1 ura5
mutant, and the ura5 serotype D MATa and
MAT strains JEC34 and JEC43. pCnTel1 differs from
pCnTel1 in that it lacks the NotI fragment containing
telomeric sequences. The dominant active Ras1 Q67L mutant was expressed
with the C. neoformans actin gene promoter and was
introduced with the hygromycin B resistance or URA5 gene as
a marker. The cloned MF1 gene (plasmid pCnTel1::MF1) (41) was expressed in the
serotype D strain JEC34 (MATa ura5) and the
isogenic gpa1 mutant strain BAC20 (MATa gpa1::ADE2 ura5).
Haploid fruiting assays. For haploid fruiting assays, the Ras1 Q67L mutant protein was expressed by introducing linear DNA fragments containing the mutant RAS1 gene, linked to the hygromycin resistance or URA5 gene as a marker, by biolistic transformation. Isolates containing the mutant allele for Ras1 Q67L were identified by PCR of genomic DNA with primers flanking the RAS1 gene and by XbaI cleavage to detect the Q67L mutation. Haploid fruiting was assayed by incubating spotted suspensions of cells on filament agar at 24°C for up to 4 weeks.
For confrontation assays, isolated colonies were streaked on the surface of filament agar as lines with sterile toothpicks. In some cases, a sterile dialysis membrane was interposed between the cell types by inserting it with sterile forceps into an agar cut that was sealed with molten agar.Virulence test. Virulence was evaluated using a rabbit model of cryptococcal meningitis (2, 45). Cells of the isogenic wild-type strain (H99) and the gpb1 mutant strain were grown for 48 h in liquid YPD medium and resuspended in 15 mM phosphate-buffered saline. New Zealand White male rabbits (four in a group for each strain) weighing 2 to 3 kg were administered cortisone acetate (2.5 mg/kg of body weight) intramuscularly 1 day prior to inoculation of C. neoformans and then daily for 14 days. Twenty-four hours following initial steroid treatment, rabbits were anesthetized with xylazine and ketamine intramuscularly and inoculated intracisternally with 0.3 ml of cell suspension (3 × 108 cells/ml). Rabbits were sedated on days 4, 7, 10, and 14 postinoculation, and cerebrospinal fluid (CSF) was withdrawn. Cell cultures were performed by plating dilutions of CSF (in phosphate-buffered saline) on YPD medium, incubating the plates at 30°C for 3 days, and counting viable colonies.
Nucleotide sequence accession number. The GPB1 gene sequence has been submitted to GenBank under accession no. AF091120.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification of the C. neoformans G-protein subunit GPB1.
Previous studies revealed that the G protein GPA1
(54) regulates mating and virulence in C. neoformans (2). To further address the role of G
proteins in mating and physiology, we identified a heterotrimeric
G-protein subunit from C. neoformans.
subunits and used as primers in low-stringency PCRs with a C. neoformans cDNA library or C. neoformans genomic DNA as
a template. Primers encompassing two conserved peptides, IYALHW and
AGYDDY, amplified a partial G cDNA homolog from the serotype D
strain B-3501. This cDNA clone was sequenced and then used to probe a Southern blot of genomic DNA isolated from the serotype A
MAT strain H99 and from the congenic serotype D strains
JEC20 (MATa) and JEC21 (MAT). The
GPB1 gene was present in a single copy in both mating types
and serotypes (data not shown).
The complete GPB1 genomic locus was cloned from a
size-selected genomic library. Sequence analysis revealed an open
reading frame of 1,059 nucleotides encoding a 352-amino-acid protein
(GenBank accession no. AF091120). Four introns were identified by
sequence comparison with a cDNA clone from strain H99. The predicted
GPB1 protein shares marked identity with G-protein subunits from other organisms, including G subunits from humans (68%),
Drosophila melanogaster (67%), C. parasitica (70%), Schizosaccharomyces pombe (40%),
and S. cerevisiae (38%) (Fig.
1).
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Disruption of the C. neoformans GPB1 gene. The GPB1 gene was disrupted by inserting the ADE2 gene into the GPB1 open reading frame, and the resulting gpb1::ADE2 disruption allele was introduced into the ade2 strain M049 by biolistic DNA transformation and homologous recombination. Genomic DNA was extracted from candidate gpb1::ADE2 strains (18). PCRs with primers flanking the ADE2 gene insertion were used to identify gpb1 mutations and generate a 550-bp product from the GPB1 allele and a 3,450-bp product from the gpb1::ADE2 allele.
In total, six gpb1::ADE2 mutant strains were identified from 306 adenine-prototrophic transformants by PCR analysis. In subsequent analyses, independent gpb1 mutations conferred the same phenotypes. Southern blot analysis confirmed that the GPB1 gene had been replaced by the gpb1::ADE2 disruption allele by homologous recombination at the GPB1 locus in all six mutant strains (Fig. 2). The wild-type GPB1 gene is located on a 4.9-kb HindIII fragment and a 1.6-kb NotI-XbaI fragment. In the gpb1::ADE2 mutant, the wild-type 4.9-kb HindIII fragment is replaced by 2.9- and 5.0-kb HindIII fragments (Fig. 2). In addition, the 1.6-kb NotI-XbaI wild-type GPB1 locus is missing from the gpb1::ADE2 mutant, having been replaced by 4.5- and 5.1-kb NotI-XbaI fragments (Fig. 2).
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GPB1 is required for mating in C. neoformans.
We
tested whether GPB1 regulates mating in C. neoformans.
MAT and MATa strains of C. neoformans mate when cocultured on nutrient-limiting medium
(25). Mating consists of conjugation tube formation, cell
fusion, and filamentation (1). Subsequent nuclear migration
results in the formation of dikaryotic filaments that differentiate to
form terminal basidia, in which nuclear fusion, meiosis, and
sporulation occur. When one or both parents are sterile, few or no
filaments or spores are produced.
serotype A strain (H99)
yielded abundant filaments and basidiospores when crossed with the
MATa serotype D strain JEC20 (Fig.
3). In contrast, no filaments or spores
were ever observed when any of the independent gpb1 mutant MAT strains were mated with their MATa
mating partners (Fig. 3). Reintroduction of the wild-type
GPB1 gene into the gpb1 mutant strain restored
filamentation and spore production to the wild-type level (Fig. 3).
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GPB1 and the G subunit GPA1 play different roles in mating.
Several findings suggest that the G protein GPA1 and the G
protein GPB1 function in distinct pathways to regulate mating. First,
the gpb1 mutation confers an absolute mating defect,
whereas, following prolonged incubation, gpa1 mutants
eventually mate to a limited extent with a wild-type mating partner,
forming filaments, basidia, and recombinant basidiospores (Fig. 3)
(2). Second, cAMP suppresses the mating defect of
gpa1 mutants, but not that of gpb1 mutants (Fig.
3) (2). Third, no interaction between GPA1 and GPB1 was
detected in the two-hybrid system (data not shown) (see Materials and Methods).
subunit GPA1 is not
required for pheromone sensing. First, in confrontation assays, the
congenic MAT strain JEC21 and the MATa
strain JEC20 both produced conjugation tubes in response to pheromone
secreted by their mating partners (Fig.
4A). Most importantly, when the wild-type
MAT strain JEC21 was grown in confrontation with a gpa1 MATa mutant strain (BAC20), both the
gpa1 mutant and the wild-type strain produced conjugation
tubes (Fig. 4A). Second, when a plasmid expressing the MF1 pheromone
was introduced into wild-type and gpa1 mutant
MATa strains, both produced conjugation tubes (Fig.
4B). The response of gpa1 mutants to pheromones was somewhat
reduced from that of the wild type, but taken together these findings
indicate that GPA1 is not required for pheromone sensing. In an assay
that detects cell fusion during mating (MAT ura5 strains were coincubated with MATa
lys1 strain JEC30 on V8 agar, and prototrophic
self-filamenting heterokaryons were detected by replica plating to YNB
medium), the gpb1 mutation prevented cell fusion whereas the
gpa1 mutation reduced but did not block fusion (data not
shown). In a mating assay in which recombinant basidiospores were
quantified (MAT prototrophic strains were mated with
MATa ura5 lys1 strain JEC53 on
V8 agar, and LYS1 ura5 recombinants were selected
on 5-fluoroorotic acid-lysine medium), no recombinant basidiospores
were produced by the gpb1 mutant whereas the gpa1
mutant produced a reduced number of basidiospores.
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GPB1 is not required for melanin or capsule production or
virulence.
The G protein GPA1 regulates the production of the
virulence factors melanin and capsule in response to nutrient
limitation (2). To determine whether the functions of GPB1
and GPA1 are distinct, we tested whether the gpb1 mutation
alters virulence factors or virulence.
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GPB1 regulates mating upstream of a MAP kinase cascade.
Our
findings suggested that the G subunit GPB1 activates a signaling
pathway that regulates mating in parallel with the GPA1-cAMP-regulated nutrient-sensing pathway. We tested whether the G protein GPB1 regulates a MAP kinase cascade during mating in C. neoformans.
subunit, two other MAP kinase cascade
components have been identified in C. neoformans: a MAP kinase homolog, CPK1 (R. Davidson and J. Heitman, unpublished data), and a homolog of the STE12 transcription factor (61, 67). We tested whether CPK1 or STE12 functions downstream of GPB1 by epistasis, using cloned genes under the control of the C. neoformans GAL7 promoter, which is induced by galactose
and repressed by glucose (62).
When the gpb1 mutant strain was transformed with the
GAL7-CPK1 gene fusion, mating with a MATa
strain was restored on galactose filament agar but not on glucose (Fig.
6A). Thus, expression of the CPK1 MAP
kinase suppresses the gpb1 mating defect, providing evidence
that GPB1 functions upstream of this MAP kinase. The
GAL7-CPK1 gene fusion did not restore mating in
gpa1 mutants (data not shown), indicating that CPK1
functions downstream of GPB1 but not of GPA1.
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gene
fusion did not restore mating of the gpb1 mutant strain on
glucose or galactose filament agar (data not shown). The functions of STE12 likely involve haploid fruiting and not mating, because STE12 overexpression stimulates haploid fruiting (61)
whereas ste12 mutations block haploid fruiting but not
mating (67).
GPB1 stimulates conjugation tube formation in MAT
and MATa cells.
We next tested whether GPB1
plays an active signaling role upstream of the MAP kinase cascade,
analogous to that of the G complex in S. cerevisiae
(50). During mating in C. neoformans, the mating
partners secrete pheromones that trigger the formation of conjugation
tubes in the opposite cell type (1, 41; R. Davidson
and J. Heitman, unpublished data). We tested whether GPB1
overexpression stimulates conjugation tube formation in cells not
exposed to pheromones.
and MATa serotype D
strains. Growth on galactose filament agar induced the formation of
conjugation tubes in both MATa and MAT
strains (Fig. 6B). Conjugation tubes produced in response to
GPB1 overexpression were similar to those observed in
confrontation assays or in MATa cells in response to
expressed or synthetic MF1 pheromone (1, 41) (Fig. 4).
MATa cells produced more conjugation tubes than did
MAT cells, suggesting that the mating responses of the
two cell types differ (Fig. 6B).
GPB1 and MATa cells regulate monokaryotic
fruiting.
Mating of MATa and MAT
cells of C. neoformans is regulated by both pheromones and
nitrogen starvation. In contrast, in response to nitrogen starvation
alone, MAT haploid strains differentiate, forming
monokaryotic filaments, basidia, and spores by haploid fruiting
(62). This filamentous differentiation shares some features
with pseudohyphal growth in S. cerevisiae (15). Components of the mating pheromone response pathway are required for
pseudohyphal growth, whereas mating pheromones, pheromone receptors,
and the coupled heterotrimeric G protein are not (35). We
therefore hypothesized that the G protein GPB1 would not be required
for haploid fruiting in C. neoformans.
gene
fusion (Fig. 7A) and the GAL7-CPK1 gene fusion (data not shown) suppressed the haploid fruiting defect of gpb1
mutants on galactose filament agar. Thus, GPB1 is required for
monokaryotic fruiting and functions upstream of CPK1 and STE12.
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protein is required
for haploid fruiting if this process normally occurs in response to
nitrogen limitation. We found that when MAT cells are
grown in confrontation with MATa cells, monokaryotic fruiting of the MAT cells is dramatically stimulated and
abundant filaments, basidia, and basidiospores are produced (Fig. 7B). In contrast, a much lower level of monokaryotic fruiting is observed when MAT cells are grown in isolation or when
MAT cells are grown in confrontation with
MAT cells (Fig. 7B). The response of MAT
cells to confronting MATa cells does not require cell-cell or cell-filament contact, and it occurs before any of the
projecting filaments touch the confronting cells. Moreover, monokaryotic fruiting was still observed when a dialysis membrane with
a molecular mass cutoff of 3,800 Da was interposed between MAT and MATa cells (data not shown). The
C. neoformans mating pheromones are predicted to diffuse
through this membrane.
By microscopic observation and nuclear staining with the DNA-specific
dye DAPI (4',6'-diamidino-2-phenylindole), it was determined that the
filament cells are linked by unfused clamp connections and are
monokaryotic, hallmarks of monokaryotic fruiting. In addition, micromanipulation and mating type tests confirmed that basidiospores produced by MAT cells in response to confronting
MATa cells are all MAT and are thus
products of asexual monokaryotic fruiting (data not shown). Our
findings indicate that monokaryotic fruiting of MAT cells
is stimulated by MATa cells, possibly in response to
MATa pheromones sensed by a receptor coupled to GPB1.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have identified the gene encoding a heterotrimeric G-protein
subunit, GPB1, from C. neoformans. GPB1 is required for mating and plays a role in the pheromone response in both
MAT and MATa cells by activating a MAP
kinase cascade leading to conjugation tube formation and cell fusion.
Two distinct signal transduction pathways regulate mating: one involves
pheromone sensing and requires GPB1, and the second senses nutrients
via the G protein GPA1-cAMP pathway and is also required for
virulence factor production and pathogenicity. These signal
transduction cascades coordinately regulate mating in C. neoformans, analogous to the role of the MAP kinase and G-cAMP
signal transduction cascades in development in other organisms,
including pseudohyphal growth in S. cerevisiae and mating in
Schizosaccharomyces pombe. We found a novel role for the
pheromone-sensing G subunit GPB1 in haploid fruiting in C. neoformans. We have also discovered that monokaryotic fruiting of
MAT cells is dramatically stimulated by
MATa cells, suggesting that this differentiation
cascade may function in mating. Finally, we have shown that
gpb1 mutant strain virulence is similar to that of wild-type
strains, indicating that this component of the mating pathway does not
play a prominent role in virulence.
C. neoformans GPB1 G and GPA1 G subunits have
distinct functions.
Our studies support a model in the which the
GPB1 G subunit and the GPA1 G subunit function in two different
signal transduction cascades that regulate different steps in mating.
Several observations indicate that GPB1 functions in the pheromone
response pathway and regulates early steps in mating involving
conjugation tube formation and cell fusion. First, gpb1
mutants are completely sterile and exhibit a profound defect in a cell
fusion assay. Second, GPB1 overexpression stimulates conjugation tube
formation. Third, overexpression of the MAP kinase CPK1 suppresses the
mating defect of gpb1 mutant strains, whereas cAMP does not.
Later in mating, the pheromone response pathway likely also plays a
second role involving the fusion of the clamp cells during filament formation.
1
pheromone gene (Fig. 4). In quantitative mating assays, the
gpa1 mutation reduces but does not block cell fusion and
also reduces filamentation and the production of recombinant
basidiospores. The nutrient-sensing GPA1-cAMP cascade is required
for melanin and capsule production and virulence, whereas GPB1 is not.
These findings support a model in which the GPA1 G and GPB1 G
subunits are components of two different signaling cascades. We propose
that GPA1 and GPB1 are components of two different G proteins and
function in distinct signaling cascades, one that senses nutrients via
a cAMP pathway (GPA1) and another that senses mating pheromones and
signals via a MAP kinase cascade (GPB1).
MAP kinase signaling in MAT and
MATa cells.
Our findings reveal that the G
subunit GPB1 regulates conjugation tube formation in both
MATa and MAT cells. The GPB1
gene is present in both MAT and MATa
strains and is expressed in cells of both mating types. This is in
contrast to other MAP kinase cascade components recently identified in C. neoformans, including STE11, STE12, and STE20
homologs, which are encoded by the MAT locus and are
specific to MAT cells (61; B. Wickes
and J. Edman, personal communication; P. Wang and J. Heitman,
unpublished results). These findings raise the conundrum of how
signaling occurs in MATa cells during mating if
several components are present only in MAT cells. Our findings suggest that the MAP kinase cascade functions during mating in
both MAT and MATa cells. We propose that
mating in both cell types is regulated by GPB1 signaling via two
divergent versions of a conserved MAP kinase cascade: one, containing
components encoded by the MAT locus, that supports mating
and can also function in haploid fruiting and virulence, and another,
with components encoded by the MATa locus, that plays
a more restricted role in mating, does not support haploid fruiting,
and remains to be identified. Such a model may be related to the
situation in the yeast S. cerevisiae, which expresses two
related MAP kinases with divergent functions: Fus3, which is
specialized for mating of haploid cells, and Kss1, which regulates
pseudohyphal differentiation of diploid cells (7, 38).
G-protein signaling roles in other yeasts.
Our studies on
G-protein function are relevant to previous studies of G proteins in
other yeasts. In S. cerevisiae, two G proteins regulate
responses to pheromone and nutrients (50). During
pseudohyphal differentiation, nutrients regulate the G protein Gpa2,
which signals via a cAMP cascade (24, 37). During mating,
pheromone binding to the Ste2 or Ste3 receptors recruits the G
complex (Gpa1-Ste4-Ste18), and the released Ste4-Ste18 complex
activates the MAP kinase cascade by recruiting signaling components to
the membrane (32-34, 47). The G subunit Gpa1 inhibits signaling by . C. neoformans G subunit GPB1
functions analogously to the Ste4 G subunit in S. cerevisiae mating, whereas the functions of the C. neoformans G subunit GPA1 are analogous to nutrient sensing by
S. cerevisiae GPA2.
subunit Gpa1 and
the G and G subunits Gpa2 and Gpb1 (65). Gpa1 is
required for the pheromone response during mating and, in contrast to
the situation for S. cerevisiae, plays a positive role in
activating the MAP kinase cascade. Gpa2 plays a role analogous to that
of the S. cerevisiae Gpa2 and C. neoformans GPA1
subunits, and it functions in a nutrient-sensing cAMP pathway
regulating mating (19, 43). Mutants lacking the G subunit
Gpb1 exhibit a phenotype similar to that of gpa2 mutants and
mate and sporulate under nutrient-rich conditions (23). The
sensing of pheromones by the G subunit GPB1 in C. neoformans appears to be distinct from the role for the
Schizosaccharomyces pombe G subunit in nutrient sensing.
GPB1 and MATa cells regulate haploid fruiting in
C. neoformans.
We found that the G subunit GPB1 is
also required for haploid fruiting in C. neoformans. Haploid
fruiting is a differentiation pathway whereby MAT cells
can form filaments and sporulate in response to nitrogen starvation in
the absence of a mating partner (62). Haploid fruiting
shares features with pseudohyphal differentiation in the yeast S. cerevisiae, which is regulated by a MAP kinase cascade and induced
by nitrogen limitation (15). However, the mating pheromones,
receptors, and the coupled G protein are not required for filamentation
in S. cerevisiae (35).
strains in response to nitrogen starvation alone
(61), we found that monokaryotic fruiting of C. neoformans MAT cells is markedly stimulated by
confrontation with MATa cells. This stimulation does
not require cell-cell or filament-filament contact. We propose that
MATa cells stimulate haploid fruiting of adjacent
MAT cells by secreting a peptide mating pheromone. Stimulation of monokaryotic fruiting by this pheromone may function to
disperse MAT spores to locate uncommon
MATa mating type cells at a distance. Thus, haploid
fruiting could function as a prelude to mating in an organism in which
MAT cells are more abundant than MATa cells.
We propose that haploid fruiting occurs in isolated C. neoformans MAT cells at a low level due to basal
signaling of the pheromone-responsive MAP kinase pathway in the absence
of pheromone and is then markedly stimulated by a pheromone produced by
adjacent MATa cells. MATa cells do
not undergo haploid fruiting, in part because they lack the
MAT locus-encodes transcription factor STE12 required
for haploid fruiting (67). The model in which haploid
fruiting is regulated by pheromones makes the testable prediction that
the MFa1 pheromone and its receptor are also required.
Although haploid invasive growth and diploid filamentous growth in
S. cerevisiae are not normally regulated by the G subunit Ste4, our findings may be related to studies of altered differentiation in mutant S. cerevisiae strains and the basal signaling
state of the yeast pheromone response pathway. First, in
fus3 mutant yeast strains, haploid invasive growth and
expression of filamentous reporter genes are increased, and this
requires the G subunit Ste4 and results from inappropriate
cross-talk between the pheromone-responsive MAP kinase cascade and the
filamentous growth pathway (38). Second, even in the absence
of pheromones, the S. cerevisiae pheromone response pathway
is active at a basal level. The G subunit Ste4, the kinases Ste11
and Ste7, the scaffold Ste5, and the Ste12 transcription factor are
required for basal signaling (11, 17, 50). By analogy, we
propose that signaling by the G subunit GPB1 in the absence of
pheromones supports a basal level of monokaryotic fruiting in C. neoformans that is then stimulated by pheromones.
Perspective.
The MAT locus has been linked to
virulence in C. neoformans. Our finding that gpb1
mutant strains are defective in mating but not impaired in virulence
indicates that mating is not required for virulence, and further
studies are needed to establish the link between the MAT
locus and virulence.
| |
ACKNOWLEDGMENTS |
|---|
We thank Cristl Arndt, Lora Cavallo, and Wiley Schell for assistance; Maria Cardenas for advice; Andy Alspaugh, Maria Cardenas, Cristina Cruz, Rob Davidson, Danny Lew, and Rey Sia for comments; and Andy Alspaugh, Rob Davidson, Don Nuss, and Brian Wickes for reagents and strains.
This work was supported by NIAID R01 grants AI39115 and AI42159 and program project grant P01 AI44975 from NIAID to the Duke University Mycology Research Unit. Joseph Heitman is an associate investigator of the Howard Hughes Medical Institute and a Burroughs Wellcome Scholar in Molecular Pathogenic Mycology.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Genetics, 322 CARL Bldg., Duke University Medical Center, Research Dr., Durham, NC 27710. Phone: (919) 684-2824. Fax: (919) 684-5458. E-mail: heitm001{at}duke.edu.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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