Section of Molecular Genetics and
Microbiology, School of Biological Sciences and Institute for
Cellular and Molecular Biology, The University of Texas at Austin,
Austin, Texas 78712
 |
INTRODUCTION |
In Saccharomyces
cerevisiae and Schizosaccharomyces pombe, the
CDC42 gene is essential for cell viability (15,
27) because it plays crucial roles in the regulation of cell
polarity via actin cytoskeleton organization and signal transduction
(14). The biological functions mediated by Cdc42 GTPase have
attracted broad interest, although its mechanism of regulating cellular morphogenesis is still largely obscure. Many Cdc42p target proteins have been identified in S. cerevisiae, such as Bem4
(23), Boi1 (1), Zds1 and Zds2 (2),
Bee1 (19), Bni1 (12), Gic1 and Gic2 (3,
4), and Iqg1 (11, 32), all of which are involved in
the regulation of actin cytoskeleton organization. Also, the p21-activated serine/threonine kinase family members Ste20p (24, 35) and Cla4p (8) interact with Cdc42p to regulate
gene expression and septin organization in mating, filamentous growth,
and yeast cytokinesis. Moreover, Cdc42 homologues and regulators have
been studied for several other organisms, including humans. The
biological functions for human Cdc42p are similar to those in yeast and
involve actin cytoskeleton reorganization (30),
transcriptional activation through the JNK/SAPK signaling pathway
(7, 28), and the induction of cell cycle progression through
the G1 phase (31). Some of these effects have
been implicated in cell transformation (33), host cell
pathogenesis with bacterial cytotoxicity (18), and human
immunodeficiency virus replication (21).
The dematiaceous (melanized) fungus Wangiella
(Exophiala) dermatitidis is one of many
causative agents of human phaeohyphomycosis (17). It is
considered a paradigm for studies of this emerging dermatomycosis
afflicting humans because of its wide range of clinical manifestations
and the increasingly frequent detection of it as a systemic pathogen
(25, 26). It also serves as a model for the study of black
fungi because of its well-defined polymorphism (36, 37) and
cell wall chemistry (38). Of particular interest has been
the unique transition from blastic to isotropic growth, whereby
yeast-like cells convert to enlarged and transversely septated
multicellular forms. This mimics the pathogenic process leading to the
production of sclerotic bodies in the tissues of patients infected by
the dematiaceous fungi that cause chromoblastomycosis (37).
In addition, invasive hyphal growth in W. dermatitidis is
also of interest because the CDC42 gene products of S. cerevisiae and Candida albicans have been implicated as
a regulator for their filamentous growth (20, 29). Thus,
understanding the mechanism regulating phenotypic conversions in
W. dermatitidis provides insights into the pathogenesis of
diseases caused not only by this species but also by the many other
related dematiaceous fungal pathogens of humans.
In this study, we cloned a W. dermatitidis CDC42 homologue,
WdCDC42, and confirmed its conserved GTPase function by
complementation of the S. cerevisiae cdc42-1ts
mutation. However, disruption of WdCDC42 did not result in a lethal phenotype in W. dermatitidis and affected cellular
morphologies only under certain stress conditions. Therefore, a series
of site-specific mutant alleles of WdCDC42 were generated,
which induced dominant lethal phenotypes in S. cerevisiae
similar to those reported previously (42). By a newly
established integrative transformation system for gene overexpression
in W. dermatitidis (41), the constitutively active allele but not dominant negative alleles was found to induce isotropic cell growth leading to the formation of sclerotic bodies and
also to strongly repress hyphal development. The results suggested a
new biological function of the Cdc42 homologue in this polymorphic fungal model, in which WdCdc42p negatively regulates cell polarization and coordinates with other factors to differentially control cellular phenotypic transitions.
| |
MATERIALS AND METHODS |
Strains and media.
Wild-type W. dermatitidis
strain 8658 (ATCC 34100; E. dermatitidis CBS 525.76), its
temperature-sensitive hyphal mutant Hf1, a parasexually derived diploid
(3u2m-428), and an albino strain (ALB303) were routinely cultured in
the minimal medium CDN or the complete medium CDY, as described
previously (5, 34, 41). For preparation of
transformation-competent cells, W. dermatitidis was grown in
the rich medium YPD (22). Liquid media were used for the
growth of yeast-like cells and multicellular forms of W. dermatitidis, whereas the solid CDY agar containing soluble starch
instead of glucose as the sole carbon source was used for the
stimulation of hyphal growth. For studies of gene expression under the
control of the glaA promoter in transformants, glucose in
the media was replaced by an equal amount of xylose for maintenance of
the transformants or by 1% soluble starch for the phenotypic characterizations. S. cerevisiae strains DJTD2-16A
(MATa cdc42-1 ura3 leu2 trp1 his4 gal2), kindly
provided by D. Johnson (University of Vermont, Burlington), and INVSc1
(Invitrogen, Carlsbad, Calif.) were grown in YPD or in SD medium, both
with standard compositions (22). The permissive and
restrictive temperatures for growth of the S. cerevisiae
DJTD2-16A strain and W. dermatitidis Hf1 strain were 25 and
37°C, respectively.
Plasmids and nucleic acid manipulations.
Plasmid pRS315 (42)
containing an S. cerevisiae CDC42 gene subclone was provided
by D. Johnson (University of Vermont). pCB1551 containing a
sulfonylurea resistance allele (SUR) of the
Magnaporthe grisea ILV1 gene was obtained from the Fungal
Genetics Stock Center (University of Kansas Medical Center, Kansas
City). For disruption of WdCDC42, two nonoverlapped partial
genomic sequences of WdCDC42 flanking SUR were
used to produce pYED42-827 (see Fig. 3A). For overexpression studies,
the integrative vector pYEX303 was used, which contains a hygromycin
resistance marker, a WdPKS1 fragment of the W. dermatitidis polyketide synthase gene for homologous targeting,
and the starch-maltose-inducible glaA promoter, which is
also temperature dependent in W. dermatitidis
(41).
The ZAPII cDNA library of W. dermatitidis and its
construction by our laboratory were described previously
(40). The mRNA used for construction of the library was
isolated from W. dermatitidis wild-type yeast-like cells
that were first grown at 25°C for 36 h and then shifted to
37°C for an additional 12 h of incubation. cDNA synthesis was
carried out with a ZAPII kit (Stratagene, La Jolla, Calif.).
Site-directed mutagenesis was performed using the Morph plasmid DNA
mutagenesis kit (5 Prime
3 Prime, Boulder, Colo.) and the
WdCDC42 cDNA clone 94AB1 (see Results) as the starting
template. The mutagenic oligonucleotides for generating each allele are listed in Table 1. The derived mutant
alleles were amplified by using the Expend high-fidelity PCR system
(Boehringer Mannheim) and primers 42Bgl and 42Xba (Table 1). The 680-bp
PCR fragments were then digested with BglII and
XbaI for subcloning into vector pYES2 (Invitrogen) or
pYEX303 to produce the respective plasmids (see Results). Finally, the
entire coding region sequence of each WdCDC42 mutant allele
(e.g. 14V, 19N, or 120A) was confirmed by DNA sequencing in two
directions.
DNA blots were prepared and hybridized with a 32P-labeled
probe of WdCDC42 derived by PCR with primers 5'42WD and
3'42WD (Table 1) or a probe of WdPKS1 derived from its 2-kb
BglII fragment (41). The hybridizations were
carried out at 42°C in a solution consisting of 50% formamide, 6×
SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4,
and 1 mM EDTA [pH 7.7]), 7.5× Denhardt's solution, 0.75% sodium
dodecyl sulfate, and 200 µg of denatured DNA/ml, which followed
washes with 1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate) and finally with 0.1× SSC at 25°C. For Northern blotting,
total RNA was isolated from the cells by hot acidic phenol extraction
(22). Hybridization of the RNA blots was carried out under
the same conditions as used for Southern blotting.
Immunoblot analysis.
Proteins from log-phase yeast cells of
W. dermatitidis were obtained by glass bead disruption
(22). After denaturation, the protein samples (~60 µg)
were separated by sodium dodecyl sulfate-12% polyacrylamide gel
electrophoresis and were transferred to nitrocellulose membranes. Total
protein loading was estimated by staining with 0.2% Ponceau-S (Sigma,
St. Louis, Mo.). WdCdc42p was then detected by using the rabbit
anti-yeast CDC42 polyclonal antibody sc-7172 (Santa Cruz
Biotechnology, Santa Cruz, Calif.) diluted to 1:500, followed by
incubation with a goat anti-rabbit immunoglobulin G-horseradish
peroxidase conjugate (Bio-Rad, Richmond, Calif.) diluted to 1:5,000 and
finally by reaction with the ECL system (Amersham, Piscataway, N.J.).
Expression in S. cerevisiae.
For expression of
WdCDC42 and its mutant alleles in S. cerevisiae,
the pYES2-derived plasmids were used to transform yeast strains
DJTD2-16A and INVSc1 by the alkali cation method (22). Ura+ transformants were recovered from SD medium (lacking
uracil) at 25°C. For temperature sensitivity testing, the
transformant cells were replica plated onto media containing either 2%
glucose or 2% galactose as the sole carbon source and then incubated
for 4 days at 25 or 37°C.
Transformation of W. dermatitidis.
For disruption of
WdCDC42, the linear DNA construct was prepared by digestion
of pYED42-827 with ApaI and NsiI (see Fig. 3A), whereas for expression of WdCDC42 and its mutant alleles,
the plasmids were linearized by NarI (41).
Transformation-competent yeast-like cells of W. dermatitidis
were prepared from mid-log-phase cultures washed with cold 10%
glycerol. Purified plasmid DNA was added to the cell suspensions at a
ratio of about 1 µg of DNA per 107 cells. Electroporation
was carried out with a Gene Pulser electroporation system (Bio-Rad) at
a setting of 1.45 kV, 25 µF, and 200 
. Transformants were grown in
YPD medium containing 30 µg of hygromycin (Sigma)/ml at 25°C, and
albino colonies were selected for further confirmation of plasmid
integrations by Southern analysis (see Results).
Photomicroscopy.
Photomicroscopy of W. dermatitidis cells was performed as previously described
(5). For staining of the cell wall with Calcofluor (Sigma)
or staining of nuclei with DAPI (4',6'-diamidine-2-phenylindole) (Accurate Chemical, Westbury, N.Y.), fungal cells were fixed for 3 h in 5% formaldehyde and then washed twice with 75% ethanol at room
temperature. After staining for 2 min, the samples were repeatedly
washed with saline and were finally examined and photographed by using
a Zeiss ICM 405 photomicroscope. For documentation of hyphal
microcolony growth, cell culture plates were photographed directly with
the same photomicroscope immediately after removal from incubation.
Nucleotide sequence accession number.
The genomic
sequence of the WdCDC42 gene has been given the
GenBank accession number AF162788.
| |
RESULTS |
Isolation and characterization of WdCDC42.
Using a
PCR-derived 428-bp fragment of the S. cerevisiae CDC42 gene
to generate a hybridization probe, we identified two putative CDC42-homologous clones, 94AB1 and 94AB2, by screening a
W. dermatitidis cDNA library under low stringency. These
clones were confirmed by DNA analysis to contain an identical
nucleotide sequence and a deduced amino acid sequence that was 78 to
85% identical to Cdc42p of other organisms (Fig.
1). Therefore, this cloned gene was
designated WdCDC42. Its genomic sequence was subsequently obtained by analysis of PCR amplicons derived from W. dermatitidis genomic DNA, which allowed two introns to be
identified in its coding region.
View larger version (96K):
[in this window]
[in a new window]
|
FIG. 1.
Comparison of the deduced amino acid sequences of Cdc42
homologues. Reference numbering of the sequences is according to the
numbering for the W. dermatitidis Cdc42 protein (Cdc42Wd).
The other Cdc42 proteins used for comparison are from S. pombe (Cdc42Sp), C. albicans (Cdc42Ca), S. cerevisiae (Cdc42Sc), Caenorhabditis elegans
(Cdc42Ce), Drosophila melanogaster (Cdc42Dm), Homo
sapiens (human fetal brain isoform [Cdc42Hsb] and human
placental isoform [Cdc42Hsp]), and Gallus gallus Cdc42
(Cdc42Gg). The percentages of identical amino acids are listed at
bottom right. Symbol: - at the C-terminal sequence (between 180 and
190), artificially introduced space for alignment. The consensus motif
sequences for GTP binding and hydrolysis and for C-terminal prenylation
are underlined.
|
|
To demonstrate that the cloned WdCDC42 gene is a functional
homologue of CDC42, we used the S. cerevisiae
cdc42 temperature-sensitive mutant strain DJTD2-16A
(15) in cross-species complementation studies. We also
generated a series of point mutations in the WdCDC42 cDNA by
site-directed mutagenesis. These mutations conferred specific amino
acid replacements in the GTP-binding and hydrolysis domains in the
Cdc42 GTPase (9, 40), such as Gly-14 to Val (G14V), Thr-19
to Asn (T19N), and Asp-120 to Ala (D120A) (Fig. 2A). Our results showed that the
transformants expressing wild-type WdCDC42 (but not
expressing mutant wdcdc42G14V,
wdcdc42T19N, or
wdcdc42D120A) were able to restore the growth of
the temperature-sensitive mutant at the restrictive temperature (Fig.
2B). Furthermore, overexpression of the dominant negative alleles
wdcdc42T19N and
wdcdc42D120A at 25°C in DJTD2-16A resulted in
growth retardation and led to terminal phenotypes of enlarged and
unbudded yeast cells after prolonged incubation (data not shown). In
contrast, the transformant cells overexpressing the constitutively
active allele wdcdc42G14V at 25°C produced a
morphologically heterogeneous population in which about 30% of cells
had elongated buds or were amorphous (data not shown). These dominant
mutational phenotypes were virtually identical to those previously
produced by overexpression of S. cerevisiae cdc42 mutant
alleles (15), suggesting that WdCdc42p has in vivo functions
identical to those of S. cerevisiae Cdc42p with respect to
control of cell polarity.
View larger version (50K):
[in this window]
[in a new window]
|
FIG. 2.
Generation and functional analysis of wdcdc42
mutant alleles. (A) Diagram of the point mutations introduced into
WdCDC42 by site-directed mutagenesis. The three mutant amino
acid codons are compared with the wild-type codons, and the codon
numbers correspond to their positions in WdCDC42. (B)
Expression of WdCDC42 and its mutant alleles with vector
pYES2 in the S. cerevisiae strain DJTD2-16A. The
transformants are indicated by the allele that each received: Wt42
(wild type), 14V (Gly-14 to Val), 19N (Thr-19 to Asn), and 120A
(Asp-120 to Ala). Prior to photography, these transformants were grown
for 4 days on SD-galactose agar medium at a temperature of 25 or
37°C.
|
|
Disruptions of WdCDC42 in W. dermatitidis.
To reveal the wdcdc42 deletion phenotype in W. dermatitidis, gene disruption experiments were carried out
initially in the parasexually derived diploid strain 3u2m-428
(5). One diploid transformant containing a single
wdcdc42 disruption was verified by Southern analysis (data
not shown) and then subjected to methyl benzimidazole-2-yl-carbamate-induced haploidization. To our surprise, five of the segregants, as determined by Southern analysis, contained only the disrupted allele of wdcdc42 but nonetheless grew
well at 25°C in the manner of the wild-type haploid, although their cellular morphologies varied (data not shown). Therefore, the essentiality of WdCDC42 in W. dermatitidis was
investigated further in the wild-type haploid and in the albino haploid
ALB303. The albino haploid was derived previously from the wild-type
parental strain by the targeted integration of vector pYEX303 into the genomic locus of the polyketide synthase gene, WdPKS1, which
is known to be required for melanin biosynthesis but not for viability, cell growth, or cellular morphological development in W. dermatitidis (41; unpublished data). In each
case, a DNA fragment containing SUR and partial
WdCDC42 flanking sequences was employed for targeted gene
disruptions by replacement (Fig. 3A).
Each of 20 putative disruptants from those two strains was analyzed
first by PCR (data not shown) and then by Southern hybridization (Fig.
3B). The results confirmed that WdCDC42 is a single-copy
gene in W. dermatitidis. Two independent transformants in
the former group (i.e., Bd42-53 and Bd42-60) and three in the latter
group (i.e., Ad42-41, Ad42-58, and Ad42-59) contained only the
disrupted allele of wdcdc42.
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 3.
Disruption of WdCDC42 by replacement with a
SUR selection marker. (A) Strategy for disruption of
WdCDC42 with a 5-kb linear DNA fragment containing
SUR and partial WdCDC42 sequences. Homologous
recombination resulted in the replacement of a 21-bp portion of the
WdCDC42 coding sequence (codons 76 to 83) with the 3-kb
SUR marker. (B) Southern analysis of wdcdc42
disruption transformants. DNA samples were digested by SacII
or ClaI, and the blot was hybridized with a
WdCDC42 probe. The fragments of wild-type (wt)
WdCDC42 were expected to be 5 kb when cut by
SacII and 4.5 kb when cut by ClaI, whereas a
fragment containing a SUR insertion would be 8 kb when cut
by SacII and would produce two bands of 7 and 0.5 kb when
cut by ClaI because of an introduced ClaI
restriction site at SUR. Note that Ad42-41, -58, and -59 and
Bd42-60 and -53 showed the expected patterns of band shifts and
therefore were specific disruption transformants.
|
|
By Northern analysis, both wild-type W. dermatitidis and an
ectopic transformant, Bd42-52 (as a control), showed two transcripts of
1.8 and 1.5 kb when hybridized with a WdCDC42 probe. The
expression levels of those transcripts were not obviously affected by
stress with either high temperature or low pH (Fig.
4A). However, in the wdcdc42
mutants, the 1.8-kb transcript, corresponding in size to the cloned
cDNA of WdCDC42, was completely eliminated, whereas the
1.5-kb species continued to be expressed (Fig. 4A). Therefore, the
1.5-kb band was not an alternatively spliced species of the WdCDC42 transcript but possibly encoded another member of
the Ras family.
View larger version (80K):
[in this window]
[in a new window]
|
FIG. 4.
Analysis of wdcdc42 disruptants. (A) Northern
blot analysis of the wild type (Wd8656) and wdcdc42
disruptants was performed with RNA samples prepared from log-phase
cells. The blot was hybridized with a 32P-labeled
WdCDC42 probe corresponding to the coding region of the
gene. Below the blots, rRNA bands corresponding to each lane in the
agarose gels are shown as references for relative RNA amounts. (B)
Immunoblotting analysis with an anti-yeast Cdc42 antibody, sc-7172.
Approximately 60 µg of total protein of cell lysates was loaded in
each lane, and an S. cerevisiae sample (Sc) was used as a
positive control. T, temperature.
|
|
To rule out the possibility that the wdcdc42 disruptants
contained residual WdCdc42p, an immunoblot analysis of the
wdcdc42 cell lysates was carried out using anti-yeast
Cdc42 antibody. The results showed that wild-type W. dermatitidis, the control strain Bd42-52, and wild-type S. cerevisiae produced positive bands at 21 kDa (Fig. 4B), the
expected size of the Cdc42 GTPase homologue. In contrast, none of the
wdcdc42 disruptants displayed a 21-kDa band or a truncated
form, which again confirmed that the WdCDC42 gene product
was completely eliminated in the disruption mutants of W. dermatitidis. Because a polyclonal antibody, sc-7172, was used in
the immunoblottings, several other bands, including a 29-kDa species in
W. dermatitidis and a 33-kDa species in S. cerevisiae (Fig. 4B), were also sometimes detected, but the signal intensity depended on the wash stringency of blotting. Unlike the
21-kDa band, these bands were all larger than that expected for a
Ras-like GTPase, and there were clearly no differences between those
from the wild type and those from the disruption mutants of W. dermatitidis in size or intensity. Therefore, these bands were
considered to be nonspecific species that cross-reacted with the
polyclonal antibody.
Microscopic observations showed that the cellular phenotypes of the
wdcdc42 null mutant were similar to those of a wild-type strain grown in a neutral CDY broth at both 25 and 37°C, in that they
retained a yeast form. However, the mutant cells were notably slimmer
than the wild-type control cells (Fig.
5). Also, about 6% of the mutant cells
formed a transverse septum in mother cells that contained at least two
nuclei. Moreover, when the phenotypes of the null mutant and the
wild-type strain were compared by culture in acidic (pH 2.5) CDY medium
to induce yeast-to-multicellular form (sclerotic body) transition,
about 21% of the wild-type cells produced the multicellular forms
comparable to that previously reported (16). In contrast,
approximately 62% of the null mutant cells converted to less enlarged
bicellular forms, suggesting that WdCdc42p might negatively regulate
cell polarization.
View larger version (67K):
[in this window]
[in a new window]
|
FIG. 5.
Phenotype of the wdcdc42 null mutant. The
cells of the wild-type strain and of a mutant, Bd42-60, were grown in
CDY medium at 37°C for 24 h. After fixation with 5%
formaldehyde, the samples were stained with Calcofluor or additionally
with DAPI. All cells are shown at the same magnification. Note the
transverse septum, indicated by arrows, and the presence of a nucleus
in each septated cell of the planate forms when also stained with DAPI.
Nomarski, Nomarski phase.
|
|
A constitutively active allele of WdCDC42 induced
isotropic growth and sclerotic-body formation.
The regulatory
effects of WdCDC42 in W. dermatitidis were
further investigated by overexpression of the wdcdc42 mutant
alleles (Fig. 2A) in both the wild-type strain and the
wdcdc42 null mutant Bd42-60. Transformations of W. dermatitidis with the wdcdc42 alleles under the control
of the glaA promoter were achieved by pYEX303-mediated homologous integration at the WdPKS1 gene locus (Fig.
6A). The transformants with the
integrated pYEX303 derivatives at this locus were easily identified by
their albino, instead of black, colony phenotypes, as previously
described (41). After confirmation of the targeted
integrations in the transformants by Southern blotting (Fig. 6B), their
proteins were also analyzed by immunoblotting of WdCdc42p at different
growth stages. The results indicated that the glaA
promoter-controlled expression of WdCdc42p was dramatically increased
after 24 h by the shift of cells from 25 to 37°C and that the
protein level gradually decreased during post-log phase (Fig. 6C).
Although the same patterns of expression were found among all the
transformants, the protein levels from the mutant alleles were
generally lower than that from the wild-type allele (Fig. 6C). Also, in
general the transformants derived from the wdcdc42 null
strain seemed phenotypically more sensitive to the mutations (Fig.
7) than did those derived from the
wild-type background (data not shown).
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 6.
Transformation of the W. dermatitidis cells
with WdCDC42 and its mutant alleles. (A) The integrative
vector pYEX303-derived plasmids contain WdCDC42 or a mutant
allele under the control of the promoter glaA. Prior to
electroporation, the plasmids were linearized by NarI at the
sequence of wdpks1 for targeting to the locus of
WdPKS1, a gene for the melanin biosynthetic pathway in
W. dermatitidis. (B) Southern analysis of the resulting
albino transformants. When hybridized with a WdPKS1 probe
(upper panel), the DNA digested by BamHI showed a 6-kb
WdPKS1 fragment in the wild-type strain (WT), which as
expected was replaced by a 17-kb BamHI-digested fragment
after integration with a pYEX303-derived plasmid. Also as expected, the
melanized ectopic transformant 303-6 retained the 6-kb fragment. The
same blot was also hybridized with a WdCDC42 probe (lower
panel), which confirmed that the integrated WdCDC42 alleles
overlapped the 17-kb hybridization bands, whereas the endogenous
WdCDC42 gene corresponded with the 15-kb fragments in
BamHI digestions. (C) Immunoblot with antibody sc-7172 of
WdCdc42p from transformant cells. Cell were grown in a
soluble-starch-containing medium at 25°C and were then shifted to
37°C. The number under each strain is the incubation time in hours.
|
|
View larger version (123K):
[in this window]
[in a new window]
|
FIG. 7.
Cellular morphologies of W. dermatitidis
overexpressing WdCDC42 or its mutant alleles. The albino
transformants were derived from a wdcdc42 null strain (d42)
and contained pYEX303 (d42/303), pYEX303-Wt42 (d42/Wt42), pYEX303-14V
(d42/14V), or pYEX303-120A (d42/120A). The transformant carrying
pYEX303-19N had a phenotype identical to that of the transformants
containing pYEX303-120A (data not shown). Before photography, cells
were grown in a starch-containing CDY liquid medium for 3 days at
37°C. Cell samples were then fixed with 5% formaldehyde and stained
with Calcofluor or DAPI. All cells are shown at the same magnification.
Note the transverse septa in the wdcdc42G14V
planate cell and sclerotic body, indicated by arrows. Nomarski,
Nomarski phase.
|
|
In CDY-starch liquid medium at 25°C, all transformants in the
wdcdc42 null background grew in a yeast form like the
wild-type strain, but those carrying the constitutively active allele
wdcdc42G14V often had an unusual, peanut-like
shape (data not shown). However, after a shift to 37°C, the
wdcdc42G14V transformants displayed unexpected
morphological changes: the yeast-like cells showed decreased bud
formation and increased isotropic growth (phase I) in the first day of
culture, suggesting that the G14V-altered WdCdc42 protein had induced
depolarization of cell wall expansion in this fungus. By early
stationary phase (~48 h), approximately 30% of the isotropically
enlarged cells had developed a transverse septum to become planate
divided forms; by middle or late stationary phase (~72 h), some of
the swollen cells had also produced the multiple septa characteristic
of sclerotic bodies (phase II) (Fig. 7c). Cell wall and nuclear
staining (Fig. 7g and k) showed that the transverse septations were
only in forms containing multiple nuclei. Notably, the septa were
formed when the expression of the G14V-altered protein had diminished
(Fig. 6C). Under the same growth conditions, the transformant cells containing the highly expressed plasmid-borne wild-type
WdCDC42 allele (Fig. 6C) induced only a low percentage of
phase I phenotypes (Table 2). In
contrast, expression of the wdcdc42T19N and
wdcdc42D120A alleles resulted neither in any
obvious cell polarity change (Fig. 7d, h, and l; Table 2; data not
shown) nor in a lethal effect in W. dermatitidis.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Change in the percentage of cell types induced by
transformation and expression of wdcdc42
mutant allelesa
|
|
The constitutively active allele of WdCDC42 repressed
hyphal development.
The effect of WdCdc42p activity on hyphal
growth in W. dermatitidis was determined on CDY-starch agar
at 37°C, because this fungus exhibits the hyphal phenotype more
homogeneously on solid media with less-available carbon sources or
limited nitrogen (unpublished data). Microscopic observations showed
that wdcdc42 null cells initiated apical growth and
elongation after 3 h of incubation (data not shown) and then
formed hyphal microcolonies within 24 h (Fig.
8a), suggesting that WdCDC42
was not required for hyphal growth in W. dermatitidis. In
contrast, the transformant carrying the constitutively active allele
wdcdc42G14V displayed the nonpolarized cell
expansion that resulted in large ovoid or spherical morphologies (Fig.
8c). However, the cells overexpressing a dominant negative allele
(wdcdc42T19N or
wdcdc42D120A) produced apically attached buds
initially (Fig. 8d) but still formed normal hyphae after prolonged
incubation.
View larger version (97K):
[in this window]
[in a new window]
|
FIG. 8.
Microcolony morphologies of W. dermatitidis
transformants overexpressing WdCDC42 or its mutant alleles.
The same transformant strains as shown in Fig. 7 were grown on a
starch-containing CDY agar surface for 24 h at 37°C. The
colonies were photographed directly by bright-field microscopy and are
all shown at the same magnification.
|
|
Similar phenomena were observed with overexpression of
WdCDC42 and its mutant alleles in Hf1, which contained
endogenous wild-type WdCDC42 (data not shown). The Hf1
transformants grew normally as yeasts at 25°C, but at the restrictive
temperature of 37°C, the transformants were still viable, except for
the transformant containing wdcdc42G14V, which
showed growth arrest (Fig. 9A).
Microscopic examinations of the Hf1 cells grown on CDY-starch agar at
37°C confirmed that the wdcdc42G14V
transformants were inhibited in apical polarization and could undergo
only a few cycles of cell division before lysis (Fig. 9B, panels i
through l). Although overexpression of wild-type WdCDC42
also induced growth tip enlargement, it did not completely repress
apical growth (Fig. 9B, panels e through h). Again, the transformants
containing wdcdc42T19N or
wdcdc42D120A showed no negative effects on cell
polarization or elongation in W. dermatitidis under these
conditions (Fig. 9B, panels m through p; data for
wdcdc42T19N not shown).
View larger version (68K):
[in this window]
[in a new window]
|
FIG. 9.
Effect of WdCDC42 and its mutant alleles on
cell growth and morphological development in strain Hf1. (A) The Hf1
transformants containing pYEX303-derived plasmids were grown on a
maltose-containing CDY agar medium for 4 days at 25°C or at 37°C.
(B) Cell morphologies of the corresponding Hf1 transformants carrying
pYEX303, pYEX303-Wt42, pYEX303-14V, or pYEX303-120A, which were grown
on a starch-containing CDY agar surface at 37°C. The inoculum of each
strain grown at 25°C is shown in the 0-h photomicrographs. The other
photomicrographs were taken at the time indicated at the top. All cells
are shown at the same magnification.
|
|
| |
DISCUSSION |
The zoopathogenic fungus W. dermatitidis exhibits three
distinct vegetative growth modes: blastic, apical, and isotropic, which
are primarily associated with growth in the yeast, hyphal, and
sclerotic-body morphologies, respectively (13). Cellular phenotypic conversions in W. dermatitidis are of great
interest because they are potentially relevant to the pathogenicity of this agent of human phaeohyphomycosis. In particular, the unique transition of yeast cells to sclerotic-body forms leads to the dramatic
enrichment of cell walls with known or suspected virulence factors,
such as melanin (6) and chitin (38, 39, 40). Based on cytological studies of multicellular-body formation, a
two-stage process is recognized (5, 13, 36). Stage I is
characterized by the production of greatly enlarged, unbudded unicellular forms having multiple nuclei and thickened cell walls. In
stage II, isotropic growth continues and the cells produce one or more
transverse septa. Moreover, multicellular bodies proliferate slowly
through a fission mode, indicating that this unique phenotype not only
is a part of the life cycle of W. dermatitidis but also is a
stress-resistant form that perhaps contributes its pathogenicity in
chronic infection (36). In addition, cell cycle mapping and parasexual genetic analysis of two temperature-sensitive
mcm/cdc mutations in this fungus provided clues that at
least two different genes normally responsible for bud emergence are
also involved in multicellular-body formation under stress conditions
(5, 34). We hypothesized that these mutations were possibly
in the homologues of CDC24, CDC42, or
CDC43 of S. cerevisiae (5, 34).
In this study, we describe the isolation of WdCDC42 and
functional characterization of this gene in W. dermatitidis.
By means of DNA sequence comparisons and by expression in S. cerevisiae, we confirmed that this gene is a functional
counterpart of yeast CDC42. Subsequent sequencing of the
WdCDC42 genes of the previously described mcm/cdc
strains documented that neither of mutants Mc2 and Mc3 (5,
34) had a defective WdCDC42 allele. However, of
greater significance was our finding that the biological functions of
WdCDC42 in W. dermatitidis were unexpectedly
different from those of CDC42 in S. cerevisiae
and other organisms. First, WdCDC42 was not essential for
cell viability like the CDC42 homologues in S. cerevisiae and S. pombe (15, 27); our data
clearly showed that the WdCDC42 gene disruption resulted in
loss of the corresponding gene products in the viable mutants of
W. dermatitidis. We suspect that some Ras- or Rho-homologous
gene product(s) may take the place of WdCDC42 in the
wdcdc42 null mutant, such as a closely related
RAC1 homologue that does not exist in S. cerevisiae but has been identified in W. dermatitidis
recently (unpublished data). Although several cross-reactive bands that
were larger than that of an expected Ras-like GTPase were also detected
in the lysates of both the wild-type and the wdcdc42 null
mutant cells by immunoblottings with a polyclonal anti-Cdc42 antibody,
they were most likely nonspecific signals. However, our data did not
exclude the possibility of these unknown factors having a compensatory
effect on the wdcdc42 defect in W. dermatitidis.
Second, the change of cellular morphology in the wdcdc42
null mutant was very subtle. Even in this background, overexpression of
the dominant negative alleles of wdcdc42 did not bring about the cell depolarization and inhibition of yeast-like bud formation (Fig. 7d and 8d) that has been observed in S. cerevisiae
(42). In contrast, only expression of the constitutively
active allele in W. dermatitidis induced transformant cells
to exhibit nonpolarized growth (Fig. 7c) rather than to develop
multiple buds or bud elongation (42). These results
suggested that WdCdc42p negatively regulated the cell polarization.
However, these contradictory effects by the different active states of
WdCdc42 GTP-binding protein in W. dermatitidis, compared to
those in S. cerevisiae, are not without precedence. In the
fission yeast S. pombe, overexpression of the corresponding
CDC42 mutant alleles cdc42G12V and
cdc42D118A also yielded phenotypes different
from those of S. cerevisiae. For example, these two mutant
alleles in the fission yeast are not dominant lethal, and both induce
enlarged, misshapen cells that contain only a single nucleus
(27). Moreover, overexpression of the constitutively active
allele cdc42HsG12V in HeLa cells leads to the
formation of enlarged multinucleate cells and to cytokinesis arrest
(10). These studies suggest that although Cdc42p is a highly
conserved component in the molecular machinery involved in cell
polarity control, the underlying mechanism of its contribution to
cellular morphogenesis is very diverse in different cell types and organisms.
Third, WdCdc42p appeared to play a unique regulatory role in the
coordination of cellular morphological transitions in W. dermatitidis. The wild-type protein cycling between the GTP- and GDP-binding states may be required for its normal yeast-like cell division cycle at high temperatures (Fig. 5) and under other stress conditions. When the sustained activation of this GTP protein existed,
a signal mimicked by overexpression of the mutant G14V product, the
W. dermatitidis cells initiated isotropic growth. This led
to the production of sclerotic bodies and simultaneously inhibited
hyphal growth (Fig. 7 and 8). Although these phenotypic transitions
certainly involve multiple gene regulation phenomena, they are far from
fully understood. However, the properties of WdCdc42p elucidated to
date seem to provide an important clue to growth mode switching by this
Rho-type GTPase in this fungus.
Collectively, our findings revealed that the WdCDC42 gene is
a highly conserved member of the CDC42 subfamily. Although
it was found to be nonessential for cell viability in W. dermatitidis, the WdCDC42 gene product nonetheless
seemed to play an important role in the regulation of stress-induced
fungal cellular morphogenesis. As a polymorphic fungus, W. dermatitidis most likely possesses more sophisticated Rho-type
GTPase-mediated regulatory pathways than do S. cerevisiae
and S. pombe for control of its cell growth and development.
We hope that further exploration of these pathways in this pathogen
will provide a better understanding of the molecular mechanisms of
fungal polymorphism and its contribution to pathogenesis in humans.
We thank B. Harrer for his critical suggestions, D. I. Johnson, M. Ward, and J. Sweigard for providing plasmids and strains, and S. M. Karuppayil, A. L. Mendoza, L. Zheng, Z. Wang, and
B. Feng for discussion and technical assistance.
This research was supported by a grant to P. J. Szaniszlo from the
National Institute of Allergy and Infectious Diseases (AI 33049).
| 1.
|
Bender, L.,
H. S. Lo,
H. Lee,
V. Kokojan,
J. Peterson, and A. Bender.
1996.
Associations among PH and SH3 domain-containing proteins and Rho-type GTPases in yeast.
J. Cell Biol.
133:879-894[Abstract/Free Full Text].
|
| 2.
|
Bi, E., and J. R. Pringle.
1996.
ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae.
Mol. Cell. Biol.
16:5264-5275[Abstract].
|
| 3.
|
Brown, J. L.,
M. Jaquenoud,
M.-P. Gulli,
J. Chant, and M. Peter.
1997.
Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast.
Genes Dev.
11:2972-2982[Abstract/Free Full Text].
|
| 4.
|
Chen, G. C.,
Y. J. Kim, and C. S. M. Chan.
1997.
The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae.
Genes Dev.
11:2958-2971[Abstract/Free Full Text].
|
| 5.
|
Cooper, C. R., Jr., and P. J. Szaniszlo.
1993.
Evidence for two cell division cycle (CDC) genes that govern yeast bud emergence in the pathogenic fungus Wangiella dermatitidis.
Infect. Immun.
61:2069-2081[Abstract/Free Full Text].
|
| 6.
|
Cooper, C. R., Jr., and P. J. Szaniszlo.
1997.
Melanin as a virulence factor in dematiaceous pathogenic fungi, p. 81-93.
In
V. H. Bossche, D. A. Stevens, and F. C. Odds (ed.), Host-fungus interplay. National Foundation for Infectious Diseases, Bethesda, Md.
|
| 7.
|
Coso, O. A.,
M. Chiariello,
J.-C. Yu,
H. Teramoto,
P. Crespo,
N. Xu,
T. Miki, and J. S. Gutkind.
1995.
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.
Cell
81:1137-1146[CrossRef][Medline].
|
| 8.
|
Cvrckova, F.,
C. De Virgilio,
E. Manser,
J. R. Pringle, and K. Nasmyth.
1995.
Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast.
Genes Dev.
9:1817-1830[Abstract/Free Full Text].
|
| 9.
|
Davis, C. R.,
T. J. Richman,
S. B. Deliduka,
J. O. Blaisdell,
C. C. Collins, and D. I. Johnson.
1998.
Analysis of the mechanism of the action of the Saccharomyces cerevisiae dominant lethal cdc42G12V and dominant negative cdc42D118A mutations.
J. Biol. Chem.
273:849-858[Abstract/Free Full Text].
|
| 10.
|
Dutartre, H.,
J. Davoust,
J. P. Gorvel, and P. Chavrier.
1996.
Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs.
J. Cell Sci.
109:367-377[Abstract].
|
| 11.
|
Epp, J. A., and J. Chant.
1997.
An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast.
Curr. Biol.
7:921-929[CrossRef][Medline].
|
| 12.
|
Evangelista, M.,
K. Blundell,
M. S. Longtine,
C. J. Chow,
N. Adames,
J. R. Pringle,
M. Peter, and C. Boone.
1997.
Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis.
Science
276:118-122[Abstract/Free Full Text].
|
| 13.
|
Geis, P. A., and C. W. Jacobs.
1985.
Polymorphism of Wangiella dermatitidis, p. 205-233.
In
P. J. Szaniszlo (ed.), Fungal dimorphism: with emphasis on fungi pathogenic for humans. Plenum Press, New York, N.Y.
|
| 14.
|
Johnson, D. I.
1999.
Cdc42: an essential Rho-type GTPase controlling eukaryotic cell polarity.
Microbiol. Mol. Biol. Rev.
63:54-105[Abstract/Free Full Text].
|
| 15.
|
Johnson, D. I., and J. R. Pringle.
1990.
Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity.
J. Cell Biol.
111:143-152[Abstract/Free Full Text].
|
| 16.
|
Karuppayil, S. M., and P. J. Szaniszlo.
1997.
Importance of calcium to the regulation of polymorphism in Wangiella (Exophiala) dermatitidis.
J. Med. Vet. Mycol.
35:379-388[Medline].
|
| 17.
|
Kwon-Chung, K. J., and J. E. Bennett.
1992.
Medical mycology, p. 337-555.
Lea and Febiger, Philadelphia, Pa.
|
| 18.
|
Lerm, M.,
J. Selzer,
A. Hoffmeyer,
U. R. Rapp,
K. Aktories, and G. Schmidt.
1999.
Deamidation of Cdc42 and Rac by Escherichia coli cytotoxic necrotizing factor 1: activation of c-Jun N-terminal kinase in HeLa cells.
Infect. Immun.
67:496-503[Abstract/Free Full Text].
|
| 19.
|
Li, R.
1997.
Bee1, a yeast protein with homology to Wiscott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton.
J. Cell Biol.
136:649-658[Abstract/Free Full Text].
|
| 20.
|
Liu, H.,
J. Kohler, and G. R. Fink.
1994.
Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog.
Science
262:1741-1744.
|
| 21.
|
Lu, X.,
X. Wu,
A. Plemenitas,
H. Yu,
E. T. Sawai,
A. Abo, and B. M. Peterlin.
1996.
Cdc42 and Rac1 are implicated in the activation of the Nef-associated kinase and replication of HIV-1.
Curr. Biol.
6:1677-1684[CrossRef][Medline].
|
| 22.
|
Lundblad, V.
1994.
Saccharomyces cerevisiae, p. 13.0.1-13.4.17.
In
F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 2. John Wiley & Sons, Inc., New York, N.Y.
|
| 23.
|
Mack, D.,
K. Nishimura,
B. K. Dennehey,
T. Arbogast,
J. Parkinson,
A. Toh-e,
J. R. Pringle,
A. Bender, and Y. Matsui.
1996.
Identification of the bud emergence gene BEM4 and its interactions with Rho-type GTPases in Saccharomyces cerevisiae.
Mol. Cell. Biol.
16:4387-4395[Abstract].
|
| 24.
|
Manser, E.,
T. Leung,
H. Salihuddin,
Z.-S. Zhao, and L. Lim.
1994.
A brain serine/threonine protein kinase activated by Cdc42 and Rac1.
Nature
367:40-46[CrossRef][Medline].
|
| 25.
|
Matsumoto, T.,
T. Matsuda,
M. R. McGinnis, and L. Ajello.
1993.
Clinical and mycological spectra of Wangiella dermatitidis infections.
Mycoses
36:145-155[Medline].
|
| 26.
|
Matsumoto, T.,
L. Ajello,
T. Matsuda,
P. J. Szaniszlo, and T. J. Walsh.
1994.
Developments in hyalohyphomycosis and phaeohyphomycosis.
J. Med. Vet. Mycol.
32(Suppl. 1):329-349.
|
| 27.
|
Miller, P. J., and D. I. Johnson.
1994.
Cdc42p GTPase is involved in controlling polarized cell growth in Schizosaccharomyces pombe.
Mol. Cell. Biol.
14:1075-1083[Abstract/Free Full Text].
|
| 28.
|
Minden, A.,
A. Lin,
F.-X. Calaret,
A. Abo, and M. Karin.
1995.
Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs.
Cell
81:1147-1157[CrossRef][Medline].
|
| 29.
|
Mösch, H.-U.,
R. Roberts, and G. R. Fink.
1996.
Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA
93:5352-5356[Abstract/Free Full Text].
|
| 30.
|
Nobes, C. D., and A. Hall.
1995.
Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81:53-62[CrossRef][Medline].
|
| 31.
|
Olson, M. F.,
A. Ashworth, and A. Hall.
1995.
An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1.
Science
269:1270-1272[Abstract/Free Full Text].
|
| 32.
|
Osman, M. A., and R. A. Cerione.
1998.
Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates Cdc42p effects on the actin cytoskeleton.
J. Cell Biol.
142:443-455[Abstract/Free Full Text].
|
| 33.
|
Qiu, R.-G.,
A. Abo,
F. McCormick, and M. Symons.
1997.
Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation.
Mol. Cell. Biol.
17:3449-3458[Abstract].
|
| 34.
|
Roberts, R. L., and P. J. Szaniszlo.
1980.
Yeast-phase cell cycle of the polymorphic fungus Wangiella dermatitidis.
J. Bacteriol.
144:721-731[Abstract/Free Full Text].
|
| 35.
|
Simon, M.-N.,
C. De Virgilio,
B. Souza,
J. R. Pringle,
A. Abo, and S. I. Reed.
1995.
Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway.
Nature
376:702-705[CrossRef][Medline].
|
| 36.
|
Szaniszlo, P. J.,
S. M. Karuppayil,
L. Mendoza, and R. J. Rennard.
1993.
Cell cycle regulation of polymorphism in Wangiella dermatitidis.
Arch. Med. Res.
24:251-261[Medline].
|
| 37.
|
Szaniszlo, P. J.,
L. Mendoza, and S. M. Karuppayil.
1993.
Clues about chromoblastomycotic and other dematiaceous fungal pathogens based on Wangiella as a model, p. 241-255.
In
H. V. Bossche, D. Kerridge, and F. Odds (ed.), Fungal dimorphism. Plenum Press, New York, N.Y.
|
| 38.
|
Szaniszlo, P. J., and M. Momany.
1993.
Chitin, chitin synthase and chitin synthase conserved region homologues in Wangiella dermatitidis.
NATO ASI Ser. Ser. H
69:229-242.
|
| 39.
|
Wang, Z.,
L. Zheng,
M. Hauser,
J. M. Becker, and P. J. Szaniszlo.
1999.
WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection.
Infect. Immun.
67:6619-6630[Abstract/Free Full Text].
|
| 40.
|
Wang, Z., and P. J. Szaniszlo.
2000.
WdCHS3, a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions.
J. Bacteriol.
182:874-881[Abstract/Free Full Text].
|
| 41.
|
Ye, X.,
B. Feng, and P. J. Szaniszlo.
1999.
A color-selectable and site-specific integrative transformation system for gene expression studies in the dematiaceous fungus Wangiella (Exophiala) dermatitidis.
Curr. Genet.
36:241-247[CrossRef][Medline].
|
| 42.
|
Ziman, M.,
J. M. O'Brien,
L. A. Ouellette,
W. R. Church, and D. I. Johnson.
1991.
Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.
Mol. Cell. Biol.
11:3537-3544[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
and
Top
Abstract
Introduction
Present address: Department of Molecular and Cellular Biology,
Baylor College of Medicine, Houston, TX 77030.
