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CHAPTER I
CHAPTER!
INTRODUCTION
1.1. Virulence in Fungi
With an increasing number of immunocompromised people, there has been a concomitant rise
in the number of cases of invasive fungal infections. These infections range from life
threatening invasive mycoses to the merely irritating but generally benign mucocutaneous
infections. Ideally, preventive strategies like administration of effective fungal vaccines should
be used for high-risk patients to abrogate the need for antifungal drug treatment. On the other
hand, antifungal agents for prophylactic, empiric, and therapeutic use, will likely be the primary
focus of pharmaceutical companies and clinicians in the immediate future. Antifungal vaccines
and drugs will perhaps be required to use integratively in controlling any epidemics of fungal
diseases. Presently, the challenge of medical management for mycoses has been partially met by
the use of amphotericin B, flucytosine, and a series of azoles for systemic drug treatment.
Nevertheless, new antifungal agents with fungicidal activities will be needed for the more
effective management of deep-seated fungal infections. There are at least two methods for the
identification of new antimicrobial drugs. The first is the classical screening of many classes of
synthetic or natural products against a variety of fungi by in vitro susceptibility testing. This
method has been very successful for a variety of antimicrobial agents, including antifungal
agents. The second method makes use of bio-molecular strategies and the concept of fungal
virulence for the development of drug targets. It is now possible to identify molecular targ~
that are essential to these eukaryotic microorganisms' ability to produce disease. The
identification of the unique molecular functions essential for fungal pathology could translate
into the use of these molecular targets for the design and development of new antifungal drugs
(Georgopapadakou and Walsh, 1996). The potential to exploit the knowledge on molecular
pathogenesis of fungi for antifungal agent and vaccine development is now being realized,
which is at the center of most research designs. An attempt to define a variety of parameters
specific to fungal virulence has been made in this introductory chapter.
1.2. Virulence Genes
When an approach is made to identify human fungal virulence genes, it is essential that each
investigator designs the most appropriate clinical conditions for gene expression and identify
the particular site of infection to study. The use of animal models to reproduce the infection
under controlled conditions is essential for the confirmation of virulence. An understanding of
the pathology of infection, infection specificity, and disease development is important. Certain
virulence genes may be necessary for an intravenous inoculation and the establishment of
infection, but have no impact on aerosol-produced infections. Another important consideration
when using animal models to detect virulence is the size of the inoculum. Certain genes and
their products may be important at low inocula, whereas others may be expressed at a high
burden of organisms. Therefore, the identification of important virulence genes expressed with
low numbers of fungi and that result in the development of infection may be ideal for vaccines.
On the other hand, important virulence genes expressed at high inocula or tissue burdens in the
range of 105 to 107 CFU/g of tissue or fluid may be more useful for antifungal targets. Also, the
impact of a gene disruption may simply reduce the ability of infection to reach a certain number
of organisms in a host. The outcome to the host resulting from infection with 103 to 104 CFU/g
of tissue may have little consequence in a pneumococcal infection, but a fungal infection with a
reduced number of organisms at the site of infection could tip the balance in favor of the host.
Also, careful attention to the storage of fungal strains and the use of clinical isolates should be
given. Although changes in the virulence of laboratory-passaged yeast strains may not be as
dramatic as they can be for bacterial strains, some fungi will attenuate with in vitro passage, and
thus, potential virulence genes could be lost through in vitro storage. In summary, investigators
need to carefully defme and control the particular conditions for studying virulence genes in
relationship to the complexity of the infection. The second set of genes consists of genes that
produce disease. These genes may be more common than is realized in fungi and may be useful
as antifungal targets and in vaccine development. For example, investigators may be able to
disrupt through gene replacement a possible virulence gene that will allow the organism to
persist in the host, but will have no impact on morbidity or mortality (reviewed by Perfect,
1996). This particular feature has elegantly been observed with many disrupted Candida
albicans genes responsible for virulence, which would be discussed in the following sections.
The mutants had prolonged survival compared with the period of survival of those infected with
the wild-type strain, but in many cases the numbers of C. albicans organisms of the two strains
in the kidney was similar. In other words, the fungus may survive at the site of infection but will
not produce obvious disease in the host. The commensal nature (Candida species) and potential
for dormancy of some pathogenic fungi (Cryptococcus neoformans, Histoplasma capsulatum,
Blastomyces dermatitidis, and Coccidioides immitis) would suggest that this type of virulence
genes does exist. The third set of genes consists of those that determine host range specificity
(Bowyer eta!., 1995). For instance, it has been shown that a virulence gene in a Pseudomonas
2
species can cross the plant-animal range specificity by its functional necessity for production of
disease in both hosts (Rahme et al., 1995). A fungus, such as Fusarium spp., can cause disease
in both plants and humans. Therefore, the principle of host range determinants should be
carefully considered in studies of fungal virulence in humans. Much of the work on fungal
virulence genes has been and will be performed in carefully controlled animal models.
Therefore, it is necessary to check the importance of identified virulence genes and their
disruptions in several animal models, with different inocula, and with different modes of
infections to ensure the general importance of the identified genes. The identified gene's
expression at sites of human infections should also be checked by studying expression of
reporter genes.
1.3. Strategies for Virulence Gene Identification
The experimental strategies for determination of virulence genes as molecular targets for
antifungal drugs and vaccines can be separated into two groups. The first group of strategies is a
direct effort to identify a specific biochemical or structural target and to identify the essential
genes in the pathway(s). The second strategy is a more indirect method, which sets certain
environmental conditions and allows the organism to direct and identify the importance of a
certain virulence gene(s) through its differential expression(s) under these in vivo signals. Both
strategies are presently being used with the Cryptococcus neoformans model (Perfect, 1995).
The first strategy uses biochemical and structural genes known to be unique to fungi in an
attempt to specifically and selectively disrupt them and determine their effects on virulence.
This directed strategy can be subdivided into investigation of the genes essential for growth
both in vitro and iri vivo or of the genes necessary for producing only in vivo growth and
disease. The first group represents a series of genes, which have been the focus of intense
research as antifungal drug targets. The fungal cell membrane target, which has a unique
component, ergosterol, as the primary sterol, has successfully been used for antifungal drug
development. Disruption of the genes in this sterol pathway generally makes the yeast less
virulent (Iwata et al., 1990), and antifungal drugs against this targeted pathway have been
particularly successful, including the azoles, the morpholines, and the allyalmines. Another
important target is the intact fungal cell wall, which is controlled by a certain number of glucan
and chitin synthase genes and their products, which are necessary for a fully virulent fungus
(Bulawa, 1993). Several prototype groups of compounds that act against this target, including
the echinocandin B congeners and nikkomycins, have already been identified. Areas of higher
risk for finding fungal virulence genes that are unique compared with those in mammals include
3
those associated with biochemical (Cole, 1996) or signal transduction pathways. On the other
hand, there is precedence for finding unique targets and drugs in basic fungal metabolic and
genetic machinery. These drugs include flucytosine for yeast infections and trimethoprim
sulfamethoxazole for pneumocystosis. In fact, even these targets can be related to virulence. For
example, flucytosine-resistant C. albicans strains have been shown to be less virulent than
susceptible strains (Fasoli et al., 1990). Several important pathogenic fungal genes in this area
have been identified. For example, genes for topoisomerases (Perfect, 1995), myristoylation
(Lodge et a/., 1994), amino acid and folate utilization (Tang et a/., 1994), and transcriptional
and translational factors (i.e., elongation factor 110 (Colthurst et al., 1991; Myers et al., 1992)
have been identified in several fungal pathogens. These types of genes stretch even the broad
definition of virulence genes since they are simply necessary for growth of the organism and
thus are essential for causing disease. However, as these genes are further identified and
studied, there will likely be significant differences in structure and function between these
fungal genes and their mammalian counterparts that can be exploited in drug development
(Perfect, 1995). The second group of genes in the directed strategy represents those, which are
required specifically for in vivo growth. Progress in the study of this type of fungal virulence
gene has also been made in fungal pathogens. Examples include the in vivo importance of the
gene encoding phosphoaminoimidazole carboxylase (ADE2) in C. albicans and Cryptococcus
neoformans (Kirsch and Whitney, 1991; Perfect eta/., 1993), a capsule gene (CAP 59) which is
essential for capsule production in Cryptococcus neoformans (Chang and Kwon-Chung, 1994),
the a mating type locus for Cryptococcus neoformans (Kwon-Chung et al., 1992), and the
calcineurin A gene for Cryptococcus neoformans (Odom et a/., 1996). These genes are not
necessary for in vitro growth under certain conditions, but in vivo they are essential for the
production of infection. This direct strategy for identifying virulence genes has· great appeal
because there is an initial knowledge base for understanding the mechanisms involved.
However, it requires careful validation ofthe gene's importance in vivo and careful exploitation
of any differences in the genes between the fungi and the mammalian hosts. The second strategy
for the identification of fungal virulence genes is more indirect in its approach but is potentially
very powerful because it allows pathobiology to direct investigations into the importance of
genes. The hypothesis to this strategy is simply that environmentally regulated genes and their
regulators are essential to the survival of the fungus. The identification of these uniquely
expressed genes will thus identify virulence genes that can be used for drug targets or vaccine
epitopes. In this era of genome analysis, a series of methods that can be used to analyze gene
expressions by capturing and quantitating cell transcripts under certain conditions have now
been developed. These new molecular advances for other systems can now be adapted for use
4
with pathogenic fungi. The first techniques used eDNA libraries from cells as probes for
differential hybridization to genomic libraries. These techniques have been used with H.
capsula tum (Keath et a/., 1989) and Cryptococcus neoformans (Rude and Perfect, 1994) to
identify regulated genes. eDNA library subtraction techniques have further refined this strategy
(Duguid et al., 1988), and with the isolation of small amounts of RNA from organisms at the
. site of infection, a differential PCR display technique may be particularly useful for capturing
unique transcripts (Liang and Pardee, 1992). Recently, two new techniques, serial analysis of
gene expression (Velculesev et al., 1995) and quantitative monitoring of gene expression
patterns with a complementary DNA microassay (Schena et al., 1995), have also been used, and
they could be adapted for pathogenic fungal genes to assess quantitative comparisons of the
expressed genes and potentially to identify their importance in vivo. Another system for
capturing highly expressed genes at the site of infection is an in vivo expression technology that
was developed for bacteria and that is very useful for the identification and validation of
virulence genes in bacteria such as Salmonella spp. (Mahan et al., 1993), and it can also be
adapted to fungi. In this system in vivo-expressed promoters are identified by their ability to
tum on an essential gene in purine metabolism to allow viability in vivo. Since ADE2 has been
shown to be essential in vivo for Cryptococcus neoformans (Perfect et al., 1993), a similar
strategy can be used to isolate in vivo regulated promoters and their genes in Cryptococcus
neoformans. All of these differentially expressed genes identified by any of the methods
described above do not ensure importance. First, these identified genes will need to be
reconfirmed for their actual differential expression by Northern blotting. Second, through site
directed disruption of these genes, mutants will need to be evaluated for the magnitude of their
importance on virulence in relevant animal models. Finally, a virulence gene(s) associated with
an attenuated phenotype may also be isolated by combining techniques of restriction enzyme
mediated integration to randomly disrupt genes and signature-tagged mutagenesis, which
specifically labels each disrupted gene to identify virulence genes by negative selection in vivo
(Hensel et al., 1995). Much ofthe focus on virulence genes has been on their use as targets for
drugs, but they could also be used as vaccine epitopes. In fact, in bacteria the products of
virulence genes have been most successfully developed into vaccines. A new exciting
technology, expression library immunization, could be used to identify fungal virulence genes
through their effects on inducing immunity in the host. Expression library immunization has
been used successfully to protect against a mycoplasma infection (Barry et al., 1995). With this
technology, which elevates the creation of nucleotide vaccines to a discovery method, the
inoculation of fungal DNA libraries into mice could protect the host against a challenge from
the whole organism. In.this strategy a library of eDNA from the fungus and under a mammalian
5
promoter is transfected into host tissue, in which fungal proteins can be expressed in the host
and the immune system can respond. When a protective library is found, actual protective
clones from this library can be identified by the use of sib selection strategy to identify the
protective genes producing these epitopes (Perfect, 1996). The proteins encoded by these genes
may then be useful in vaccine development, but the genes can also be evaluated for their
contribution to the intrinsic virulence of the fungus and its interaction with host immunity.
1.4. The Pathogenic Fungus Candida albicans- A Major Health Threat
Awareness of Candida albicans, the human fungal pathogen, has risen during recent years.
Although infections by C. albicans can be relatively mild and superficial (Fig.l.l.), systemic
mycoses often occur in immunocompromised patients, or even as a consequence of long-term
therapy with broad-spectrum antibiotics or of chemotherapy (reviewed by Odds, 1988).
Effective antifungal agents which are free of side-effects are urgently needed. There is hope that
recently developed techniques of manipulating C. albicans and the sequencing of its genome
will lead to a thorough understanding of the virulence and biology of this fungal pathogen, thus
offering the possibility of a knowledge-based approach to novel antifungal agents. Experimental
work on C. albicans has long been hampered by its asexuality, its diploidy and its non-canonical
codon usage (CUG encodes serine) (reviewed by Scherer and Magee, 1990; Santos eta/., 1996).
Molecular genetic techniques have overcome these difficulties, and today host strains and
transformation vectors are available and an efficient gene-disruption protocol has been
developed (Fonzi and Irwin, 1993; Morschhauser et al., 1999; Wilson et al., 1999). Reporter
genes encoding B-galactosidase (Lac4p ), luciferase, and green fluorescent protein (GFP) are
available (Leuker et al., 1992; Srikantha et al., 1996; Cormack et a/., 1997). The Stanford
genome sequencing project is expected to complete 10-fold coverage of the C. albicans genome
in the year 2000 (http://www-sequence.stanford.edu/group/candidal). Now, what are the factors
that determine the virulence of C. albicans? In the past, possible virulence traits have been
suggested, which were characterized on a phenomenological level, but studies could not prove
their role in pathogenesis. There are several predisposing factors for C. albicans to alter from a
state of a relatively quiescent communalism, to an aggressive pathogenic lifecycle. C. albicans
acts as an alert opportunist in the presence of these factors. Natural factors, like infectious,
idiopathic, congenital, and other debilitating diseases or a digression from the natural
physiological status inclusive of a hormonal variation can cause an impaired state of immune
function which is a prerequisite for candidiasis. Dietary factors, like excess or deficiency of
6
A.
B.
Fig.l.l. Clinical Features of Oral Candidiasis in Man. A. Pseudomembranous Candidiasis. B. Chronic Hyperplastic Candidiasis of Dorsal and Lateral Tongue. (Photos Courtesy Dr. M. I . Burzynski)
certain nutrients may alter the endogenous microbial flora. Mechanical factors, like trauma or
occlusive injury can alter the microenvironment, medical factors like drugs used to depress the
immune activity after surgery, and medication, which alters the host defenses against specific
infections are all causes for this predisposition towards candidiasis (Odds, 1988). The
characteristics held responsible for pathogenicity of this organism will be discussed in this
chapter. The unique feature possessed by the pathogenic strains of Candida in being able to
utilize the aminosugar N-acetylglucosamine (GlcNAc) certainly demanded attention. We
suspected a correlation between this feature and virulence, and were successful in adding this
trait to the list of virulent properties possessed by C. albicans, discuss~ in Chapter 3.
1.4.1. Virulence Determinants of C albicans
C. a/bicans needs to use several attributes which are potential pathogenicity parameters, to
establish pathogenesis in the host. In an effort to understand this process, various physiological
and biochemical activities of C. albicans have been studied over the years. These include
factors related to species and strains, adherence, dimorphism, toxin and enzyme production, and
cell surface composition. C. albicans virulence is a function of a multiplicity of factors working
jointly to overcome the host defences. A lack or debility in any of these parameters will reflect
negatively on its infectivity and make it difficult for Candida to establish itself, particularly in a
healthy individual (Ghannoum and Abu-Elteen, 1990).
1.4.1.1. Adherence
C. albicans maintains a commensal relationship with human hosts probably by adhering to
mucosal tissue in a variety of physiological conditions. The adherence of yeasts to oral mucous
cells is one of the main characteristics· of pathogenicity. Strains of C. a/bicans isolated from the
buccal mucosa of IllY -infected patients in the initial stages of AIDS, adhered more to oral
mucous cells than the isolated strains from subjects without mv infection. An interesting
breakthrough came with the discovery of ALAi gene in C. a/bicans. Adherence due to ALAi
gene in C. a/bicans comprises of two sequential steps. Initially C. albicans attaches itself to
extracellular matrix (ECM) protein coated magnetic beads in small numbers (the attachment
phase). This is followed by a relatively slower step in which cell to cell interactions
predominate (the aggregation phase). Neither of these phases is observed in Saccharomyces
cerevisiae, but expression of ALAi gene from a high copy vector results in both attachment and
7
aggregation. The adherence of C. albicans and S. cerevisiae, overexpressing ALAI, to a number
of protein ligands, occurs over a broad pH range, is resistant to shear forces generated by
vortexing, and is unaffected by the presence of sugars, high salt levels, free ligands, or
detergents. Adherence is, however, inhibited by agents that disrupt hydrogen bonds. The
similarities in the adherence and aggregation properties of C. albicans and S. cerevisiae
overexpressing ALAI suggest a role in adherence and aggregation for ALAi-like genes in C.
albicans (Gaur et al., 1999).
In a landmark finding, Gale et al., in 1997 reported a single gene INTI linked to adhesion,
filamentous growth, and virulence in C. albicans. Inti p is a C. albicans surface protein with
limited similarity to vertebrate integrins. INTI expression in S. cerevisiae was sufficient to
direct the adhesion of this normally nonadherent yeast to human epithelial cells. Furthermore,
disruption of INTI in C. albicans suppressed hyphal growth, adhesion to epithelial cells, and
virulence in mice. INTi links adhesion, filamentous growth, and pathogenicity in C. albicans,
and Int lp may be an attractive target for the development of antifungal therapies.
1.4.1.2. Phospholipase
Microbial pathogens like bacteria, parasite, and pathogenic fungi, use secretion of enzymes such
as phospholipase, as a genetic strategy to invade the host and cause infection. Phospholipases
are important pathogenicity determinants in C. albicans. They play a significant role in
damaging cell membranes and invading host cells. High phospholipase production is correlated
with an increased ability of adherence and a higher mortality rate in animal models (Mayser et
al., 1996). Extracellular (secreted) phospholipase activities that have been reported from C.
albicans include phospholipases A, B, and C, and D. Phospholipase A and lysophospholipase
activities have been found in the cell wall of yeast cells and hyphae. Enzyme activity was more
in the walls of older yeast cells than of younger cells and was more prominent at the tip of
growing hyphae. When yeast cells and germ tubes were grown in the same medium but at a
different pH, the specific activity of extracellular phospholipase A was similar for yeast cells
and germ tubes, while that of lysophospholipase was higher for the yeast form (Goyal and
Khuller, 1992).
The virulence of strains deleted for the C. albicans phospholipase B gene Caplbl, for
hematogenously disseminated candidiasis, was significantly attenuated, compared with the
isogenic wild-type parental strain. Although deletion of CaPLBJ did not produce any detectable
effects on candida! adherence to human endothelial or epithelial cells, the ability of the Caplbi
null mutants to penetrate host cells was dramatically reduced. Thus, phospholipase B may well
8
contribute to the pathogenicity of C. albicans by abetting the fungus in damaging and traversing
host cell membranes, processes which likely increase the rapidity of disseminated infection
(Leidich et a!., 1998). Another evidence procured by Ghannoum, 1998, claims that a
phospholipase-producing strain caused more fatality in mice, while the phospholipase-deficient
null mutant strain was avirulent. These data prove that phospholipase B is essential for Candida
virulence, and paves the way for studies directed at determining the mechanism(s) through
which phospholipase modulate virulence in this organism.
1.4.1.3. Acid Proteinase
Aspartyl proteinases are secreted by pathogenic species of C. albicans in vivo during infection.
(Staib et al., 2000). This enzyme is also secreted in vitro when the organism is cultured in
presence of exogenous protein (usually BSA) as nitrogen source. The C. albicans isolates which
adhered most strongly to buccal epithelial cells had the highest relative proteinase activities and
were most pathogenic (Ghannoum and Abu-Elteen, 1986). It was found that the fungal isolates
from HIV-infected symptomatic patients secreted, on average, up to eight fold more proteinase,
than the isolates from uninfected or HIV-infected, but asymptomatic, subjects. This differential
property was stably expressed by the strains even after years of maintenance in stock cultures.
Moreover, representative high-proteinase isolates were significantly more pathogenic for mice
than low-proteinase isolates of C. albicans (De Bemardis eta/., 1996).
The secreted aspartyl proteinases of C. albicans are thought to contribute to virulence through
their effects on Candida adherence, invasion, and pathogenicity. The role of Candida secreted
aspartyl proteinase referred to as Sap (SAP gene and Sap protein), has been studied by a number
of laboratories as a potential virulence factor of C. albicans. As a protease, the enzyme may
have a spectrum of substrates, depending upon the host organ, e.g., skin or blood that is
colonized or infected. Virulence genes like SAP are differentially activated during infection. C.
albicans can colonize or infect virtually all body sites because of its high adaptability to
different host niches, which involves the activation of appropriate sets of genes in response to
complex environmental signals. An in vivo expression technology was used that is based on
genetic recombination as a reporter of gene expression to monitor the differential activation of
individual members of a gene family encoding secreted aspartic proteinases (Saps) at various
stages of the infection process. It is shown that SAP expression depends on the type of infection,
with different SAP isogenes being activated during systemic disease as compared with mucosal
infection. In addition, the activation of individual SAP genes depends on the progress of the
infection, some members of the gene family being induced immediately after contact with the
host, whereas others are expressed only after dissemination into deep organs. In the latter case,
the number of invading organisms determines whether induction of a virulence gene is
necessary for successful infection.
So far, nine distinct SAP genes (SAP 1 to SAP9) have been identified. The levels of the Sap 1,
Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several C.
albicans strains (White et al., 1995), including strain W0-1, which alternates between two
switch phenotypes, white (W) and opaque (0) (Soli, 1992). These studies revealed that the
specific Sap isoenzyme produced is determined by the cell type (strain) whereas the level of Sap
production is affected by environmental factors, and they showed that both the yeast-to
mycelium transition and phenotypic switching can determine which of the Sap isoenzymes is
produced. SAPJ and SAP3 levels were regulated during the phenotypic transition between W
and 0 forms. SAP2 was the dominant transcript in the yeast form, and its expression was
autoinduced by peptide products of its own enzymatic activity and repressed by amino acids
(Hube et al., 1994). SAP4 and SAP6 expression was observed only at neutral pH during
morphogenetic conversion from yeast to hypha induced by serum. Expression of SAP7 was not
detected under any ofthe experimental conditions used throughout the study. SAPB is the third
gene of the family to be expressed in the opaque phenotype (SoU, 1992).
Implication of Sap proteins in virulence has also come from recent studies by Hube, Sanglard
and colleagues. The authors constructed strains harboring disruptions in a number of SAP genes,
including SAPJ, SAP2, and SAP3 (Hube et al., 1997) and a triple-knockout of SAP4, SAP5, and
SAP6 (Sanglard et al., 1997). In all cases, mutants showed decreased virulence in an animal
model of disseminated candidiasis. Interestingly, Sap4, Sap5, and Sap6, are produced by C.
albicans cells after phagocytosis by macrophages. A sap4, sap5, sap6 null mutant was killed
more effectively by 53% after contact with macrophages, than the wild-type strain. (Von
Zepelin et al., 1998). However, expression of Sap2p as a sole putative virulence factor did not
causeS. cerevisiae to become virulent and constitutive overexpression of SAP2 did not augment
virulence of C. albicans in experimental oral or systemic infection (Dubois eta/., 1998).
,.
1.4.1.4. pH and Pathogenicity
C. albicans has to survive at host environment of diverse pH range. The environmental pH acts
as a manipulator for many physiological functions including morphogenesis. It has been shown
that pH can alter the expression of the pathogenic trait also. C. albicans PHRJ gene which is
expressed at ambient pH at 5.5 or higher (neutral to alkaline pHs), and PHR2, expressed at an
ambient pH below 5.5, play a role in morphogenesis (Saporito-Irwin eta/., 1995). The virulence
10
of the organism also is affected in this pattern, when either or both of the genes are disrupted.
Deletion of PHRJ, results in pH-conditional defects in growth, morphogenesis, and virulence,
evident at neutral to alkaline pH, but absent at acidic pH. Conversely, a phr2 null mutant
exhibited pH-conditional defects in growth and morphogenesis analogous to those of phr 1
mutants, but manifests at acid rather than alkaline pH values. Engineered expression of PHRJ at
acid pH in a phr2 mutant strain and PHR2 at alkaline pH in a phr 1 mutant strain complemented
the defects in the opposing mutant. Deletion of both PHRJ and PHR2 resulted in a strain with
pH-independent, constitutive growth and morphological defects (Ghannoum et a/., 1995;
Muhlschlegel and Fonzi, 1997). When these strains were tested for pathogenicity in various
niches of the host with different pH (systemic pH is near neutrality and vaginal pH is around
4.5), the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The phrl
null mutant was avirulent in a mouse model of systemic infection, but uncompromised in its
ability to cause vaginal infection in rats. The virulence phenotype of a phr 2 null mutant was the
inverse. The mutant was virulent in a systemic infection model, but avirulent in a vaginal
infection model. Heterozygous mutants exhibited partial reductions in their pathogenic
potential, suggesting a gene dosage effect (De Bernardis et a/., 1998). Another pH regulatory
gene of C. albicans whose maximal expression occurs at neutral pH, with no expression
detected below pH 6.0, has been cloned (Sentandreu et al., 1998). This gene was designated as
PRAJ, for pH regulated antigen. The protein predicted from nucleotide sequence was 299 amino
acids long, with motif characteristics of secreted glycoproteins. The predicted surface
localization and N- glycosylation of the protein were demonstrated directly by cell fractionation
and immunoblot analysis. The PRAJ protein was homologus to surface antigens of Aspergillus
species, which react with serum from aspergillosis patients, suggesting that the PRAJ protein
may have a role in the host-parasite interaction during candidal infection.
1.4.1.5. Integrins
The existence of integrin-like proteins in C. albicans has been postulated because monoclonal
antibodies to the leukocyte integrins, bind to blastospores and germ tubes, recognize a candidal
surface protein, and inhibit candidal adhesion to human epithelium. The gene a INTi has motifs
common to human integrins, and a-lntlp is surface localized in C. albicans. Expression of
a!NT 1 led to the production of germ tubes in haploid S. cerevisiae and in the corresponding
stel2 mutant. Studies of alntlp reveal a role for integrin-like proteins in adhesion and in
STE12-indcpendent morphogenesis. Disruption of INTi in C. albicans suppressed hyphal
11
growth, adhesion to epithelial cells, and virulence in mice. INTI links adhesion, filamentous
growth, and pathogenicity in C. albicans (Gale et al., 1996).
1.4.1.6. High-affinity Iron Permease: An Essential Virulent Factor of C albicans
Two high-affinity iron permease genes, CaFTRI and CaFTR2, have been isolated (Ramanan
and Wang, 2000). CaFTRI expression was induced under iron-limited conditions and repressed
when iron supply was sufficient, whereas the expression of CaFTR2 was regulated in a reversed
manner. Mutants lacking CaFTRI, but not CaFTR2, exhibited a severe growth defect in iron
deficient medium, and were unable to establish systemic infection in mice. Thus, CaFTRI
mediated iron-uptake mechanism constitutes a virulence factor of C. albicans. It could also be a
target for the development of anti-candida therapies (Ramanan and Wang, 2000).
1.4.2. GlcNAc Catabolic Pathway of C albicans
The metabolism of N-acetyl-D-glucosamine (GlcNAc) by C. albicans has attracted interest,
since the strains which are associated with the disease candidiasis, were able to grow on N
acetylglucosamine (GlcNAc) as the sole carbon source (Singh and Datta, 1979b). GlcNAc is
also capable of inducing cellular morphogenesis of C. albicans. This fungus frequently causes
infections in the gastrointestinal, respiratory, and genital tracts. The mucous membranes at the
site of infection are rich in aminosugars. Although aminosugars are not present in free form,
they are constituents of glycoproteins such as mucin. The organism has an efficient catabolic
system for the uptake and subsequent catabolization of N-acetylglucosamine into fructose-6-
phosphate, which is then fed into glycolysis. The response of the organism to induction by
GlcNAc involves dramatic changes in the enzyme levels ofthe aminosugar catabolic pathway.
Investigations into the GlcNAc catabolic pathway had begun by the study on the induction and
regulation of N-acetylglucosamine kinase (Bhattacharya et al., 1974) and N-acetylglucosamine-
6-phosphate deacetylase (Rai and Datta, 1982) in C. albicans. GlcNAc is transported by a
membrane bound permease and is sequentially metabolised by GlcNAc kinase, GlcNAc-6-
phosphate deacetylase and GlcNAc-6-phosphate deaminase (Singh and Datta, 1979a). Activities
of these enzymes are absent in cells grown on glucose. The pathway in C. albicans was found
not to be repressed by glucose (Singh and Datta, 1978), though Niimi et al., 1987, found that
glucose does repress the catabolism ofGlcNAc in C. albicans, giving an explanation that strain
variations could be responsible for the report. Synthesis of N-acetylglucosamine catabolic
enzymes, namely permease qtigh-affmity uptake system), kinase, and deaminase, was induced
12
by N-acetylglucosamine (Singh and Datta 1979a; Gopal et a/., 1982) during germ tube
formation, and was dependent on concomitant new protein synthesis, as the inducer operated at
a transcriptional level. As a related branch of the pathway, N-acetylmannosamine catabolism
was also found to be inducible, by either aminosugar N-acetylmannosamine or N
acetylglucosamine, and evidence was found that both the pathways converged at GlcNAc
(Biswas et a/., 1979). N-acetylmannosamine could also induce the enzymes for N
acetylglucosamine utilization (Sullivan and Shepherd, 1982). However, there is evidence that
the germ tube formation induced by GlcNAc, and N-acetylglucosamine metabolism, may be
mutually exclusive events. The level of activity of the N-acetylglucosamine catabolic enzymes
in germ tube stage is lower as compared to yeast phase cells. A strain of C. albicans that did not
form germ tubes was endowed with a pronounced ability for induction of N-acetylglucosamine
catabolic enzymes (Natarajan eta/., 1984). The purification ofGlcNAc-6-phosphate deaminase,
the terminal enzyme of the GlcNAc catabolic pathway, and cloning of the eDNA (Natarajan and
Datta, 1993), and genomic DNA of NAGJ (Kumar et a/., 2000), has thrown more light on
GlcNAc catabolism in C. albicans. The deaminase is induced over 100 fold by GlcNAc, and it
was also found that GlcNAc induced NAGJ at the transcriptional level (Natarajan and Datta,
1993).
1.5. Convergent Pathways for Utilization of Aminosugars in Escherichia coli
Aminosugars are versatile components of the cell surface structures of bacteria. They form the
essential backbone of the peptidoglycan in both gram-positive and gram-negative bacteria and
are also constituents of the outer membrane lipopolysaccharide (LPS) layer and the
polysaccharide capsules of gram-negative bacteria. The g/mS-encoded amidotransferase,
glucosamine (GleN) synthase, is responsible for the de novo synthesis of aminosugars in
Escherichia coli, producing GlcN-6- P from fructose-6-phosphate and glutamine. The pathway
for the conversion of GlcN-6-phosphate to UDP-N-acetylglucosamine (GlcNAc), the first
dedicated precursor of the cell wall components, has been recently elucidated (Mengin-Lecreulx
and Heijenoort, 1996). UDP-GlcNAc serves as aminosugar donor in several transferase
reactions in the synthesis of peptidoglycan, the core and lipid A moieties of the LPS,
enterobacterial common antigen, some 0 antigens of gram-negative bacteria, and the teichoic
acids of gram-positive bacteria (Raetz, 1998). Some of the UDP-bound amino sugar in enteric
bacteria is subsequently converted to N-acetylmannosamine (ManNAc) and N
acetylmannosaminuronic acid for incorporation of the latter into the enterobacterial common
antigen. In addition to having a structural role, the amino sugars are particularly useful to
13
bacteria as energy sources since they supply both carbon and nitrogen. Both GleN and GlcNAc
are phosphoenolpyruvate-dependent phosphotransferase system (PTS) sugars in E. coli, and the
proteins that mediate:thein-uptake (nag£ and~man*YZ) or degradation (nagBA·Yhave· been·
purified and characterized (Calcagno eta/., 1984; Erni and Zanolari, 1985; Mukhija and Erni,
1996). The genes encoding the GlcNAc-specific PTS transporter (nagE), and the enzymes that
convert GlcNAc-6-phosphate to GlcN-6-phosphate and fructose-6-phosphate (nagBA), are
arranged in divergent operons controlled by the nagC-encoded repressor (Plumbridge and Kolb,
1993). In addition, the genes for the biosynthesis (glmS) and degradation (nagB) of GlcN-6-
phosphate are expressed in a coordinated manner so that in the presence of aminosugars, the
catabolic enzymes are induced and the expression of glucosamine synthase is decreased
(Plumbridge, 1995). The aminosugars are ubiquitous and abundant in nature (e.g., chitin is a P-1,4-linked homopolymer of GlcNAc), and are present in a range of both simple and complex
biopolymers. Most glycoconjugates (glycoproteins and glycolipids) of mammalian cell surfaces
contain aminosugars, including sialic acid residues, where the oligosaccharide chains of these
conjugates are important ligands for cellular recognition. The sialic acids are a series of N-and
0-substituted derivatives of N-acetylneuraminic acid (NAN A), a compound which is formed by
the condensation ofManNAc and pyruvate (Fig.l.2.). Certain bacteria are capable of degrading
the complex oligosaccharide chains of glycoconjugates, and many bacteria, including enteric
bacteria like E. coli K-12, can use sialic acid as a source of carbon and nitrogen (Vimr and
Troy, 1985a, b). In contrast to this widespread catabolic pathway in bacteria, relatively few,
mostly pathogenic species, are able to synthesize sialic acid for subsequent incorporation into
surface structures, e.g., the capsular polysialic acid virulence factors of E. coli Kl and Neisseria
meningitidis (Vimr et a/., 1995). Although the E. coli genes encoding a sialic acid transporter
(nanD and N-acetylneuraminate lyase (nanA) have been sequenced and characterized both
genetically and physiologically (Aisaka et a/., 1991; Rodriguez-Aparicio et a/., 1987; Vimr and
Troy, 1985a, b), the subsequent fate of the ManNAc liberated by the lyase has not been
determined for any bacterium.
Unlike the two common aminosugars GleN and GlcNAc, ManNAc is not efficiently used as a
carbon source by wild-type E. coli K-12. The efficient utilization of ManNAc depends on the
two mutations, mlc (transporter) and ama (epimerase), to activate the otherwise cryptic pathway
(Plumbridge and Vimr, 1999). Mlc has been shown to be a repressor for this operon, and an mlc
mutation enhances manXYZ expression threefold (Plumbridge, 1998). The ManXYZ transporter
shows a wide substrate specificity, transporting glucose, mannose, GleN, and GlcNAc, it is thus
not surprising that it can also transport ManNAc. Neither ptsG (PTS transporter of mannose
and glucose) nor nagE (GlcNAc-specific transporter) can substitute for manXYZ. It had been
14
Sialic acid
NANA NANA ManNAc + pyruvate
1~--ManNAc --+-------+-----------. ManNAc-6-P
GlcN-1-P
GlcN-6-P GlcNAc.,.1-P
EX1ERIOR INTERIOR Fru-6-P UDP-GlcNAc
Fig.1.2. Pathway for the Metabolism of GlcNAc and Proposed Pathway for the Degradatio of ManNAc and NANA (Sialic acid) in Escherichia coli. GlcNAc is transported by both the manXYZ-encoded transporter and its own specific tranporter encoded by nagE, producing intracellular GlcNAc-6-P, which is degraded by the nagA and nagl encoded enzymes. The biosynthetic pathway producing UDP-GlcNAc for incorporation into eel wall components involves the glmS, glmM, and glmU gene products. ManNAc is taken up by th4 manXYZ transporter, producing intracellular ManNAc-6-P. NANA is taken up as the free sugar by a sugar-cation symporter encoded by nanT. Inside the cell, NANA is cleaved by the aldolase encoded by nanA to give ManNAc and pyruvate. It is proposed that intracellular ManNAc is phosphorylated to ManNAc-6-P, which is subsequently converted to GlcNAc-6-P, the substrate of the nagA encoded deacetylase.Thus, the pathways for degradation ofNANA, ManNAc, and GlcNAc converge at the level ofGlcNAc-6-P. Adopted from Plumbridge and Vimr, 1999.
established many years ago, by the work of Roseman's laboratory, that ManNAc is a PTS
sugar; it was one of the first sugars used to demonstrate the PTS, and mutations in the ptsH and
pis/ genes eliminated this phosphorylation- (Saier et·al:,- 1976):"'-The,~.first-purified~enzyme II.
preparation was capable of phosphorylating ManNAc, suggesting that it was predominately the
manXYZ-encoded complex (Kundig and Roseman, 1971). It is perhaps surprising that a three
fold increase in manXYZ expression can produce such a significant increase in growth rate on
ManNAc, unless the metabolic equipoise of this aminosugar is such that a small increase in
uptake, coupled with derepression of nanE, is sufficient to stimulate ManNAc catabolism to an
extent, comparable with the observed growth of mlc ama double mutants. At present it is not
known if the sole role of the mlc mutation in allowing growth on ManNAc is to enhance
manXYZ expression, or whether it also affects some other genes involved in ManNAc
utilization. The second mutation ama, required for good growth on ManNAc, occur at high
frequency in the mlc background. The ama mutation can be replaced by a plasmid carrying an
ORF from downstream of the nanAT genes (yhc.J). The frequently occurring ama mutations are
regulatory mutations (either inactivating a repressor or providing a cis-acting promoter
mutation}, which allow the expression of nanAT and the previously uncharacterized
downstream genes of this operon. The pathway for ManNAc metabolism (Fig.1.2.) has been
deduced by Plumbridge and Vimr, 1999. Transport of ManNAc by the manXYZ PTS transporter
produces ManNAc-6-phosphate. The requirement for the nagBA gene products strongly
suggests that ManNAc-6-phosphate is converted to GlcNAc-6-phosphate, which is subsequently
degraded to GlcN-6-phosphate and then to fructose-6-phosphate and ammonia via the nagA
encoded GlcNAc-6-phosphate deacetylase and nagB-encoded GlcN-6-phosphate deaminase,
respectively. The missing step in the pathway for ManNAc degradation, and thus the function
potentially supplied by the ama mutation or yhc.J on a plasmid, should be the conversion of
GlcNAc-6-phosphate to ManNAc-6- phosphate. GlcNAc-6-phosphate deacetylase is incapable
of degrading ManNAc-6-phosphate. The step for which no gene product is assigned for the
utilization of ManNAc, is the conversion of ManNAc-6-phosphate to GlcNAc-6- phosphate,
and that the yhc.J gene immediately downstream of the nanT is a candidate to supply this
ManNAc-6-phosphate epimerase function.
In yeast, ManNAc was shown to induce GlcNAc catabolic pathway enzymes, and not utilized as
a growth substrate (Sullivan and Shepherd, 1982). Biswas eta/., 1979, also detected the
presence of a ManNAc epimerase and GlcNAc kinase. It is quite possible that, uptake of the
free sugar was probably followed by its conversion to the GlcNAc epimer for induction.
The nan operon in E. coli potentially contains five genes: nanAT, the putative nan£ epimerase
gene, yhc.J (described above}, plus two other downstream genes (Fig.1.2.). The first of these
15
other genes, yhcl, encodes a protein homologous to NagC, Mlc, and other proteins of the so
called ROK (repressor, ORF, and kinase) family (Titgemeyer eta/., 1994). This family consists
of two· classes:· of proteins: the·otranscriptional regulatory proteins exemplified- by NagC and
XylR, and the somewhat smaller proteins encoding sugar kinases which are missing the N
terminal helix-tum-helix DNA binding domain present in the transcription factors. The yhcl
encoded protein belongs to this latter class and would be expected to encode a sugar kinase.
Yhcl is also homologous to the ManNAc kinase domain of the bifunctional UDP-GlcNAc 2-
epimerase/ManNAc kinase from mammalian liver (Hinderlich eta/., 1997; Stasche eta/., 1997)
and includes (from residues 15 to 30) one oftwo phosphate binding (ATPase) regions found in
a range of hexose kinases. The nanA-encoded aldolase (NANA lyase) generates free
intracellular ManNAc and pyruvate from sialic acid. It is tempting to speculate that the substrate
for the putative yhcl-encoded kinase is this internally liberated ManNAc, thus generating
ManNAc-6-phosphate, the substrate of the epimerase predicted to be encoded by the upstream
gene. The gene was named nanK. The existence of the putative genes for the ManNAc kinase
and epimerase function within the nanAT operon allows the metabolic pathway for use of sialic
acid to converge with that of ManNAc and GlcNAc at the common intermediate GlcNAc-6-
phosphate (Fig.l.2.). Evidence that the metabolism of sialic acid does pass via the pathway of
GlcNAc utilization is that growth on sialic acid, like that of ManNAc, replaces the aminosugar
requirement of glmS strains, requires the nagBA genes, and results in strong induction of the
nagE and nagB operons as measured by a nagB-lacZ fusion. Sialic acid in Streptococci
viridians induces the nag degradative genes (Byers eta/., 1996). It shows that in E. coli and
probably other bacteria, ManNAc-6-phosphate is the substrate of the epimerase. In H.
injluenzae, the nagBA genes are clustered with the nanA gene (encoding a putative NANA
lyase), as well as homologues of the two genes proposed as the ManNAc kinase and epimerase
genes, yhcl and yhc.J (Plumbridge and Vimr, 1999). The gene order in Haemophilus injluenzae,
yhc.J, yhcl, nanA, nagB, nagA, is somewhat different from that in E. coli, but suggests a
remarkable grouping of related functions. H injluenzae can grow on sialic acid, but is unable to
use GlcNAc as a carbon source (Macfadyen et a/., 1996), implying that the nagBA genes may
be present solely for the degradation of the GlcNAc-6-phosphate generated intracellularly from
sialic acid (Macfadyen eta/., 1996).
GlcNAc is expected to be present in both free-living and animal environments, whereas sialic
acid is found only in the host. In contrast to the efficient use of these two sugars, ManNAc
metabolism seems to rely on part of the sialic acid pathway, which itself converges with the
GlcNAc pathway. The fact that reasonable growth rates on ManNAc requires mutations in two
regulatory loci to increase the expression of the transporter (mlc) and the epimerase (ama)
16
suggests that E. coli is not specially adapted to use ManNAc per se but degrades this sugar only
as part of the sialic acid dissimilatory pathway. The proposed pathway for sialic acid utilization
requires that ManNAc, generated intracellularly from the action of the nanT and nanA gene
products, be a substrate for phosphorylation by the nanK (yhcl) and epimerization by the nanE
(yhcJ) gene products. Expression of the operon appears to be subject to negative control by a
repressor "ncoded by the nanR (yhcK) gene located upstrean1 of the operon. Previous results
(Vimr and Troy, 1985a, b) now suggest that sialic acid induces the nan operon by binding to
NanR (Plumbridge and Vimr, 1999). Since NanR lacks obvious homology to other known sialic
acid binding proteins, determining the exact interaction of NANA with the repressor will
provide new insight into the structure and function of sialic acid recognition macromolecules.
1.6. Dimorphism in Fungi
Despite the evolutionary divergence, fungi are more closely related to animals, than to plants,
algae, bacteria, or archea, and thus share important features with mammalian cells. This is
perhaps most apparent in the signaling cascades that regulate cell function. The yeast
Saccharomyces cerevisiae expresses at least three members of the G protein-coupled family of
serpentine receptors, which are in tum coupled to a heterotrimeric G protein, and a G protein
alpha-subunit homolog. Mating in yeast cells is regulated by a mitogen-activated protein (MAP)
kinase cascade that is highly conserved with MAP kinase cascades in mammalian cells. Finally,
the signal transduction components that are targeted by the immunosuppressive drugs
cyclosporin A, FK506, and rapamycin are remarkably conserved from yeasts to humans. Thus,
studies on signal transduction in S. cerevisiae and other genetically tractable fungi promise to
reveal common conserved mechanisms of signal transduction. Recent studies have revealed that
the yeast S. cerevisiae undergoes a dimorphic transition to filamentous growth in response to
nutritional· signals in the environment, particularly nitrogen limitation. Filamentous growth
occurs in both haploid and diploid cells in different environments, and may play novel roles in
the life cycle of this organism. At least two conserved signal transduction cascades that regulate
filamentous growth have been defined, and remarkably related signaling pathways also operate
during differentiation of other fungi, including pathogens of both plants and animals. These
recent findings suggest that S. cerevisiae is an excellent model system, with great potential to
provide insights into signaling in other fungi. The signal transduction cascades that regulate
yeast filamentous growth and the related signaling pathways also operate in the fission yeast
Schizosaccharomyces pombe, in human fungal pathogens Candida albicans and Cryptococcus
neo.formans, in plant fungal pathogens Ustilago maydis, Magnaporthe grisea, Cryphonectria
17
parasitica, and in model filamentous fungi Aspergillus nidulans and Neurospora crassa. These
studies showed a high degree of conservation between divergent organisms and illustrate
conser-ved. basic.principles:::in: the: molecular. -determinants:of life· (reviewed .. by bengeler et a/.,
2000).
1.6.1. Filamentous Growth in Saccharomyces cerevisiae
In response to nitrogen limitation and abundant fermentable carbon source, diploid cells of S.
cerevisiae undergo dimorphic transition to a filamentous growth form referred to as
pseudohyphal differentiation (Gimeno et a/., 1992). Filamentous growth represents a dramatic
change in the normal pattern of cell growth in which the cells become elongated, switch to a
unipolar budding pattern, remain physically attached to each other, and invade the growth
substrate. This alternative growth form may enable this nonmotile species to forage for nutrients
under adverse conditions.
1.6.1.1. Conserved MAP Kinase Cascade for Hyphal Growth in Fungi
At least two conserved signaling pathways regulate yeast filamentous growth. The first cascade
involves components of the MAP kinase pathway that is also required for mating in haploid
yeast cells in response to pheromones (Cook eta/., 1997; Liu eta/., 1993; Madhani eta/., 1997)
(Fig.l.3.). The components ofthis MAP kinase cascade required for filamentous growth include
the Ste20, Stell, Ste7, and Kssl kinases, and the Stel2 transcription factor. In addition, another
transcription factor Tecl, forms a heterodimer with Stel2, that regulates Tecl itself and
additional targets, such as the cell surface flocculin Flo11, required for agar invasion and
filamentation (Gavrias et al., 1996; Lo and Dranginis, 1998; Madhani and Fink, 1997). The
pheromones, pheromone receptors, and subunits of the pheromone-activated heterotrimeric G
protein are dispensable for filamentous growth and are not expressed in diploid cells (Liu et a/.,
1993). Thus, S. cerevisiae is able to use common components and yet couple them into two
different pathways that sense two different environmental signals and give rise to two
completely different developmental fates: mating in haploid cells in response to pheromone, and
filamentous growth in diploid cells in response to nitrogen limitation and other environmental
signals (Fig.l.3.). Signaling specificity is achieved by at least four different specialization in
this signaling pathway (Fig.l.3.). First, the MAP kinase cascade is activated by the py subunits
of the pheromone-activated heterotrimeric G protein during mating of haploid cells. During
18
Mating Pseudohyphal~o~h
Fig.1.3. Signaling Specificity During Mating and Pseudobyphal Growth in Saccharomyces cerevisiae. Atleast four mechanisms are involved in signaling specificity. First, pheromone activates a G protein in haploids, whose fly subunits activate the MAP kinase cascade, and these components are not expressed in diploid yeast cells, and do not regulate filamentous growth. Second, the Ste5 scaffold tethers the components during mating, but does not play a role in filamentous growth. Third, the MAP kinase has diverged and specialized: Fus3 to regulate mating and Kss 1 to regulate filament formation. Finally, Stel2 homodimers or heterodimers with Mcm 1 activate pheromone response element-regulated genes in mating or with Tecl to regulate filamentation response element driven genes during diploid filamentous growth.
filamentous growth, the MAP kinase pathway is activated by a different mechanism involving
the Cdc42, Ras2, and 14-3-3 proteins Bmh1 and Bmh2 (Mosch and Fink, 1997; Mosch et al,
1996·~ Roberts, et:aL; -1997};--,Seeond;-the·eomponents-::of·the.,-MAP-kinaseccascade·.-are tethered
together by the scaffold protein Ste5 during mating, whereas Ste5 is not required during
filamentous growth, and another protein may serve this scaffolding function. It has been
suggested that the Spa2 protein, which physically interacts with many of the MAP kinase
cascade components, might function as the scaffold during filamentous growth (Roemer et a/.,
1998). The third level of specialization is at the level of the MAP kinase itself. In S. cerevisiae,
the Fus3 and Kss1 kinases have diverged, so that Fus3 is specialized to regulate mating and
actually inhibits invasive growth, whereas Kss 1 is specialized to regulate invasive and
filamentous growth (Cook et a/., 1997; Madhani et al., 1997). In addition, Kss1 has both
positive and negative regulatory roles during filamentous growth (Bardwell et a/., 1998a, b;
Madhani and Fink, 1997, 1998a, b), in part by relieving repression of Ste12 by the Dig1 and
Dig2 proteins (Cook eta/., 1996). Finally, during mating Ste12 interacts with the Mcm1 protein
to activate transcription of genes containing pheromone response elements, whereas in diploid
cells Ste12 forms a heterodimer with Tecl that activates transcription of genes with
filamentation response elements (Madhani and Fink, 1997). In this way, one common protein,
Ste12, can yield two different patterns of appropriate transcriptional responses in haploid and
diploid cells.
1.6.1.2. Nutrient-Sensing cAMP Pathway
A second signaling pathway functions in parallel with the MAP kinase pathway to regulate
pseudohyphal differentiation. This second pathway is a nutrient-sensing pathway and involves a
novel G protein-coupled receptor, Gprl, the G proteins Gpa2 and Ras2, adenylyl cyclase, cyclic
AMP (cAMP), and cAMP-dependent protein kinase (Ansari eta/., 1999; Kubler eta/., 1997;
Lorenz and Heitman, 1997; Lorenz eta/., 2000b; Pan and Heitman, 2000; Robertson and Fink,
1998; Thevelein and de Winde, 1999; Xue eta/., 1998) (Fig.l.4.). InS. cerevisiae, three genes
encode the catalytic subunits of cAMP-dependent protein kinase, which play redundant roles in
vegetative growth but specialized roles in filamentous growth. The Tpk2 catalytic subunit
positively regulates filamentous growth by regulating the transcription factor Flo8, which in
tum regulates Flo11 statement (Pan and Heitman, 2000). Flo11 is a glycosyl
phosphatidylinositol (GPI)-linked cell surface protein that is required for pseudohyphal and
haploid invasive growth. In particular, Flo11 plays a role in mother-daughter cell adhesion,
which is required for the integrity of pseudohyphal filaments (Lambrechts et a/., 1996; Lo and
19
Dranginis, 1998). Flo8 was previously shown to be required for pseudohyphal differentiation,
and the common lab strain S288C harbors a naturally occurring jlo8 mutation that prevents
filamentous differentiation (Liu eta/., 1996); Tpk2calso. inhibits.a.transcriptional repressor, Sfl1,
which also regulates Flo11 statement (Robertson and Fink, 1998). The Tpk1 and Tpk3 catalytic
subunits play a negative role in regulating filamentous growth, possibly by a feedback loop that
inhibits cAMP production (Nikawa et al., 1987). Yeast cells express only two heterotrimeric
Ga. protein subunits: Gpa1, which plays a well-established role in mating, and Gpa2, which was
discovered in 1988 by low-stringency hybridization with a mammalian Ga. subunit but whose
physiological function was unknown for many years (Nakafuku et al., 1988). Gpa2 was
subsequently discovered to be required for yeast filamentous growth, but it signals in a pathway
distinct from the MAP kinase cascade (Kubler eta/., 1997; Lorenz and Heitman, 1997). Fil
amentous growth of gpa2 mutant strains is restored by exogenous cAMP, suggesting that Gpa2
regulates a cAMP signaling pathway regulating filamentous growth. In fact, earlier studies had
suggested that Gpa2 might play a role in regulating cAMP production in response to
extracellular glucose (Nakafuku et a/., 1988). The Gprl G protein-coupled receptor was
subsequently identified by a two-hybrid screen with the Ga. protein Gpa2 (Xue et al., 1998; Yun
eta/., 1997). This is one of only a few examples in which integral membrane proteins have been
studied in the two-hybrid system; in this case, the C-terminal soluble tail of Gpr1 was found to
interact with the coupled Ga. protein Gpa2. The Gprl receptor was found to play a role
important for vegetative growth, and gpr 1 mutations are nearly synthetically lethal with ras2
mutations (Xue et al., 1998). Recent evidence suggests that the ligand of the Gprl receptor may
be glucose and other structurally related fermentable sugars. When yeast cells are starved for
glucose, addition of glucose triggers a rapid and transient increase in intracellular cAMP levels
(reviewed by Thevelein and de Winde, 1999). Most interestingly, both the Gprl receptor and
the Ga. protein Gpa2 are required for cAMP production in response to addition of glucose
(Colombo et al., 1998; Kraakman et al., 1999; Lorenz eta/., 2000b; Yun eta/., 1998). The Gpr1
receptor is coupled to the heterotrimeric G protein a. subunit Gpa2 and is also required for
pseudohyphal differentiation and plays a role in nutrient sensing (Ansari et a/., 1999; Lorenz et
a/., 2000b; Pan and Heitman, 1999; Tamaki et a/., 2000). Early studies suggested Gpa2 might
stimulate cAMP production by adenylyl cyclase. Consistent with this, cAMP stimulates
pseudohyphal differentiation and suppresses the filamentation defects of both gprl and gpa2
mutant strains (Kubler et al., 1997; Lorenz and Heitman, 1997; Lorenz et al., 2000b; Pan and
Heitman, 1999). Dominant activated Gpa2 mutations also suppress the pseudohyphal defect of
mutant strains lacking the Gprl receptor, supporting the hypothesis that Gpa2 signals
20
D
-----
l
I
downstream of Gpr 1. Interestingly, several observations suggest that the Gpr 1- Gpa2 receptor
system plays a dual role in sensing both fermentable carbon sources and limiting nitrogen
source. First, statement of the GPRJ receptor gene is dramatically induced by nitrogen
starvation (Xue et a/., 1998). Second, cAMP or dominant active alleles of Gpa2 signal
filamentous growth even when nitrogen levels are increased to levels that repress differentiation
of wild-type strains (Lorenz and Heitman, 1997). Third, Gpa2 has been shown to bind to and
inhibit the meiotic regulatory kinase Ime2 specifically under nitrogen-limiting conditions
(Donzeau and Bandlow, 1999). These findings suggest that the sensitivity of this carbon-sensing
mechanism is enhanced under nitrogen starvation conditions and that Gpa2 may receive input
from other sources that sense nitrogen levels.
Interestingly, a recent report has suggested that the yeast phospholipase C homolog is in a
physical complex with the Gpr 1 receptor, and is required for association of the Ga. subunit
Gpa2 with the receptor, and for pseudohyphal differentiation (Ansari eta/., 1999). Plc1 cleaves
PIP2 to produce inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Flick and Thorner,
1993). Recent studies have revealed two inositol kinases, lpk1 and lpk2, which phosphorylate
IP3 and its products (Saiardi eta/., 1999, 2000~ York eta/., 1999). IP3 is phosphorylated to IP4
and IP5 by the dual-specificity kinase lpk2, which regulates transcription (Odom et a/., 2000),
and the lpkl kinase then phosphorylates IP5 to produce IP6, which regulates mRNA export
from the nucleus (York eta/., 1999). Some studies have suggested that glucose regulates a
pathway involving Ras and Plcl in phosphatidylinositol metabolism and regulation of the
plasma membrane H 1 -ATPase (Brandao eta/., 1994~ Coccetti eta/., 1998~ K.aibuchi eta/.,
1986). Further studies will be required to eludicate the role of Pic 1 in yeast filamentous growth.
In summary, a second signaling pathway comprising the Gprl receptor, the Gpa2 Ga. protein,
and cAMP regulates pseudohyphal growth in parallel to and independently of the MAP kinase
pathway (Fig.l.4.).
The target of cAMP inS. cerevisiae is the cAMP-dependent protein kinase PKA. Yeast and
mammalian PKA are similar, and both enzymes consist of a regulatory and a catalytic subunit.
In yeast cells, the PKA regulatory subunit is encoded by a single gene, BCYJ, and three
catalytic subunits are encoded by the TPKJ, TPK2 and TPK3 genes (Cannon and Tatchell,
1987~ Toda eta/., 1987a, b). In both S. cerevisiae and mammals, PKA in resting cells is an
inactive tetramer composed of two regulatory subunits bound to two catalytic subunits. In
response to external signals that increase intracellular cAMP levels, cAMP binds to the
regulatory subunit and triggers confonnational changes that release the active catalytic subunits.
The yeast cAMP-dependent protein kinase is required for vegetative growth. Triple mutants
lacking the Tpkl, Tpk2, and Tpk3 catalytic subunits are inviable, whereas mutant strains
21
Glucose
cAMP t-j Pdel, zjj sizo j
r-----------, ~
~ I S~71
~
~ I Tp~j;pk2
I ?, Flo8 I
~~ j Unipolar ! ,_1 _F...;._lo_l.;;....l_,•
1 budding 1 c__t~
Cell adhesion Agar invasion
l Cell elon~ation J
Pseudohyphal differentiation
Fig.1.4. Signal Transduction Cascades Regulating Pseudohyphal Differentiation in Saccharomyces cerevisiae. Two parallel signaling cascades regulating filamentous growth are depicted: a nutrient sensing cAMP-PKA pathway, and the MAP kinase cascade. AC, adenylyl cyclase.
expressing any one of the three Tpk subunits are viable. These findings led to the model that the
three PKA catalytic subunits are largely redundant for function. Recent studies reveal that
cAMP..,dependenL protein,-~kinase,,,_plays. a, centraL- role:.dn. ,regulating,- yeast pseudohyphai
differentiation (Pan and Heitman, 1999; Robertson and Fink, 1998). First, mutations of the PKA
regulatory subunit Bcyl dramatically enhance filamentous growth (Pan and Heitman, 1999).
Second, the PKA catalytic subunits play distinct roles in regulating filamentous growth: the
Tpk2 subunit activates filamentous growth, whereas the Tpkl and Tpk3 subunits primarily
inhibit filamentous growth (Pan and Heitman, 1999; Robertson and Fink, 1998). The unique
activating function ofthe Tpk2 subunit is linked to structural differences in the catalytic region
of the kinase and not to differences in gene regulation or the unique amino-terminal region of
the protein (Pan and Heitman, 1999). Genetic epistasis experiments support a model in which
Tpk2 functions downstream of the Gpr 1 receptor and the Ga. protein Gpa2 (Lorenz et a/.,
2000b; Pan and Heitman, 1999). Importantly, activation of PKA by mutation of the Bcyl
regulatory subunit restores pseudohyphal growth in mutants lacking elements of the MAP
kinase pathway, including ste12, tecl, and ste12 tecl mutants (Pan and Heitman, 1999; Rupp et
a/., 1999). Thus, the MAP kinase and PKA pathways independently regulate filamentous
growth. Further analysis reveals that the PKA pathway regulates the switch to unipolar budding
and invasion, whereas the MAP kinase pathway is required for cell elongation and invasion
(Fig.l.4.). Recent studies have defined a role for the PKA pathway in activating pseudohyphal
growth via transcriptional regulation of the cell surface flocculin Floll by the Flo8 transcription
factor (Pan and Heitman, 1999; Rupp et al., 1999). The transcriptional repressor Sfll was also
found to interact with the Tpk2 catalytic subunit in a two-hybrid screen (Robertson and Fink,
1998). Tpk2 appears to enhance Flo 11 statement by inhibiting the repression activity of Sfll.
How PKA regulates either Flo8 or Sfll is not yet understood in molecular detail. The FLOJJ
gene promoter is extremely large, 3,000 bp, and is regulated by a complex set of transcription
factors that includes Stel2ffecl, Flo8, Sfll, Msnl/Mssl0/Phd2, and Mssll (Gagiano et a/.,
1999; Rupp et a/., 1999). Taken together, these findings reveal an intimate role for the cAMP
dependent kinase in the regulation of yeast dimorphism. Two other proteins that appear to
regulate the Gpr1-Gpa2- cAMP signaling pathway under certain physiological conditions have
recently been identified. One is the low-affinity cAMP phosphodiesterase Pdel (Ma et a/.,
1999). Yeast cells express two different cAMP phosphodiesterases: a high-affinity form called
Pde2, and a low-affinity form called Pdel. Interestingly, pdel mutations dramatically enhance
production of cAMP in response to glucose readdition, whereas pde2 mutations have little effect
(Ma eta/., 1999). This is in contrast to pseudohyphal differentiation, where pde2 mutations
enhance filamentous growth (Kubler eta/., 1997; Lorenz and Heitman, 1997; Pan and Heitman,
22
1999). The effects of pdel mutations on filamentous growth are not yet known. The Pdel
enzyme has a single PKA consensus phosphorylation site and is a phosphoprotein in vivo, and
phosphorylation in crude extracts leads to modest increases in enzyme activity (Ma et a/.,
1999). These findings suggest that Pde I could be part of the feedback loop that limits cAMP
excursions in response to glucose signaling. A second novel regulator of the pathway is a
regulator of G protein signaling (RGS) protein homolog, Rgs2, which binds to the Gpa2-GTP
complex and stimulates GTP hydrolysis. rgs2 mutations enhance cAMP production in response
to glucose, whereas overstatement of Rgs2 attenuates this response. In this regard, Rgs2
functions analogously to the RGS protein Sst2, which stimulates GTP hydrolysis of the Gpal
GTP complex to attenuate the pheromone response in haploid yeast cells. A role for Rgs2 in
pseudohyphal differentiation has not yet been established. In summary, studies on pseudohyphal
differentiation have resulted in the elucidation of several novel signaling features of the PKA
pathway. First is the identification of a novel G protein-coupled receptor, Gprl, which may be
the first known G protein-coupled receptor whose ligand is a nutrient. Interestingly, Gprl is
conserved in C. albicans and S. pombe, and the coupled G protein is known to be present in
many different fungi. Second, the Ga protein Gpa2 has sequence identity with other Ga
subunits of heterotrimeric G proteins, and it appears to be coupled to a classic G protein
coupled receptor. However, no associated J3y subunits have been identified. A number of
candidate J3 and y subunits have been mutated, but none conferred phenotypes related to
pseudohyphal growth (Lorenz and Heitman, 1997). We propose that Gpa2 may function either
as a solo Ga subunit or in complex with other proteins such as Plcl and Ras2. In either case,
this represents a new paradigm for signaling from a G protein-coupled receptor via a very
unusual type of G protein. Finally, a very interesting finding is the specialized role of the PKA
catalytic subunits in regulating filamentous growth. The three catalytic subunits of PKA were
thought to be functionally redundant because the essential vegetative function can be satisfied
by statement of any one of the three. In contrast, Tpk2 plays a specialized role to activate
filamentous growth, whereas Tpk1 and Tpk3 play an inhibitory role. The positive function of
Tpk2 involves regulation of the Flo8 and Sfll transcription factors. The negative role of Tpk 1
and Tpk3 may involve the known role of PKA in a feedback loop that inhibits cAMP
production (Nikawa et a/., 1987). Studies to localize the catalytic and regulatory subunits of
PKA under different nutritional conditions have just begun (Griffioen et a/., 2000) and may
provide insights into the unique and specialized roles ofTpk2 compared to Tpk1 and Tpk3.
1.6.1.3. Cross Talk between MAP Kinase and cAMP Pathways
Recent studies have revealed several examples--of cross talk between the· MAP kinase and·
cAMP signaling pathways in the regulation of filamentous growth. First, the small G protein
Ras2 plays a dual signaling role, activating both the MAP kinase signaling pathway and
adenylyl cyclase (Lorenz and Heitman, 1997; Masch and Fink, 1997; Masch et al, 1996, 1999).
Second, both the MAP kinase and the PKA signaling pathways converge to regulate the large,
complex promoter of the FLO 11 gene (Pan and Heitman, 1999; Rupp et a/., 1999), which
encodes a cell surface protein required for pseudohyphal growth (Lambrechts eta/., 1996; Lo
and Dranginis, 1998). Third, cAMP inhibits the statement of MAP kinase-regulated reporter
genes (Lorenz and Heitman, 1997), and high levels of cAMP or PKA activity enhance
filamentous growth but give rise to filaments comprised of round rather than elongated cells
(Pan and Heitman, 1999). Because the MAP kinase pathway regulates cell elongation, these
observations suggest that the PKA pathway may antagonize signaling by the MAP kinase
pathway at some level. Recent findings have suggested that during haploid invasive growth,
PKA may also positively regulate signaling by the MAP kinase cascade at a level downstream
of the Ste20 kinase (Masch et al., 1999). The Stel2 transcription factor has several consensus
PKA phosphorylation sites, and overstatement of Ste 12 can in some cases suppress
pseudohyphal defects, whereas the constitutive active Stell-4 mutant does not (Lorenz and
Heitman, 1998), suggesting that Stel2 could represent a target of the PKA pathway that is both
positively and negatively regulated, depending on cell type.
1.6.1.4. Haploid Invasive Growth
Pseudohyphal growth occurs in diploid cells in response to the presence of an abundant
fermentable carbon source and limiting nitrogen source. A related morphological process,
termed haploid invasive growth, has also been described, which shares some features with
diploid pseudohyphal growth (Roberts and Fink, 1994). During diploid filamentous growth,
some cells invade the agar to produce chains of cells that cannot be removed by vigorous
washing. Although haploid strains do not undergo pseudohyphal differentiation under standard
conditions~ haploid strains derived from the Sl278b background can invade the agar when
grown on rich medium for an extended period oftime. Many of the same signaling components
that regulate diploid filamentous growth are also required for haploid invasive growth,
including several components ofthe MAP kinase pathway (Ste20, Stell, Ste7, Stel2, and Tecl)
(Roberts and Fink, 1994). The two related MAP kinases play opposing roles, andfos3 mutants
24
are hyperinvasive whereas kssl mutants have a defect in agar invasion. Components of the PKA
pathway also regulate filamentous growth, including Gpr 1, Gpa2,. Ras2, Tpk2, Flo8, and Flo 11
(Lo and Dranginis, 1998; Lorenz et a/., 2000b; Mosch et a/., 1999;-Pan and Heitman, 1999).
Taken together, these findings suggest that haploid invasive growth shares many features with
diploid filamentous growth. In contrast to diploid filamentous growth, which occurs on
nitrogen-limiting medium, haploid invasive growth normally occurs on rich growth media,
including YEPD (yeast extract-peptone-dextrose) medium. It has been suggested that nutritional
limitation might occur beneath colonies and stimulate haploid invasive growth, even on rich
medium. When yeast cells begin to exhaust nutrients in rich medium, cellular proteins and
amino acids are catabolized to produce nitrogen, resulting in the production of short-chain
alcohols derived from amino acids, which are called fuse! oils. Recent studies reveal that several
short-chain alcohols, including isoamyl alcohol and butanol, dramatically stimulate
pseudohyphal differentiation of haploid yeast strains (Dickinson, 1996; Lorenz eta/., 2000a).
How these alcohols are sensed is not yet known, but these or other metabolic products could
regulate differentiation under certain culture conditions. Other recent findings reveal that mating
pheromones regulate invasive and filamentous growth of haploidS. cerevisiae strains.
In the wild, most strains of S cerevisiae are diploid, and the haploid state of the life cycle
essentially represents gametes that are short-lived in nature. An elaborate pattern of axial
budding has evolved in haploid yeast cells and is thought to promote more rapid mating and
diploidization following meiosis. Thus, the main activity of haploid yeast strains would appear
to be locating a mating partner. Recent studies using genome arrays reveal that pheromone
induces several genes that are known to be induced during filamentous growth, including a
hydrolytic enzyme encoded by the PGUJ gene (Madhani et a/., 1999; Roberts et a/., 2000).
Remarkably, low concentrations of mating pheromones were found to increase agar-invasive
growth (Roberts eta/., 2000). These observations suggest that haploid invasive growth may be a
mechanism by which yeast cells can locate mating partners at a distance, and yeast cells are
known to be responsive to gradients of mating pheromone. During invasive growth, the haploid
cells become elongated, switch from an axial pattern of budding to bipolar and unipolar
budding, and invade the agar (Roberts eta/., 2000; Roberts and Fink, 1994). Thus, while the
role of filamentous growth in diploids may be to forage for nutrients, the role in haploid cells
may be to forage for mating partners. Haploid invasive growth can occur to some extent in the
absence of a mating partner. This may be attributable to a basal level of signaling in the absence
of ligand. Alternatively, low concentrations of pheromone may result from cells that have
switched mating type in the culture or in which repression of the silent mating type cassettes is
inefficient, as is known to be the case in older yeast cells. The finding that the pheromone
. 25
receptors and coupled G protein are not required for standard haploid invasive growth indicates
that pheromones may not normally be involved. Pheromone-induced invasive growth requires
Stef;2 buhs -independent~o£,..'Fedco(Reberts·-et-a/-:;-2000)i-·whereas"'haploidoinvasive-growth:in-the ·
absence of pheromone requires both Stel2 and Tecl (Roberts and Fink, 1994). Interestingly,
filamentous differentiation of haploid S. cerevisiae cells that occurs in response to mating
pheromones is analogous to the recent discovery that filamentation and sporulation (haploid
fruiting) ofMATa. cells ofthe human fungal pathogen C. neoformans are dramatically induced
by factors secreted by MATa mating partner cells (Wang eta/., 2000). Taken together, these
studies illustrate that mating and filamentous growth are linked, which is perhaps most apparent
in the basidiomycetes in which mating results in a filamentous dikaryon. These observations on
the activation of filamentous growth by pheromone may be related to the previous finding that
filamentation reporter genes are inappropriately activated by basal signaling of the pheromone
response pathway infos3 mutant haploid cells (Madhani and Fink, 1998). This example of cross
talk requires the pheromone-activated GP subunit Ste4, occurs via misactivation of Kss 1 on the
Ste5 scaffold, and can be further enhanced by mating pheromone. Another example of cross talk
between the MAP kinase and cAMP signaling pathways occurs in haploid cells responding to
pheromone. Normally, when glucose is readded to yeast cells that have been starved for
glucose, cAMP levels increase (reviewed by Thevelein and de Winde, 1999). In contrast, in
haploid cells that have first been exposed to mating pheromone, readdition of glucose fails to
stimulate cAMP production (Arkinstall eta/., 1991). The pheromone receptor Ste2 and the P
subunit of the heterotrimeric G protein (Ste4) are required for inhibition of cAMP production by
pheromone, but further-downstream components of the pheromone-signaling pathway are not. It
shows that a constitutively activated Ras2 mutant (Val-19) restored cAMP production in the
presence of pheromone, and pheromone failed to inhibit the modest increase in cAMP in
response to glucose that still occurs in a gpa2 mutant strain (Papasavvas et a/., 1992). Taken
together, these findings support a model in which pheromone-stimulated release of the fly
subunit of the heterotrimeric G protein inhibits activation of adenylyl cyclase by the Ga. subunit
Gpa2. Although Gpa2 does not appear to have its own dedicated py subunit, this cross talk
between the pheromone-regulated G protein and Gpa2 could serve to inhibit signaling via the
PKA pathway under certain conditions, such as high levels of pheromone, to promote mating
and inhibit filamentous growth. This pathway can only operate in haploid cells, and not during
diploid filamentous growth, because the pheromones, pheromone receptors, and J3y subunits are
only expressed in haploid cells.
26
1.6.1.5. Other Signaling Pathways
Other proteins that-regulate yeasHilamentous growth' have' been' identified that do not appear to
be components of either the MAP kinase or the cAMP signaling pathway. These include the
ammonium transporter Mep2, which is required for filamentous differentiation in response to
ammonium-limiting conditions (Lorenz and Heitman, 1998a,b), and the transcription factors
Phd1, Sok2, and Ash1 (Chandarlapaty and Errede, 1998; Gimeno and Fink, 1994; Pan and
Heitman, 2000; Ward et a/., 1995). How limiting nitrogen sources are sensed during
pseudohyphal differentiation is not understood in detail. Mep 2 ammonium permease is required
for filamentous differentiation and plays a unique role not shared with the related Mep 1 and
Mep3 ammonium permeases (Lorenz and Heitman, 1998a, b). The mutant mep2 failed to
differentiate, but have no growth defect on medium limiting for ammonium, leading to the
hypothesis that the Mep2 protein may function to both transport and sense ammonium ions
(Lorenz and Heitman, 1998b). The Gln3 transcriptional activator and the Gln3 repressor Ure2
that regulate the nitrogen catabolite response (NCR) are also required for pseudohyphal growth.
Both g/n3 mutants unable to induce the NCR response and ure2 mutants in which the NCR
response is constitutive fail to differentiate, suggesting that the ability both to induce and to
repress the NCR genes involved in nitrogen source utilization is required for filamentous·
differentiation. The GATA family transcription factor Ashl represses HO endonuclease
statement in haploid daughter cells, restricting mating type switching to haploid mother cells.
Ash1 is also required for pseudohyphal differentiation and is localized to daughter cell nuclei in
filament cells (Chandarlapaty and Errede, 1998), where it may function to regulate Floll
statement (Pan and Heitman, 2000). The transcription factor Sok2 was recently found to
regulate a transcription factor cascade involving Phdl, Swi5, and Ashl, which in turn regulates
statement of proteins and enzymes (Floll, Egt2, and Ctsl) involved in mother-daughter cell
adhesion and separation (Pan and Heitman, 2000). Several additional proteins also appear to
inhibit filamentous growth, including the Elm 1 protein kinase homolog and the B subunit of
protein phosphatase 2A (Cdc55) (Blacketer et a/., 1993). Recent studies suggest that these
proteins regulate Cdc28 via the Hsll-Hsl7-Swe1 regulatory cascade and that the Cdc28-cyclin
complexes regulate filamentous growth (Edgington et a/., 1999). In addition, several recent
studies indicate that both the Gl cyclins Clnl, Cln2, and Cln3 and the Clb2 mitotic cyclin may
play roles in filamentous growth (Ahn et a/., 1999; Loeb et a/., 1999a, b). clnl and cln2
mutations inhibit filamentous growth, whereas cln3 mutations enhance filamentation. Mutations
in the F-box component ofthe Skpl-Cdc53-F box protein (SCF) ubiquitin ligase complex, Grr1,
dramatically enhance the stability of Clnl and Cln2 and enhance filamentous growth. Epistasis
27
Budding Yeast
Pseudohyphae
Germ Tubes
Fig.1.5. Cell Morphology of Candida albicans.
Fig.1.6. Different Colony Morphologies of Candida albicans.
analysis suggests that Cdc28-Clnl/2 may act at an early step in the MAP kinase cascade (Loeb
eta/., 1999a). In addition, the Cdc28-Clnl /2 complex can phosphorylate Ste20, and c/nl and
c/n2 mutations are synthetically lethal with mutations in the CLA4 gene, which encodes a Ste20
homolog (Oehlen and Cross, 1998; Wu eta/. , 1998). These findings imply that the MAP kinase
cascade may be regulated at the level of Ste20 by Cdc28 in complex with G 1 cyclins . Further
studies will be required to understand these additional regulatory elements in molecular detail.
1.6.2. Signal Transduction in Candida albicans
C. a/bicans is the most common human fungal pathogen and causes a wide range of superficial
mucosal diseases as well as life-threatening systemic infections in immunocompromised
patients (Odds, 1988). This fungus is dimorphic, and can grow as a budding yeast (blastospores)
or switch to a filamentous form forming hyphae or pseudohyphae (Figs .l.5. and 1.6.), under a
variety of environmental conditions (Odds, 1988). The ability to undergo these reversible
dimorphic switches is essential for the virulence of this pathogen (Lo et a/., 1997). C. a/bicans
is a diploid organism for which the sexual cycle has only very recently been discovered (Hull et
a/. , 2000; Magee and Magee, 2000) . The characterization of a filamentous state in the baker' s
yeast S. cerevisiae has made it possible to use this as a model to compare regulation of
filamentation in C. a/bicans and S. cerevisiae (Gimeno eta/., 1992).
1.6.2.1. MAP Kinase Signaling Pathway
Multiple pathways have been shown to regulate the morphogenic transition between budding
and filamentous growth in C. albicans (Fig.l.7 .). A transcription factor, Acpr/Cphl , which is
homologous to the Ste12 transcription factor that regulates mating and pseudohyphal growth in
S. cerevisiae, has been identified (Malathi eta/., 1994; Liu eta/., 1994). InS. cerevisiae, Ste12
is regulated by a conserved MAP kinase cascade, and a related MAP kinase cascade has been
identified in C. a/bicans. The cascade consists of the kinases Cst20 (homologous to the p21-
activated kinase [PAK] kinase Ste20), CaSte7 /Hst7 (homologous to the MAP kinase kinase
Ste7), and Cekl (homologous to the Fus3 and Kss1 MAP kinases) (Clark eta/. , 1995; Kohler
and Fink, 1996; Leberer eta/. , 1996; Singh eta/., 1997; Whiteway eta/., 1992). Null mutations
in any ofthe genes in the MAP kinase cascade (Cst20, Hst7, or Cek1) or the transcription factor
Cph 1 confer a hypha! defect on solid medium in response to many inducing conditions;
however, all of these mutants filament normally in response to serum (Csank et a/. , 1998;
28
...
e~ ~ 0 cAMP
~ 1 Tpk2
l l Acpr
Filamentation and Virulence
Fig.1.7. Signaling Cascades Regulating Virulence of Candida albicans. Two parallel signaling cascades involving a MAP kinase and a cAMP-PKA signaling pathway regulate filamentation and virulence of this human fungal pathogen.
Kohler and Fink, 1996; Leberer et al., 1996). Interestingly, although a cekl MAP kinase mutant
strain forms morphologically normal filaments in response to serum, it has a minor growth
defect on serum-containing~medium (Csank eta/., 1998). The cekl mutant strain also has a
virulence defect that may be attributable to this growth defect (Csank eta/., 1998, Guhad et al.,
1998b). This indicates that the Cek1 MAP kinase may function in more than one pathway or
that deletion of the gene causes aberrant cross talk between distinct MAP kinase cascades,
similar to the altered signaling that occurs in afos3 mutant of S. cerevisiae. The other elements
of the pathway have small but varied effects on virulence. cst20 mutant strains have a modest
virulence defect in a mouse model of systemic candidiasis (Leberer eta/., 1996). However, hst7
and cphl mutant strains are able to cause lethal infection in mice at rates comparable to wild
type strains (Leberer et al., 1996; Lo et al., 1997). In addition to these components, a MAP
kinase phosphatase, Cppl, has been identified which regulates filamentous growth in C.
albicans (Csank et a/., 1997). Disruption of both alleles of the CP P 1 gene derepresses hyphal
production and results in a hyperfilamentous phenotype. This hyperfilamentation is suppressed
by deletion of the MAP kinase Cek1 (Csank eta/., 1997). cppl mutant strains are also reduced
for virulence in both systemic and localized models of candidiasis (Csank eta/., 1997; Guhad et
a/., 1998a).
1.6.2.2. Efgl Transcription Factor
In addition to the transcription factor Acpr/Cph 1, and the MAP kinase module that regulates it,
a second transcription factor Efg1, that regulates filamentous growth in C. albicans has been
identified (Fig.l.7.) (Stoldt eta/., 1997). Efg1 is a conserved basic helix-looP,_-helix type protein
that is homologous to the Phd1 transcription factor of S. cerevisiae. Similar to Phd1,
heterologous overexpression of Efg 1 can induce pseudohyphal growth in S. cerevisiae (Stoldt et
al., 1997). Overstatement of Efg1 can also induce filamentous growth in C. albicans (Stoldt et
al., 1997). An efgl mutant strain has a moderate but not complete defect in hyphal growth in
response to many environmental conditions (Lo eta/., 1997). efgl mutant strains also show an
aberrant morphology in the presence of serum, forming exclusively pseudohyphae instead of the
true hyphae that are produced by wild-type strains (Lo et al., 1997). An efgl cphl double
mutant strain has a much more severe defect in filamentous growth and does not filament under
almost any conditions tested, including the presence of serum (Lo et a/., 1997). In addition,
while the efgl mutant strain has a minor reduction in virulence and the cphl mutant has little or
no defect, an efgl cphl double mutant strain is essentially avirulent (Lo et al., 1997). Thus,
29
Cph1 and Efg1 define elements of two separate pathways that together are essential for both
filamentation and virulence in C. albicans.
1.6.2.3. cAMP-PKA Pathway
A PKA-dependent pathway has been shown to regulate filamentous growth of C. albicans under
some conditions (Fig.l.7.). An increase in cAMP levels accompanies the yeast to hyphal
transition, and inhibition of the cAMP phosphodiesterase induces this transition (Sabie and
Gadd, 1992). Moreover, two cell-permeating PKA inhibitors, myristoylated protein kinase
inhibitor (myrPKI) amide and the small-molecule PKA inhibitor H-89, both block hyphal
growth induced by N-acetylglucosamine, but not in response to serum (Castilla et a/., 1998).
More recently, a PKA gene, TPK2, was identified and shown to be required for hyphal
differentiation in response to a number of conditions, including the presence of serum
(Sonneborn et a/., 2000). Hyphal formation in the tpk2 mutant strain was not completely
eliminated, however, and the defect was less apparent during growth at 37°C (Sonneborn eta/.,
2000). Overstatement of the Tpk2 catalytic subunit also induced hyphal growth under normally
non-inducing conditions both in liquid culture and on solid medium (Sonneborn et a/., 2000).
This is consistent with a model in which PKA acts positively in one of two or more pathways
regulating dimorphism. The phenotypes of a tpk2 mutant strain were suppressed by
overstatement of either the MAP kinase Cek 1 or the transcription factor Efg 1 (Sonneborn et a/.,
2000). On the other hand, overstatement of Tpk2 could suppress the hyphal defect of a cekl
mutant strain but not that of an efgl mutant strain (Sonneborn eta/., 2000). Efg1 has a single
PKA consensus phosphorylation site, which may be required for its transcriptional activation
activity (Sonneborn eta/., 2000). These findings suggest that the Efg1 transcription factor may
lie downstream of cAMP and the PKA homolog Tpk2, in one oftwo major pathways regulating
hyphal morphogenesis in C. albicans. Interestingly, while Efg1 plays a prominent role in
regulating filamentation in C. albicans, mutation of the related protein Phd1 confers only a
minor defect inS. cerevisiae filamentation (Gimeno and Fink, 1994; Lo eta/., 1997; Ward et
a/., 1995).
1.6.2.4. Function of Ras
In S. cerevisiae, the Ras2 protein regulates pseudohyphal growth and functions upstream of
both the MAP kinase and cAMP-PKA pathways and, in conjunction with Rasl, is essential for
viability. A single Ras homolog, Rasl, has been identified in C. albicans, which is not essential
30
for survival (Feng eta/., 1999). The rasl mutant strains have a severe defect in hyphal growth
in response to serum and other conditions (Feng eta/., 1999). In addition, while a dominant
negative,Rasl~mutation {RaslAl6)~caused, a~defect~in. filamentation~ ac dominant- active: Ras.I.
mutation (Ras1Vl3) enhanced the formation of hyphae (Feng eta/., 1999). The rasl mutant
strains exhibit a filamentation defect similar to that of efgl cphl mutant strains, and evidence
from S. cerevisiae indicates that Ras2 lies upstream of both the cAMP-PKA and MAP kinase
pathways regulating pseudohyphal growth (Lorenz and Heitman, 1997~ Masch and Fink, 1997~
Pan and Heitman, 1999~ Roberts et a/., 1997). These fmdings are consistent with a model in
which Ras I lies upstream of the related pathways in C. albicans and regulates hyphal
morphogenesis. However, these studies indicate that additional pathways may also control
filamentation in C. albicans, because even the rasl and efgl cphl mutant strains are able to
form hyphae under some conditions (Feng et al., 1999~ Riggle eta/., 1999).
1.6.2.5. Repressing Environments and Factors
Besides low pH, low temperature, and high cell density, it is known that high glucose
concentrations downregulate hypha! development of C. albicans in liquid media. Glucose
repression of morphogenesis is also observed initially during growth on solid media, although
glucose consumption permits filamentation after several days of growth. On solid media, high
osmolarity also inhibits hypha formation (Alex et a/., 1998). The presence of easily utilizable
nitrogen sources such as ammonium salts, modulates hypha! development negatively to some
degree (Ernst, 2000). In the infected host, inhibition of filamentation by y-interferon occurs at
contact of C. albicans with lymphocytes (Kalo-Klein and Witkin, 1990~ Levitz and North,
1996). Some chemical inhibitors of filamentation are known including diaminobutanone and
some antifungals at low concentrations, such as amphotericin and azole antifungals, these
compounds inhibit hypha! development by as yet unknown mechanisms (Hawser eta/., 1996~
Martinez eta/., 1990). The Tupl transcription factor may be involved in constituting the hypha
repressed state in the presence of glucose and other non-inducing conditions. In S. cerevisiae,
the Tup 1 protein regulates about 60 genes involved in glucose regulation, oxygen stress
response and DNA damage. A C. a/bicans homologue of Tuplp was identified that is 67%
identical to S. cerevisiae Tuplp (Braun and Johnson, 1997). Tuplp contains seven conserved
WD40 repeats at the C terminus, which could anchor Tuplp to some of its DNA-binding
proteins, and an N-terminal domain that could interact with a homologue of Ssn6p, as in S.
cerevisiae (Keleher et al., 1992~ Komachi and Johnson, 1997). A homozygous C. albicans tupl
31
mutant grew m filamentous form in all media tested~ filaments on most media had the
characteristics of pseudohyphae, but in some media had the appearance of true hyphae.
Pseudohyphae:of a-tupl-. mutant,. unlike pseudohwhae .. produced .. by EEGL.overexpression
(Stoldt et a/., 1997) could not be induced to form germ tubes or true hyphae by the addition of
serum (Braun and Johnson, 1997). Tuplp had activities besides repression of filamentation,
because tupl mutants failed to grow at 42°C, grew faster on glycerol and had misshapen cell
walls compared to the wild-type. In epistasis experiments most, but not all of the filamentation
phenotype induced by the tupl mutation, was abolished by the presence of an efgl mutation,
while a cphl mutation had very little effect. A comparison of a tupl ejgl mutant with a tupl
efgl cphl mutant showed a slight influence of the cphl mutation on hyphal morphogenesis
(Braun and Johnson, 2000). An analysis of transcript levels of hypha-specific genes including
HYRJ, ALSJ, HWPJ and ECEJ showed no differences between tupl and tupl cphl mutants,
only the HWP 1 transcript was lowered slightly in the tupl efgl cphl mutant compared to the
tupl efgl mutant. These results indicate that Efglp is the main, and Cphlp a minor contributor
to the tupl hyphal phenotype. Genes repressed by Tuplp have been identified recently, of which
some are expressed in a filament-specific manner.
In S. cerevisiae, the Rap 1 protein acts as both a transcriptional silencer and a structural protein
at telomeres by binding to a sequence designated the RPG box (Drazinic et a/., 1996). A C.
albicans protein was identified, Rbflp, which is not homologous to Raplp, but binds to the
RPG box of S cerevisiae (Ishii et a/., 1997). Rbflp contains two glutamine-rich regions
embedding a region with weak similarity to bHLH domains, which binds to RPG sequences.
Homozygous rbfl null mutants grew in filamentous form in all media tested; the filaments
formed had the characteristics ofpseudohyphae rather than true hyphae (Ishii eta/., 1997~ N.
Ishii, M. Watanabe andY. Aoki, unpublished). Thus, Rbflp seems to be involved exclusively in
pseudohyphal, but not truehyphal growth. In fact, Rbflp so far seems to be the only component
in C. albicans that exclusively determines pseudohyphal growth. On an interesting aside, the
. authors reported that three alleles of RBFJ were present in the standard disruption strain CAI4.
Aneuploidy or triploidy had been previously demonstrated in other C. a/bicans strains, such as
strain SGY-243 (Gow eta/., 1994; Delbriick et a/., 1997), but not in strain CAI4. Besides
derepression of filamentous growth, the rbfl knockout strain showed significantly slower
growth and increased sensitivities to high temperature, high osmolarity and hydrogen peroxide
compared to the wild-type strain. Virulence of the rbfl mutant in the mouse model of systemic
infection was significantly attenuated (N. Ishii, M. Watanabe and Y. Aoki, unpublished).
Recently, by screening for sequences that mediate Rbflp-dependent transcriptional regulation,
32
target genes were identified in the heterologous hostS. cerevisiae. Among the genes identified
as Rbflp targets was the WH1 1 gene, which in phenotypic switching between a white and an
opaque.phenoty.pe is. specifically expressedin the white phase-(Soll, 1997); theJevel of WH1 1
transcripts is reduced in homozygous rbf1 mutants compared to wild-type cells (N. Ishii, M.
Watanabe, S. Fukuchi, M. Arisawa and Y. Aoki, unpublished). The Sir2 protein represses
hyphal formation, which is consistent with the role of Sir2p as a repressor in S. cerevisiae
(although it is unrelated to pseudohyphal growth in this species) (Perez-Martin et a/., 1999).
Another repressing factor is the Rad6 protein, which besides contributing to UV protection,
represses hyphal growth under inducing conditions by an unknown pathway; its deficiency
under non-inducing conditions generates a pseudohyphal phenotype (Leng eta/., 2000).
1.6.2.6. Downstream Effectors of Efgl
Although there seems to be a clear link between dimorphism and virulence in C. albicans, it is
difficult to unambiguously establish this because it is possible that the signaling elements also
regulate other cellular processes. One of the ways to dissect the relative contributions of these
various components to the virulence of the organism is to identify the downstream effectors of
these signaling molecules. One of the genes recently shown to be regulated by Efg1 is HWP 1,
which encodes a protein of unknown function that is homologous to GPI-anchored proteins
(Sharkey eta/., 1999). An hwp1 mutant strain is reduced for hyphal growth under a variety of
different environmental condition (Sharkey eta/., 1999) and statement ofHwp1 is reduced in an
efg1 mutant and increased in a tupl mutant (Sharkey et a/., 1999). Thus, Efg1 and Tup1
regulate at least some of the same target genes and may be acting coordinately to modulate
dimorphism in C. a/bicans. Ece1, which is expressed during cell elongation, is also not
expressed in an efgl mutant (Sharkey eta/., 1999). However, Ece1 is not regulated by Tup1,
and an ecel!ecel mutation does not affect filamentous growth despite being regulated by Efg1
(Sharkey et a/., 1999). Efg1 has also been shown to regulate other morphogenic events,
including chlamydospore production and phenotypic switching (Sonneborn et a/., 1999a, b).
Because Efg1 is a complex signaling molecule that regulates a variety of morphogenic
processes, ·it cannot yet be determined to what degree each of these functions of Efg 1
contributes to virulence. Recent studies of single and multiple mutant strains lacking Tup 1,
Cph1, and Efgl reveal that each makes a unique contribution to the regulation of genes involved
in filamentous growth, suggesting that multiple pathways rather than a single central regulatory
pathway are responsible (Braun and Johnson, 2000). Further analysis of the downstream
effectors of each of these transcription factors will be required to determine the relative
33
contribution of the processes controlled by these proteins to the overall virulence of the
organism.
1.6.2. 7. Embedded/Microaerophilic Conditions
Although homozygous efgl mutants have a drastic block in true hyphal formation under most
standard induction conditions, considerable filamentation occurs in certain other environments
(Brown eta/., 1999; Sonneborn eta/., 1999a). A limited supply of oxygen, as occurs under a
coverslip during induction of chlamydospores, allows wild-type cells to form filaments, which
is enhanced in efgl mutants (Sonneborn eta/., 1999a). Similarly, growth of wild-type colonies
embedded in agar stimulates filamentation, which still occurs in homozygous efgl cphl mutants
(Riggle eta/., 1999; A. Giusani and C. Kumamoto, unpublished). Thus, there appears to exist an
Efg1p-independent pathway of filamentation in C. albicans, which is operative under
microaerophilic/embedded conditions. The filaments produced under microaerophilic conditions
have the characteristics of mostly pseudohyphae in EFGJ wild-type strains and of mostly true
hyphae in efgl mutants (Sonneborn et a/., 1999a), under embedded conditions mostly true
hyphae were produced (Brown eta/., 1999). Interestingly, the alternative filamentation pathway
not only is independent of Efglp, but it is even repressed by it to some degree. The enhanced
filamentation in efgl mutants does not depend on the Cph1 MAP kinase, because a homozygous
efgl cphl strain is as hyperfilamentous as the homozygous efgl mutant (Sonneborn et a/.,
1999a). It is possible that agar embedding generates microaerophilic conditions, which activate
the same Efg1p-independent pathway of morphogenesis under both conditions. The putative
transcription factor Czfl is probably an important element of the alternative pathway of
filamentation in C. a/bicans (Brown eta/., 1999). The central portion of Czflp contains four
clusters of glutamine residues and the C terminus contains a cysteine-rich region similar to zinc
finger elements. There is no direct homologue of Czflp in the genome of S. cerevisiae.
Overexpression of CZFJ stimulates filamentous growth, but only under embedded conditions
and in certain media lacking glucose. Homozygous czfl null mutants filament normally under
standard induction conditions, but they are defective in hyphal development when embedded in
agar. This defective phenotype occurs only during embedding in certain media, such as complex
medium containing sucrose or galactose as carbon sources at 25°C, but not at 37°C, or in media
containing strong inducers including serum and GlcNAc. These characteristics suggest that
factors other than Czflp contribute to filamentation under embedded conditions. The defective
phenotype of a czfl mutant is exacerbated by the presence of a cphl mutation, which by itself
34
shows defects in the types of media used for monitoring the czfl phenotype. Thus, although the
cphl mutant phenotype does not appear to be specific for embedded conditions, it worsens
filamentatien~defects:-caused""by-the:,czfl ,mutation, Hyperlilamentation~,of~efg L sing.le.and-efgl
cphl double mutants suggests that Efg1p is a negative modulator of the Czflp pathway under
microaerophilic/embedded conditions. Thus, these data once again suggest that positive and
negative functions are combined in the Efg1 protein. Conceivably, Efg1p and Czflp collaborate
to allow filamentation in different host environments, as in the blood (serum) and during the
passage of tissues or within host cells, at low oxygen partial pressure. It is also possible that
Efg 1 p and Czfl p trigger the formation of different types of hyphae, each of which are equipped
with different sets of proteins required for viability and virulence in specific host niches, such as
the blood and at limiting oxygen concentrations within cells or within organs.
1.6.2.8. pH
It is well established that a pH around neutrality (pH -6·5) favours hypha! development of C.
albicans in vitro, while a low pH (pH -6·5) blocks hypha! formation and stimulates growth of
the yeast form (Buffo eta/., 1984). The Prr2 transcription factor, which has homologues in other
fungi, has a central role in pH-dependent regulation by inducing the expression of alkaline
expressed genes and repressing acid-expressed genes at alkaline pH (Ramon eta/., 1999). prr2
mutants are unable to induce the alkaline-specific gene PHRJ and to repress the acid-specific
gene PHR2, which both encode cell-surface proteins required for growth and cell polarization
(Fonzi, 1999). Although prr2 mutants were able to form hyphae in liquid serum media, they
were defective on a solid medium containing serum. Under still weaker induction conditions,
such as in some liquid media and on solid Spider medium, the prr 2 mutant was clearly defective
in hypha! growth. Thus; Prr2p is needed for the full morphogenetic potential of C. albicans,
while PRR2 overexpression allows filamentation under non-inducing conditions (A. El Barkani
and F. A. Muhlschlegel, unpublished). Prr2p contains a zinc-finger domain that is conserved in
the orthologous protein PacC of Aspergillus nidulans and which is known to recognize the 5'
GCCAAG-3' sequence. Although such sequences occur in the promoter region of the alkaline
induced PHRJ gene they are not required for transcriptional activation of PHRJ (Ramon et a/.,
1999). It is not yet clear if repression of acid-expressed genes is directly or indirectly regulated
by Prr2p. Because the expression of PRR2 depends on the Prrl protein it was not surprising that
prrl mutants had similar phenotypes to prr2 mutants (Porta eta/., 1999). Interestingly, forced
expression of one downstream target of the Prrl p/Prr2p pathway, PHRJ, only partially
reconstituted the morphogenetic defect of both prr mutants (Porta et a/., 1999). This result
35
suggests that components other than Phrl p are involved in morphogenetic control by the Prr2
transcription factor.
1.6.2.9. Kinases in Search of Transcriptional Pathways
A number of genes encoding conceptual protein kinases of C. albicans have been identified
whose disruption generates a morphogenesis-defective phenotype, but whose input and output
pathways are unknown. The SLNJ, COSJ and HKJ genes encode possible two-component
histidine kinases containing sensor and regulator domains. In vitro autophosphorylation activity
has been shown for the Slnlp and Coslp proteins (Yamada-Okabeet al., 1999). InS. cerevisiae,
activation of Slnlp occurs at normal osmolarity and leads to phosphorylation (and thereby
inactivation) of the Sskl regulator. Although the C. albicans Slnl and Sskl proteins are the
direct homologues of S. cerevisiae Slnl and Ssk1 proteins, they are not essential in sensing
hyperosmolarity. However, hyphal development of slnl and sskl null mutants is blocked on
starvation-type media and is severely impaired on serum agar, while filamentation is normal in
all liquid media (Nagahashi et al., 1998; Yamada-Okabe et al., 1999; Calera et al., 2000).
Interestingly, while growth of the sskl mutant on SLAD medium did not allow formation of
hyphae, invasive growth was stimulated significantly compared to the wild-type strain (Calera
eta/., 2000). A similar phenotype, i.e. a filamentation defect and hyperinvasive growth on solid
media, was observed for C. albicans hogl mutants, which lack a homologue of the S. cerevisiae
Hog1p MAP kinase, which in this species is a downstream target of Ssklp (Alonso-Monge et
a/., 1999). Although till date it has not been resolved ifthe Slnlp, Ssklp and Hoglp proteins are
in a common pathway, and it can be speculated that Hog1p downregulates the MAP kinase
pathway responsible for filamentation upstream of Cst20p (Ste20p), as occurs inS. cerevisiae
(O'Rourke and Herskowitz, 1998). Thus, the hyperinvasive characteristics of sskl and hogl
mutants is possibly related to activation ofthe Cph1p transcription factor. The COSJ (NIKJ) and
HKJ gene products, which lack transmembrane regions, have no direct homologues in S.
cerevisiae. The Cos1 protein is a homologue of the Neurospora crassa Nik1 histidine kinase,
which in this fungus is involved in hyphal growth and protects against osmotic stress. Null
mutants lacking COSJ or HKJ alleles have no defect in osmoprotection, but they are
significantly defective in hyphal formation cin solid media (starvation-type or medium
containing serum), but not in liquid media (Alex et al., 1998; Yamada-Okabe et al., 1999). In
addition, the Hk1 histidine kinase is needed to prevent flocculation of hyphae (Calera and
Calderone, 1999a). Interestingly, deletions of SLNJ or COSJ alleles in a hkl mutant restored
filamentation and virulence, suggesting that Sln1p and Coslp act upstream of Hklp, via a
negative regulator (Yamada-Okabe eta/., 1999). Possibly histidine kinase pathways including
36
the Slnlp and Coslp pathways downregulate hypha! development on agar surfaces and within
agar. In S. cerevisiae, one function of the protein kinase C (PKC) pathway is to control the
expression of genes encoding cell-wall components and presumably the vectorial transport of
secretory vesicles (Banuett, 1998), thus, it could be expected that mutation of the gene encoding
the C. a/hi cans PKC homologue would significantly affect morphogenesis. A homozygous pkcl
null strain showed a cell-lysis defect, which was osmotically remediable; however, normal
hyphal morphogenesis occurred in stabilized liquid serum media (Paravicini et a/., 1996).
Because high osmolarity inhibited hypha! formation of wild-type cells on solid media, an effect
of the pckl mutation on Spider media could not be clarified. A gene encoding a downstream
target of PKC, the MAP kinase Mkclp, was also shown not to be absolutely necessary for
morphogenesis (Navarro-Garcia eta/., 1995, 1998). Homozygous mkcl mutants were less viable
and had cell-wall defects, and they were more sensitive to some cell-wall inhibitors. On Spider
media, hyphal development was blocked, but again in the presence of serum and other inducers,
filamentation occurred. Cyclin-dependent protein kinases (Cdk) regulate cell-cycle progression
in eukaryotes. A C. albicans gene (CLNJ) encoding a Gl-type cyclin homologue has been
isolated and both alleles were disrupted (Loeb eta/., 1999b). Besides slightly retarded growth
the mutants were filamentation-defective on solid Spider and serum media, in liquid media an
effect was seen only in Spider medium, not with serum as the inducer. The observed
filamentation defects were not apparent immediately after inoculation of solid or liquid media,
but c/nl mutants appeared to revert to yeast growth more rapidly than wild-type cells. Although
a gene encoding a homologue of the S. cerevisiae Cdc28 protein kinase has been isolated
(Sherlock eta/., 1994), it is not yet clear if this is the Cdk protein which is activated by Cln1p.
Recently, a gene encoding a Cdk homologue CRKJ has been identified, which has a major effect
on hypha! growth (J. Chen, S. Zhou, Q. Wang, X. Chen, T. Pan and H. Liu, unpublished).
Hyphal formation under all standard induction conditions, in liquid and on solid media, was
severely defective, but not blocked completely. Ectopic expression of CRKJ promoted hyphal
growth under non-inducing conditions. Such relatively drastic effects on morphogenesis were
hitherto observed only for mutants lacking the Efg1, Ras1 and Tpk2 proteins. There is no
information yet on regulators or targets ofCrk1p.
1.6.2.10. Targets ofTranscription Factors
Several genes have been identified whose expression is induced during (true) hyphal induction.
Such genes include the HYRJ, ALS3, HWP 1 and ECEJ genes, most of which encode cell-wall
proteins (Bailey eta/., 1996; Birse eta/., 1993; Hoyer et al., 1998; Staab eta/., 1999). In
addition, the EFGJ transcript is transiently downregulated during the initial phases of
37
filamentation (Stoldt eta/., 1997). Till date there is no evidence that any of the above discussed
pathways and transcription factors directly regulate these hypha-specific genes. None of the
sequence-motifs~found,_in their promoter' regions, which·ohave'' been" proposed- for. the above
transcription factors, including an E box (Efg1p), an FRE element (Acprp/Cphlp) and a PacC
element (Prr2p ), have yet been proven to be essential for gene regulation. Clearly, the absence of
expression of a hypha-specific gene in a filamentation-negative mutant, e.g. lacking the
transcription factor Efg 1 p, could be due to direct or indirect effects on gene regulation. In S.
cerevisiae many signaling pathways converge on the promoter of the FLO 11 gene, whose gene
product is similar to Hwp 1 p (31% identity) and the Als protein family (Rupp et a/., 1999),
recently, it has been shown that the FL011 promoter is regulated by the heterologous Cph1p,
Efg1p and Mcm1p proteins (S. Rupp, unpublished). Expression of HWP1 does not appear to be
stringently coupled to hypha formation, since in a prr 1 mutant HWP 1 is induced in some media
in the absence offilamentation (Porta eta/., 1999).
Studies in different fungi have converged to define two broadly conserved signal transduction
cascades that regulate fungal development and virulence. One is a MAP kinase cascade that
mediates responses to pheromone. The second is a nutrient-sensing G<X protein-cAMP-PKA
cascade. These pathways function coordinately to regulate mating, filamentation, and virulence.
How these pathways are organized differs in detail, likely as organisms have evolved to live
predominantly as haploid or diploid, to mate under nutrient-rich or -poor conditions, or to
respond to host signals as they infect plants or animals. What is most striking is how the general
mechanisms of signal sensing are conserved. These signaling cascades have much to teach us
about conserved principles of signal transduction that are likely also applicable to more
complex multicellular eukaryotes such as vertebrates and mammals. These signaling cascades
also represent excellent targets for novel antifungal drugs.
38