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

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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