CA2269633A1 - Candida albicans proteins associated with virulence and hyphal formation and uses thereof - Google Patents

Abstract

The present invention relates to Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, associated with virulence and hyphal formation and uses thereof, such as to design screening tests for inhibitors for the treatment of pathogenic fungi infections and/or inflammation conditions. The invention also relates to an in vitro screening test for compounds to inhibit the biological activity of at least one protein selected from the group consisting of CaCla4p, Cst20p, CaCdc42p and CaBemlp, which comprises: a) at least one of said proteins; and b) means to monitor the biological activity of said at least one protein; thereby compounds are tested for their inhibiting potential.

Description

C,ANDIL~A ,AIrBICANS PROTEINS ASSOCIATED KITH VIRULENCE AND
RYPHAL FORMATION AND USES THEREOF' ' HACKGROL~D OF' THE INVENTION
(a) Field of the Invention ' The invention relates to Candida albicans pro-teins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, associated with virulence and hyphal formation and uses thereof, such as to design screening tests for inhibi-tors for the treatment of pathogenic fungi infections and/or inflammation conditions.
(b) Description of Prior Art Candida albicans is the major fungal pathogen in humans, causing various forms of candidiasis. The incidence of infections is increasing in immunocom promised patients. This fungus is diploid with no sex-ual cycle and is capable of a morphological transition from a unicellular budding yeast to a filamentous form.
Extensive filamentous growth leads to the formation of a mycelium displaying hyphae with branches and lateral buds. In view of the observation that hyphae seem to adhere to and invade host tissues more readily than does the yeast form, the switch from the yeast to the filamentous form probably contributes to the virulence of this organism (for a review see Fidel, P. L. &
Sobel, J. D. (1994) Trends Microbiol. 2, 202-205). The molecular mechanisms by which morphological switching is regulated are poorly understood.
Like C. albicans, bakers yeast Saccharomyces cerevisiae is also a dimorphic organism capable of switching under certain nutritional conditions from a budding yeast to a filamentous form. Under the control of nutritional signals, diploid cells switch to pseudo hyphal growth (Gimeno, C. J. et al. (1992) Cell 68, 1077-1090), and haploid cells to invasive growth (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985).
The similarities between the dimorphic switching of S. cerevisiae and C. albicans suggest that these morphological pathways may be regulated by similar mechanisms in both organisms. In S. cerevisiae, mor-phological transitions are controlled by signaling com-ponents that are also involved in the mating response of haploid cells (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science 262, 1741-1744). The switch to pseudohyphal growth requires a transcription factor encoded by the STE12 gene, and a mitogen-activated protein (MAP) kinase cas-cade including Ste7p (a homolog of MAP kinase kinase or MEK), Stellp (a MEK kinase homology and Ste20p (a MEK
kinase kinase) (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science Z62, 1741-1744). The MAP kinases involved in this response are as yet unknown ( Roberts , R . L . & Fink , G .
R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al.
(1993) Science 262, 1741-1744).
Members of the Ste20p family of serine/threonine protein kinases are thought to be involved in trigger-ing morphogenetic processes in response to external signals in organisms ranging from yeast to mammalian cells. Two of these kinases, Ste20p and Cla4p, are well characterized in S. cerevisiae (Leberer, E. et al.
(1992) ENIHO J. 11, 4815-4824; Cvrckova, F. et al.
(1995) Genes Dev. 9, 1817-1830). Ste20p is required for pheromone signal transduction (Leberer, E. et al.
(1992) EMBO J. 11, 4815-4824) and for filamentous growth in response to nitrogen starvation (Roberts, R.
L. & Finks G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science 262, 1741-1744), and shares an essential function with Cla4p during budding tCvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). Ste20p and Cla4p interact with the small G-protein Cdc42p, and this interaction is required for viability of S. cere-visiae cells. Ste20p also interacts with the SH3 ~ 5 domain protein Bemlp, and this interaction plays a role in morphogenetic processes (Leeuw; T. et al. (1995) Science 270, 1210-1213).
Here we show that Cst20p, a C. albicans homolog of the Ste20p protein kinase, is required for hyphal growth of C. albicans under certain in vitro condi tions. We also show in a mouse model for systemic can-didiasis that Cst20p plays a role in virulence, as judged from significantly prolonged survival of mice infected with CST20 deleted cells. Our results suggest that Cst20p acts in a regulatory pathway which is involved in hyphal growth of C. albicans.
We also demonstrate that CaCla4p, a C. albicans homolog of the Cla4p protein kinase, is required for hyphal formation in vitro in response to serum, and in vi vo in a mouse model for systemic candidiasis. We also show that CaCla4p is required for efficient colo-nization of kidneys with C. albicans cells after infec-tion of mice and essential for virulence in the mouse model.
SUI~IARY O~' THE INVENTION
One aim of the present invention is to provide Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, and their uses thereof.
One aim of the present invention is to provide the nucleotide and amino acid sequences of CaCla4p, . Cst20p, CaCdc42p and CaBemlp.
Another aim of the present invention is to pro . vide screening tests for inhibitors of CaCla4p, Cst20p, CaCdc42p and CaBemlp or of their interactions.

The term "fungi" when used herein is intended to mean any fungi, pathogenic or not, which show hyphal induction using kinases, such as C. albicans, Saccharo- -myces cerevisiae, Aspergillus, Ustilago maydis, and all the species of the fungal genera Aspergillus, Blastomy-ces, Candida,.- Cladosporium, Coccidioides, Cryptococcus, Epidermophyton, Exophilia, Fonsecaea, Histoplasma, Madurella, Malassezia, Microsporum, Paracoccidioides, Penicillium, Phaeoannellomyces, Phialophora, Scedospo-rium, Sporothrix, Torulopsis, Trichophyton, Trichospo-ron, Ustilago, Wangiella, Xylohypha, among others.
In accordance with the present invention there is provided an in vitro screening test for compounds to inhibit the biological activity of at least one protein selected from the group consisting of CaCla4p, Cst20p, Cdc42p and Bemlp, which comprises:
a) at least one of the proteins; and b) means to monitor the biological.activity of at least one protein;
thereby compounds are tested for their inhibiting potential.
In accordance with another embodiment of the present invention, the inhibition of the interactions between CaCla4p and CaCdc42p is determined.
In accordance with another embodiment of the present invention, the inhibition of the interactions between Cst20p and CaCdc42p is determined.

Figs. lA to 1D illustrate photomicrographs which show that C. albicans CST20 gene complements defects in pseudohyphal growth of ste20/ste20 S. cerevisiae dip- -loid cells.
Figs. 2A to 2C show the morphology of S. cere visiae MATa cells (strain YEL306-lA) deleted for STE20 and CLA4, and transformed with plasmids expressing CLA4 ( Fig . 2A ) , STE20 ( Fig . 2B ) and C. albi cans CST20 ( Fig .
2C).
Figs. 3A to 3C show the nucleotide (SEQ ID N0:5) and predicted amino acid sequences of CST20 (SEQ ID
N0:6).
Fig. 4A is the deletion of CST20 in C. albicans.
Fig. 4B is the Southern blot analysis with a CST20 fragment from EcoRI to XbaI as a probe.
Figs. 5A to 5J show colonies of C. albicans cells grown for 5 days at 37°C on solid "Spider" medium containing mannitol. Wild type strain SC5314 (A), ura3/ura3 cst20d/cst20d::URA3 strain CDH22 (B), ura3/ura3 cst20d/cst20d: : CST20: : URA3 strain CDH36 (obtained by reintegration of CST20 into strain CDH25 by homologous recombination using linearized plasmid pDH190) (C), ura3/ura3 cst20d/cst20d strain CDH25 transformed with plasmids pYPBl-ADHpt (D) and pYPBl-ADHpt-HST7 (L), ura3/ura3 hst7d/hst7d strain CDH12 transformed with plasmids pVEC (F), pVEC-HST7 (G), pYPBl-ADHpt (H), and pYPBl-ADHpt-HST7 (I), and ura3/ura3 cphl/cphl strain CDH72 [ura3/ura3 derivative of strain JKl9l transformed with pYPBl-ADHpt-HST7 (J).
Photomicrographs of representative colonies were taken with a 2x lens (bar=2mm).
Figs. 6A to 6C illustrate virulence assays. Sur-vival curves of mice ( n=10 for each C. albicans strain at each inoculation dose) infected with 1 x 106 (A) and 1 x 105 (B) cells of C. albicans strains SC5314 (wild type), CAI4 (ura3/ura3), CDH22 (ura3/ura3 cst20d/cst20d .:URA3) (C) Staining of mouse kidney sections with periodic acid Schiff's stain 48 hours after infection with cst20d/cst20d::URA3 mutant strain CDH22 (a). Some hyphal cells are indicated with arrows (bar=0.1 mm).

WO 98!18927 PCT/CA97/00809 Figs. 7A to 7B illustrate the nucleotide (SEQ ID
N0:7) and predicted amino acid (SEQ ID N0:8) sequences of CaCLA4 . ' Fig. 8A illustrates the deletion of CaCLA4 in C.
albicans.
Fig. 8B illustrates the Southern blot analysis with the CaCLA4 fragment from PstI to XbaT as a probe.
Fig. 8C illustrates the Northern blot analysis with the CaCLA4 fragment as a probe. PCR with the divergent oligodeoxynucleotides OEL109 and OEL110 was used to delete the coding sequence of CaCLA4. A hisG-URA3-hisG cassette was then inserted, and homologous recombination was used in a two-step procedure to replace both CaCLA4 alleles.
Fig. 9 illustrates virulence assays. Survival curves of mice (n=15 for each C. albicans strain) infected with 1 x lOS cells of C. albicans strains SC5314 (wild-type), CDH77 (CaCLA4/cacla4d), CLJ1 (cacla4d/cacla4d) and CLJ5 (CaCla4d/cacla4d) trans-formed with the control plasmid pVEC and plasmid pVEC-CaCLA4 carrying the CaCLA4 gene.
Fig. 10 illustrates the staining of mouse kidney sections with periodic acid Schiff's stain 48 h after infection with C. albicans strains SC5314 and CLJ1.
Fig. 11 illustrates the nucleotide (SEQ ID N0:9) and predicted amino acid (SEQ ID N0:10) sequences of CaCdc42p.
Figs . 12A to 12B illustrate the nucleotide ( SEQ
ID N0:11) and predicted amino acid (SEQ ID N0:12) sequences of CaBemlp.
DETAILED DESCRIPTION OF THE INVENTION
The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65P~ family of protein kinases.

WO 98!18927 PCT/CA97/00809 - .~
Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid "Spider" media. However, hyphal devel-opment was not impaired in some other media. Cells . 5 deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelia! formation in asexual C. albi cans.
The CaCZA4 gene of C. albicans was cloned by functional complementation of the growth defect of S.
cerevisiae cells deleted for the STE20 and C_.LA4 genes .
CaCZA4 encodes a homolog of the Ste20p family of ser-ine/threonine protein kinases with pleckstrin homology and Cdc42p binding domains in the amino-terminal non-catalytic region. Deletion of both alleles of CaCZA4 in C. albicans caused defects in hyphal formation in vitro in synthetic liquid and solid media, and in vivo in a mouse model for systemic candidiasis. The deletions reduced the invasion of C. _ albicans cells into kidneys after infection into mice and completely suppressed virulence in the mouse model. Thus, hyphal formation of C. albicans mediated by the CaCla4p protein kinase may contribute to the pathogenicity of this dimorphic fun-gus.
The CaBEMl and CaCDC42 genes of C. albicans were cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM1 and CDC42 genes, respectively. CaBEMl encodes an SH3 domain protein with homology to Bemlp, and CaCDC42 encodes a small G-protein with homology to members of the Rho-family of G-proteins.

g -MATERIALS AND METHODS
Yeast manipulations The yeast form of C. albicans was cultured at 30°C in YPD medium. Hyphal growth was induced at 37°C
on solid "Spider" media (Liu, H. et al. (1994) Science 266, 1723-1726) containing 1~ (w/v) nutrient broth, 0.2~ (w/v) K2HP04, 2$ (w/v) agar and 1$ (w/v) of the indicated sugars (pH 7.2 after autoclaving). Cells were grown in liquid "Spider" media at 30°C to station-ary phase, and then incubated for 5 days at 37°C on solid "Spider" media at a density of about 200 cells per 80 mm plates. All media were supplemented with uridine (25 ~tg/ml) for the growth of Ura- strains.
Germ tube formation was induced at 37°C in either 10$
fetal bovine serum (GIBCO/BRL) on liquid "Spider" media containing the indicated sugars at an inoculation den-sity of 107 cells per ml.
Yeast manipulations were performed according to standard procedures.
Isolation of CST20 The CST20 gene was isolated from a genomic C. albicans library constructed in plasmid YEp352 from genomic DNA of the clinical isolate WO1 (Boone, C. et al. (1991) J. Bacteriol. 173, 6859-6864). A plasmid carrying an amino-terminally truncated version of CST20 missing the first 918 nucleotides of coding sequence was isolated by screening for suppressors of defects in basal FUSI::hTIS3 expression and mating in S. cerevisiae strain YEL64 which was disrupted in STE20. A fragment from nucleotides 958 to 1,252 of CST20 was amplified by the polymerase chain reaction (PCR) and used as a probe to isolate a full length clone by colony hybridization to the C. albicans genomic library transformed into E.
coli strain MC1061. Both DNA strands were sequenced by .. g the dideoxy chain termination method. The full length clone was subcloned between the SacI and HindIII sites of the S. cerevisiae centromere plasmid pRS316 to yield plasmid pRL53.
Isolation of CaGZA4 The S. cerevisiae MATa strain YEL257-lA-2 deleted for STE20 and ChA4 and carrying plasmid pDH129 with CLA4 under control of the GA.L1 promoter was trans-formed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al. (1991) J. Bacte-riol. 173, 6859-6864). Transformants were grown on selective medium in 4~ galactose and then replica-plated to selective medium containing 2$ glucose to select for plasmids that were able to support growth in the absence of Cla4p and Ste20p. By screening 1,600 transformants, we isolated plasmid YEp352-CaCLA4 carry-ing an insert of 5.6 kb with an open reading frame of 2,913 by capable of encoding a homolog of Cla4p. Sub-cloning indicated that this open reading frame was responsible for complementation. Both DNA strands were sequenced by the dideoxy chain termination method.
Molecular cloning of CaCDC42 The S. cerevisiae MATa strain DJTD2-16A carrying the cdc42-Its mutation was transformed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boons, C. et al. (1991) J. Bacteriol. 173, 6859-6864).
Transformants were grown on selective medium at room temperature. Colonies were then replica-plated to selective medium and grown at 34°C. By screening 2,000 " transformants, we isolated plasmid YEp352-CaCDC42 car rying an open reading frame of 573 by capable of encod ing a homolog of Cdc42p. Both DNA strands were sequenced by the dideoxy chain termination method. Sub cloning of various restriction endonuclease fragments indicated that the open reading frame was responsible for complementation of the temperature-sensitive growth defect caused by the cdc42-lts mutation.
Molecular cloning of Ca8~11 The S. cerevisiae MATa strain YEL220-lA deleted for BEM1 and carrying plasmid pGAL-BEMl with BEM1 under control of the GAL1 promoter was transformed with the genomic C. albicans library constructed in the S. cere-visiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al. (1991) J. Bacteriol. 173, 6859-6864).
Transformants were grown on selective medium in 4~
galactose and then replica-plated to selective medium containing 2$ glucose to select for plasmids that were capable of supporting growth of Bemlp-depleted cells.
We isolated plasmid YEp352-CaBEMl carrying an open reading frame of 1,905 by fulfilling this criterion and capable of encoding a homolog of Bemlp. Both DNA
strands were sequenced by the dideoxy chain termination method, and subcloning of various restriction endonu-clease fragments indicated that this open reading frame was responsible for complementation.
Construction of C. albicans strains and plasmids To construct a CST20 null mutant, an EcoRI to SacI fragment from nucleotide positions 989 to 4,134 of CST20 was subcloned into the Bluescript KS(+) vector (Stratagene) to yield plasmid pDH119. A plasmid that contained CST20-flanking sequences from nucleotides 989 to 1,674, and 3,423 to 4,134 joined with BamHI sites, was then created by PCR using the divergent oligodeoxynucleotide primers ODH68 (5'-CGGGATCCAGACCAACCACTCGAACTACT-3' (SEQ ID NO:1) and ODH69 (5'-CGGGATCCGAAGGTGAACCACCATATTTG-3' (SEQ ID
N0:2); newly introduced BamHI sites are underlined) and plasmid pDH119 as a template. The amplified DNA was cleaved with BamHI and ligated with a 4 kb BamHI to BglII fragment of a hisG-URA3-hisG cassette derived ~ from plasmid pCUB-6 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to yield plasmid pDHl83. This ~ 5 plasmid was linearized with XhoI and SacI and trans formed into the Ura- C. albicans strain CAI4 (Fonzi, W.
A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to par-tially replace the coding region of one of the chromo-somal CST20 alleles with the hisG- URA3-hisG cassette by homologous recombination. Ura+ transformants were selected on Ura- medium, and integration of the cas-sette into the CST20 locus was verified by Southern blot analysis. Spontaneous Ura- derivatives of two of the heterozygous disruptants were selected on medium containing 5-fluoroorotic acid. These clones were screened by Southern blot hybridization to identify those which had lost the URA3 gene by intrachromosomal recombination mediated by the hisG repeats. This pro cedure was then repeated to delete the remaining func tional allele of CST20.
A similar procedure was employed to delete the CaCST20 gene. A 4.6 kb XbaI fragment of YEp352-CaCZA4 was subcloned into the pBluescript KS(+) vector (Stratagene) to yield plasmid pDH205. A plasmid that contained CaCLA4 flanking sequences joined with BglII
sites was then created by PCR using the divergent oligodeoxynucleotide primers _QEL109 (5'-GAAGAT TTGTAATCAATGTTCCCGTGGA-3' (SEQ ID N0:3) and OEL110 (5'-GAAGATCTCATCGTGATATTAAATCCGAT-3' (SEQ ID
N0:4); newly introduced BglII sites are underlined) and plasmid pDH205 as template. The amplified DNA was ' cleaved with BglII and ligated with a 4 kb BamHI-BglII
fragment of a hisG- URA3-hisG cassette derived from plasmid pCUB-6 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to yield plasmid pDH210. This plasmid was linearized with PstI and SacI and trans-formed into the Ura- C. albicans strain CAI4 (Fonzi, W.
A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to replace the coding region of one of the chromosomal CaCLA4 alleles with the hisG-URA3-hisG cassette by homologous recombination. Ura+ transformants were selected on Ura- medium, and integration of the cas-sette into the CaCLA4 locus was verified by Southern blot analysis. Spontaneous Ura- derivatives were then selected on medium containing 5-fluoroorotic acid.
These clones were screened by Southern blot hybridiza-tion to identify those which had lost the URA3 gene by intrachromosomal recombination mediated by the hisG
repeats. This procedure was then repeated to delete the remaining functional allele of CaC.LA4.
To reintegrate CST20 into the genome of mutant strains, the C. albicans integration plasmid pDHl90 was constructed by subcloning a KpnI to PstI fragment of CST20 into pBS-c URA3 ( pBluescript KS ( + ) into which the C. albicans URA3 gene was cloned between the NotI and XbaI sites of the polylinker). The integration plasmid was then linearized with NsiI and transformed into C.
albicans to target integration into the NsiI site of the CST20d::hisG fusion gene. Integrations were selected on Ura- medium and confirmed by Southern blot analysis.
The C. albicans CST20 expression plasmid pDH188 was constructed by subcloning a SacI to PstI fragment of CST20 into plasmid pVEC carrying a C, albicans autonomously replicating sequence and URA3 as selectable marker. The C. albicans plasmid pVEC-CaCLA4 was constructed by subcloning the KpnI to SacI insert of YEp 352-CaCZA4 into plasmid pVEC.

Northern blot analyses Northern blots of total and poly (A)+ RNA from C. albicans cells were performed as described (Leberer, E. et al . ( 1992 ) EMBO J. 11, 4815-4824 ) . Signals were ~ 5 quantified by 2-D radioimaging.
Animal experiments Eight week-old, male CFW-1 mice (Halan-Winkel-mann, Paderborn, Germany) were inoculated with 1 x 105 or 1 x 106 cells by intravenous injection. Survival curves were calculated according to the Kaplan-Meier method using the PRISM'"'' program (GraphPad Software Inc., San Diego) and compared using the log-rank test.
A P value <0.05 was considered significant.
To quantify colony-forming C. albicans units in kidneys, mice were sacrificed by cervical dislocation 48 hours after injection and kidneys were homogenized in 5 ml phosphate buffered saline, serially diluted and plated on YNG medium (0.67$ yeast nitrogen base, 1$
glucose, pH 7.0). Histological examination of kidney sections was done with periodic acid Schiff's stain.
RESULTS
Isolation and characterisation of CST20 A C. albicans homolog of the S. cerevisiae STE20 gene was cloned by functional complementation of the pheromone signaling defect of S. cerevisiae cells that were deleted for the STE20 gene. The mating defect of the STE20 deleted S. cerevisiae strain YEL20 was fully complemented by introduction of the centromeric plasmid pRL53 carrying full length CST20 (mating efficiency was 81t9~ in cells expressing CST20, compared with 85t8~ in . cells expressing STE20; n=3). Similarly, detects in growth arrest and morphological changes in response to . pheromone were completely cured by transformation with the CST20 plasmid.

As shown in Fig . 1, nitrogen deficiency-induced pseudohyphae formation, which is blocked by disruption of STE20 in diploid cells (Liu, H., Styles, C. & Fink, G. R. (1993) Science 262, 1741-1744), was restored by introduction of the CST20 plasmid. Colonies of the diploid STE20 wild type strain L5266 ( 4 ) ( Fig . lA ) and the isogenic ste20/ste20 strain HLY492 (4) transformed with either the control plasmid pRS316 (Fig. 1B), the CST20 plasmid pRL53 (Fig. 1C), or the STE20 plasmid pSTE20-5 (9) (Fig. 1D) were grown on nitrogen starva-tion medium (2) for 5 days at 30°C. Photomicrographs were taken with a 4x objective (bar=lmm).
As illustrated in Fig. 2, the cytokinesis defect caused by deletion of CLA4, encoding an S. cerevisiae isoform of Ste20p (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830), was not complemented by CST20 (Fig.
2). However, the lethality caused by deletion of both STE20 and CLA4 (Cvrckova, F. et al. (1995) Genes Dev.
9, 1817-1830), could be rescued by CST20 (Fig. 2). The diploid strain YEL306 heterozygous for ste20d .:TRP1/STE20 cla4d::LEU2/CLA4 was transformed with plasmid pRS316 carrying either no insert, CLA4 (pRL21), CST20 (pRL53) or STE20 (pSTE20-5), and then sporulated and dissected. No viable haploid ste20d cla4d spores were obtained from transformants with the plasmid with-out insert, but were obtained from transformants with plasmids carrying CLA4 (Fig. 2A), STE20 (Fig. 2B) or CST20 ( Fig . 2C ) .
Cells were grown to mid-exponential phase in YPD
medium at 30°C. No viable ste20d cla4d segregants were obtained in medium containing 5-fluoro-orotic acid sug gesting that the plasmids were essential for viability.
Neither STE20 nor CST20 were able to suppress the mor phological defect of cla4d cells. Photomicrographs were taken by phase contrast with a 40x objective ( bar=3 0 ~.~m ) .
The open reading frame of CST20 is capable of encoding a protein of 1,229 amino acids with a pre ~ 5 dicted molecular weight of 133 kDa and a domain struc ture characteristic of the Ste20p/p65~'~ family of protein kinases (Fig. 3). Numerals at the left margin indicate nucleotide and amino acid positions (Fig. 3).
Nucleotide 1 corresponds to the first nucleotide of the initiation codon and amino acid 1 to the first residue of the deduced protein. The putative p21 binding domain has been shadowed, and the kinase domain has been boxed.
The catalytic domain present in the carboxyl terminal half of the protein has sequence identities of 76 and 56$, respectively, with S. cerevisiae Ste20p (Leberer, E. et al. (1992) EM80 J. 11, 4815-4824) and Cla4p (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817 1830). The amino terminal, non-catalytic region con tains a sequence from amino acid residues 473 to 531 with 68~ identity to the p21 binding domain of Ste20p that has been shown to bind the small GTPase Cdc42p.
This region contains the sequence motif ISxPxxxxHxxH
thought to be important for the interaction of the p21 binding domain with the GTP-bound forms of Cdc42Hs and Racl (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). The remaining non-catalytic sequences are less conserved. Unique sequences not present in Ste20p and the other members of the family are found at the amino terminus and between the p21 binding and catalytic domains.
' A CST20 transcript of 4.9 kb in size was detected in Northern blots. This transcript was pres-ent at similar levels in yeast cells grown in YPD at WO 98/1892? PCT/CA97/00809 room temperature and germ tubes induced by a tempera-ture shift to 37°C.
Isolation and characterisation of CaCl.A.~
A C. albicans homolog of the S. cerevisiae CLA4 gene was cloned by functional complementation of the growth defect of S. cerevisiae cells that were deleted for the STE20 and CZA4 genes.
The open reading frame of the CaCLA4 gene is capable of encoding a protein of 971 amino acids with a predicted molecular weight of 107 kDa and a domain structure characteristic of the Ste20p family of pro-tein kinases (Fig. 7). The catalytic domain present in the carboxyl terminal half of the protein has sequence identities of'74, 63 and 64~, respectively, with S.
cerevisiae Cla4p, S. cerevisiae Ste20p and an uncharac-terized open reading frame present in the S. cerevisiae genome, 65~ with the C. albicans Ste20p homolog Cst20p, and 61$ with rat p65P~ (Fig. 7). The amino terminal, noncatalytic region contains a sequence from amino acid residues 69 to 180 with similarity to pleckstrin homol-ogy (PH) domains and a sequence from amino acid resi-dues 229 to 292 with 63$ identity to the Cdc42p binding domain of S. cerevisiae Cla4p that has been shown to bind the small GTPase Cdc42p (Cvrckova, F. et al.
(1995) Genes Dev. 9, 1817-1830). The remaining non-catalytic sequences are less conserved.
Chromosomal deletion of CST20 Homologous recombination was used in a multistep procedure to partially delete CST20 in a URA- C. a~bi cans strain (Fig. 4A). PCR with the divergent oligode oxynucleotides ODH68 and ODH69 was used to partially delete the coding sequence of CST20. A hisG-URA3-hisG
cassette was then inserted. The deletion was confirmed by Southern blot analyses (Fig. 4B). The genomic DNA
samples digested with XhoI were from following strains:

Lane #1, CAI4 (ura3/ura3 CST20/CST20); lane 2, CDH15 ( ura3/ura3 CST20/cst20d: :hisG-URA3-hisG) ; lane 3, CDH18 ~ (ura3/ura3 CST20/cst20d::hisG); lane 4, CDH22 (ura3/ura3 cst20d::hisG-URA3-hisG/cst20d::hisG); lane . 5 5, CDH25 (ura3/ura3 cst20d::hisG/cst20d::hisG). North ern blots showed that the CST20 transcript was absent in the corresponding homozygous deletion strains.
The lateral outgrowth of hyphae from colonies grown on solid "Spider" media containing mannitol or sorbitol was completely blocked by deletion of CST20 (Fig. 5B).
Mycelial formation was drastically reduced when the media contained galactose, mannose or raffinose.
The mutant strains regained the ability to form hyphae when wild type CST20 was reintroduced by transformation with the CST20 expression plasmid pDH188 or rein-tegrated into the genome by targeted homologous recom-bination (Fig. 5C). The CST20 transcript was detected in these strains by Northern blot analysis.
Mutant strains formed hyphae when colonies were grown on "Spider" media containing either glucose or N-acetyl g.lucosamine. Normal hyphae formation was also observed on rice agar and on agar containing Lee's medium or 10~ serum. The frequency of germ-tube forma-tion in either liquid Lee's medium, 10$ serum or liquid - "Spider" media containing any of the sugars tested above, were also normal. These results indicate that Cst20p is not required for hyphae formation under all conditions but are involved in the lateral formation of mycelia on some solid surfaces.
Chromosomal deletion of CaCl~l4 Homologous recombination was used in a multistep procedure to delete both alleles of CaCLA4 in C. albi-cans (Fig. 8A). Fig. 8A shows the restriction endonu-clease map of CaCLA4. The coding sequence is indicated by the arrow. PCR with the divergent oligodeoxynucleo-tides OEL109 and OEL110 Was used to delete the coding sequence of CaCLA4. A hisG-URA3-hisG cassette was then inserted and a two-step procedure was used to delete both alleles of CaCLA4 by homologous recombination. The endonuciease restriction sites are as follows: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; P, PstI; S, SacI; X, XbaI. The deletions were confirmed by South-ern blot analyses (Fig. 8B). Southern blot analysis with a 1.1 kb CaCLA4 fragment from PstI-XbaI as a probe. The genomic DNA samples digested with EcoRI were from following strains: Lanes: 1, CAI4 (ura3/ura3 CaCLA4/CaCLA4); 2, CDH77 (ura3/ura3 CaCLA4/cacla4d .:hisG-URA3-hisG): 3, CDH88 (ura3/ura3 CaCLA4/cacla4d .:hisG); 4, CLJ1 (ura3/ura3 cacla4d::hisG-URA3-hisGlcacla4d::hisG); and 5, CLJ5 (ura3/ura3 cacla4d .:hisG/cacla4d::hisG). Northern blots showed that the CaCLA4 transcript with a size of 4.1 kb was reduced to about 40$ in heterozygous CaCLA4/cacla4d cells and was absent in homozygous cacla4d/cacla4d deletion cells (Fig. 8C). The transcript was present at about wild-type levels when the CaCLA4 gene was retransformed into the homozygous deletion cells by using an autonomously replicating plasmid carrying the CaCLA4 gene Fig. 8C).
Northern blot analysis of poly(A)+ RNA isolated from following strains grown in the yeast form in YPD at 30°C: Lanes: 1, SC5314 (wild-type); 2, CDH88; 3, CLJS
transformed with pVEC; 4, CLJ5 transformed with pVEC-CaCLA4. The blot was probed with fragments specif is for CaCLA4 (upper panel) or CaACTI (lower panel) and quan-tified by radioimaging. Numbers at the bottom of the figure depict the relative amounts of CaCLA4 transcript in relation to the amounts of CaACTI transcript (mean values of two independent experiments).

We found that viability of C. albicans cells was not affected by deleting either one or both alleles of ' CaCLA4. Mutant cells showed the same growth behavior as wild-type cells, independently whether the cells were grown under conditions favoring either the yeast or filamentous forms. However, deletion of both CaCZAg alleles generated defects in cellular morphology pro-ducing a heterogeneous population of aberrantly shaped cells that were frequently multibudded and multinucle-ated. This phenotype indicates a defect in cytokinesis resembling the phenotype of S. cerevisiae cells deleted for C.LA4 (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830).
Deletion of both CaCLA4 alleles caused defects in hyphal formation in all media and under all condi tions that we investigated. When morphological switch ing was induced in liquid media by either serum, N-ace tyl glucosamine, proline, pH increase, temperature shift, or Lee's medium, wild-type cells and cells deleted for only one or both alleles of CaCZA4 produced germ tubes after about 30 minutes. In wild-type cells and cells deleted for only one allele of CaCLA4, these germ tubes elongated and grew into long hyphae after w prolonged incubation. Cells deleted for both alleles of CaCLA4 failed to produce hyphae, however. Instead, these cells produced multiple short protrusions giving rise to an aberrant morphology.
On solid media containing either serum, rice agar or mannitol, the normal formation of mycelia was completely suppressed by deletion of both CaC.LA4 alleles. This phenotype was reversed by introducing the CaCLA4 gene on a plasmid, and deletion of only one allele had no effect.

Virulence studies To determine the role of Cst20p for virulence, mice were injected intravenously with wild type and mutant strains and monitored for survival and for fun-s gal invasion into kidneys. We found that the Ura-strain CAI4 was not pathogenic (Figs. 6A and B). How-ever, infection with Ura+ wild type cells resulted in rapid mortality with a rate that was dependent on the dose of injected cells (1 x 106 cells in Fig. 6A, and 1 x 105 cells in Fig. 6B). Survival was significantly prolonged, however, in mice infected with Ura+ cells deleted for both alleles of CST20 ( cst20d/cst20d . : URA3) . This effect, which was reproducible and sta-tistically significant, was observed at high (Fig. 6A) or low (Fig. 6B) doses of infection (with P values of 0.027 and 0.001, respectively) and correlated with col-ony-forming units per kidney ( 1. 5 x 106 for wild type cells and 7 x 105 for cst20dlcst20d::URA3 mutant cells) after 48 hours of infection with 1 x 10~ cells. These effects on virulence could be reversed by reintroducing CST20 into the strain deleted for both CST20 alleles, and were not observed in Ura+ cells deleted for only one CST20 allele. A histological examination revealed that cells deleted for both alleles of CST20, were able to form hyphae in infected kidneys (Fig. 6C).
To investigate whether CaCla4p is required for virulence, mice were injected intravenously with wild-type and mutant C. albicans strains and monitored for survival and for fungal invasion into kidneys. Infec-tions with CaCLA4 wild-type cells (strain SC5314) resulted in rapid mortality (Fig. 9). No difference in the mortality rate was observed after infection with cells deleted for only one allele of CaCLA4 (strain CDH77). All mice survived, however, after infection with cells deleted for both alleles of CaCLA4 (strain CLJ1 and CLJSpVECl). This effect correlated with a reduction in the amount of colony-forming units per kidney of infected animals and was reversed by trans-formation of the cells with a plasmid carrying the CaCLA4 gene (strain CLJSCaCLA4) (Fig. 9). A histologi-cal examination revealed that kidneys from mice injected with_either wild-type cells or cells deleted for one allele of CaCLA4 were heavily infected with C.
albicans cells that produced hyphae densely penetrating the animal tissue (Fig. 10, left panel), whereas kid-neys from mice injected with cells deleted for both CaCLA4 alleles contained small foci of aberrantly shaped cells that frequently carried multiple protru-sions (Fig. 10, right panel). The morphologies of these cells were similar to those induced by serum under in vitro conditions. Thus, the function of CaCla4p is required for morphological switching of C. albicans under in vitro and in vivo conditions and for viru-lence.
Molecular cloning of the CaCDC42 and Ca8EM1 genes A C. albicans homolog of the CaCDC42 gene was cloned by functional complementation of the tempera-ture-sensitive growth defect of S. cerevisiae cells carrying the cdc42-Its mutation. The growth defect was fully complemented by plasmid YEp352-CaCDC42. The open reading frame of the CaCDC42 gene is capable of encod-ing a protein of 191 amino acids with homology to the Rho-family of small G-proteins (Fig. 11). The highest homology is found with Cdc42p from S. cerevisiae.
A C. albicans homolog of the CaBEMI gene was cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM/
gene. This defect was fully complemented by plasmid YEp352-CaBEMl carrying the CaBEMl gene. The open read-ing frame of the CaBEMI gene is capable of encoding a protein of 635 amino acids with a domain structure characteristic of Bemlp (Fig. 12). CaBemlp contains two conserved SH3 domains which are most homologous to the SH3 domains of Bemlp, and also has homology to Bemlp outside of the SH3 domains.
Discussion In S. cerevisiae, Ste20p fulfills multiple func-tions during mating ( Leberer, E. et al . ( 1992 ) EM80 J.
11, 4815-4824), pseudohyphae formation (Liu, H., Styles, C. & Fink, G. R. (1993) Science 262, 1741-1744), invasive growth (Roberts, R. L. & Fink, G. R.
(1994) Genes Dev. 8, 2974-2985) and cytokinesis (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830).
CST20 expression in S. cerevisiae fully complements these functions. Thus, Cst20p has the potential to fulfill similar functions in C. albicans.
The yeast-to-hyphal transition of C. albicans is a morphological change that can be triggered by a wide variety of factors. Carbohydrates, amino acids, salts, and serum have been described as inducers of germ tube formation, as have pH changes, temperature increases and starvation, but no single environmental factor could be defined as uniquely significant in stimulating the morphological switch. Hence C. albicans appears capable of responding to many divergent environmental signals. Disruption of both CPHT alleles, which encode a homolog of the S. cerevisiae Stel2p transcription factor (Liu, H. et al. (1994) Science 266, 1723-1726), suppressed the lateral formation of mycelia from colo-nies grown on solid "Spider" medium, but did not block hyphal development in other media. We have shown that C. albicans mutant cells deleted for CST20 display a similar phenotype, and that the effect of these muta-tions on hyphal development is dependent on the carbon source in which the cells were grown.

These observations are consistent with the idea that several signaling pathways can trigger morphogene-. sis in C. albicans_ Furthermore, the behavior of C, albicans mutant strains deleted for either CPH1 or CST20 indicates that these pathways might operate inde pendently to activate hyphal development under differ-ing environmental conditions. C. albicans encounters a variety of different microenvironments during the development of superficial and systemic infections.
Hence, the existence of parallel morphogenetic signal-ing pathways might provide a distinct advantage to this pathogen.
Our results indicate that the pathway controlled by Cst20p is not essential for virulence in a mouse model of systemic infections. It is not inconceivable that this pathway plays a role in other forms of infec-tions, for example in the development of superficial infections of the mucosal epithelia (thrush). An as yet undefined role of Cst20p in pathogenicity outside of the Cst20 signaling pathway is suggested, however, by prolonged survival of mice infected with cst20 deleted cells. It is unlikely that this effect is caused by defects in hyphal formation since a his-tological examination of infected kidneys revealed that the CST20 deleted cells are not restricted in their capacity to form hyphae.
In S. cerevisiae, Cla4p plays a role in cytoki-nesis and shares with Ste20p an essential function for polarized growth during budding (Cvrckova, F. et al.
(1995) Genes Dev. 9, 1817-1830). Cla4p binds the Rho like small G-protein Cdc42p (Cvrckova, F. et al. (1995) ' Genes Dev. 9, 1817-1830) which is involved in control ling cell polarity during budding and in response to ' pheromone. Like Ste20p and the mammalian homolog p21 activated kinas~ (p65P~), Cla4p is able to phosphory late and activate myosin-I, a mechanism that may con-tribute to the organization of the actin cytoskeleton.
Our finding that CaCLA4 expression in S. cere-visiae completely complements the Cla4p functions sug-gents that CaCla4p may have similar properties in C. .
albicans. Thus, CaCla4p may be required for myosin-I
driven polarized growth during hyphal formation in a mechanism that may involve the C. albicans homolog of Cdc42p. Our complementation assays in S. cerevisiae suggest that CaCla4p may share an essential function with Cst20p, the C. albicans homolog of Ste20p (Figs. 6A and 6B). This notion suggests, together with our findings that null mutants of CaCLA4 are completely non-pathogenic ( Fig . 10 ) and null mutants of CST20 are reduced in virulence (Figs. 6A and 6B), that CaCla4p and Cst20p, and proteins such as CaCdc42p and CaBemlp interacting with these protein kinases, may be valid targets for the development of antifungal agents.
The present invention will be more readily un derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLF~ I
Screening test for inhibitors of CaCla4p and Cst24p An in vitro assay containing the proteins CaCla4p and/or Cst20p will be used to test compounds inhibiting their activity to render avirulent any fungi, which may be pathogenic.
The activity of the protein will be monitored to determine if the compounds tested do inhibit their bio logical activity, using myelin basic protein as a sub strate.
In cases were a selective inhibition of CaCla4p and Cst20p and not to p65P~ would be desired, com-pounds testing positive for the inhibition of both CaCla4p and Cst20p will be tested to determine if they also inhibit the protein p65P~. This would be useful . in cases of pathogenic fungi infection such as for C.
albicans were the fungi is to be rendered avirulent without affecting the normal protein of the patient p65P~.
In some cases of inflammation, it would be desirable to be provided with compounds inhibiting all three proteins, namely, CaCla4p, Cst20p and p65P~.
EXAMPLE II
Screening test for inhibitors of CaCla4p and CaCdc42p interactions An in vitro assay containing the proteins CaCla4p and CaCdc42p will be used to test compounds inhibiting their interactions.
CaCla4p may be solid phase bound and CaCdc42p will be in suspension free to interact with CaCla4p. A
labeled antibody specific to CaCdc42p will be added to the assay to determine the presence of CaCdc42p bound to CaCla4p. The compounds tested to inhibit the CaCdc42p-CaCla4p interactions, should when tested posi tive, cause only a minute quantity of CaCdc42p to bind to CaCla4p interactions.
The analogous in vitro assay will be used to test compounds that inhibit the interaction between Cst20p and CaCdc42p.
EXAMPLE III -Screening test for inhibitors of CaCla4p and CaBemlp interactions An in vitro assay containing the proteins CaCla4p and CaBemlp will be used to test compounds inhibiting their interactions.
CaCla4p may be solid phase bound and CaBemlp will be in suspension free to interact with CaCla4p. A

labeled antibody specific to CaBemlp will be added to the assay to determine the presence of CaBemlp bound to CaCla4p. The compounds tested to inhibit the CaBemlp-CaCla4p interactions, should when tested positive, cause only a minute quantity of CaBemlp to bind to CaCla4p interactions.
The analogous in vitro assay will be used to test compounds that inhibit the interaction between Cst20p and CaBemlp.
EXAMPLE IV
A two-hybrid CaCdc42p and CaCla4p interaction system in a humanized S. cerevisiae strain This screening assay is based on the assumption that the interaction of the small G-protein CaCdc42p with its cellular targets Cst20p and CaCla4p is essen-tial for viability of C albicans cells. This essential function is reasonable to assume based on work that has been performed in S. cerevisiae (Leberer E. et al.
( 1997 ) Embo J. 16, 83-97 ) . The two hybrid interaction system will use green fluorescent protein fused to the GAL1 promoter as a functional read out. This reporter gene will be integrated into a S. cerevisiae strain in which the STE20 and CLA4 genes have been replaced by the human homolog p65PAK. The CaCDC42 gene will be fused to the DNA binding domain of GAZ4, and the CaCLA4 gene will be fused to the activation domain of- GAL4.
Interaction of the two proteins will cause green fluo-rescence. Whereas inhibitors of the interaction will suppress fluorescence.
Non-specific inhibitors of the two-hybrid inter-action system will be excluded by performing a parallel screen with unrelated fusion proteins known to inter-_ 35 act. Compounds of general toxicity or inhibitors of WO 9$/18927 PCT/CA97/00809 the human homologs will also be excluded in this system because those compounds will not allow growth of the cells and therefore reduce the fluorescent readout in both parallel screens.
A two-hybrid yeast strain carrying the GAZ4-GFP
fusion gene is constructed. This strain will be deleted for the CLA4 gene using the TRP1 marker as described (Leberer E. et al. (1997) Embo J. 16, 83-97).
The STE20 gene will be replaced by the human PAK gene as described above. To replace the CDC42 gene by its human homolog, an integrating plasmid will be con-structed carrying the HsCDC42 gene fused to a URA3 blaster gene and CDC42 flanking sequences. After line-arization, the construct will be transformed into the PAK containing two-hybrid strain, and integrants will be selected on -ura medium. The URA3 gene will then be looped out on FOA medium. The various gene disruptions and gene replacements will be verified by Southern blot analyses.
The two-hybrid vectors carrying the CaCDC42 gene fused to the GAL4-DNA binding domain and the CaCZA4 gene fused to the transcriptional activation domain of GA.L4 will be constructed by standard procedures. To facilitate the interaction of the two proteins, we will use site-directed mutagenesis to create a mutation in the CAAX-box domain of CaCDC42p to prevent isopren-ylation and targeting of the fusion protein to the plasma membrane. We will evaluate and optimize the assay system and adapt the assay conditions to the scale used in microtiter plates for automated screening of compounds.

EXAMPLE V
Detection of the presence of C. albicans using probes The sequences of either one of the genes CaCLA4, CST20, CaCDC42 and CaBEMI may be used to derive probes for the detection of C. albicans using PCR techniques or hybridization assays.
- EXAMPLE VI
Use of nucleotide sequences of CaCLA4, CST20, Ca.CDC42 and CaBETSI to identify homologue from other fungi The nucleotide sequences of CaCLA4, CST20, CaCDC42 and CaBEMI may be used to identify and clone homologues fro~tr other fungi.
EXAMPLE VII
A S. cerevisiae-based screening system using Ca8te20p and the pheromone signaling pathway as drug target In this system, we will use green fluorescent protein (GFP) under transcriptional control of a phero-mone inducible promoter (FUSI) as a read out. The pheromone signaling pathway and thereby the reporter gene will be induced with pheromone in two different strains. First, in a strain in which STE20 is func-tionally replaced by the CaSTE20 gene. And second, in a strain in which STE20 is functionally replaced by the mammalian homolog PAK. Compounds that block the induc-tion of the reporter gene in the CaSTE20 strain but not in the PAK strain are expected to be specific inhibi-tors of the C. albicans kinase. This assay is very specific and is a positive selection of compounds that excludes the finding of compounds with inhibitory action against the mammalian homolog PAK or compounds of general toxicity.
The FUS1 gene, including its promoter, will be isolated by the polymerase chain reaction (PCR) from genomic DNA of S. cerevisiae and fused to the GFP gene from Aeqaoria victoria on a yeast expression plasmid.
The function of the reporter gene will be analyzed after transformation of a MATa yeast strain and induc tion with pheromone.
The STE20 gene will be replaced in a supersensi-tive sstl yeast strain by the human PAK gene using homologous recombination. For this purpose, an inte-grating plasmid will be constructed carrying the PAK
gene fused to a URA3 blaster gene and STE20 flanking sequences. The construct will be linearized and trans-formed into yeast, and integrants will be selected on -ura medium. The URA3 gene will then be looped out on FOA medium to gain back the ura3 marker. Correct inte-gration of the PAK gene will be confirmed by Southern blot analysis.
The humanized strain will then be transformed with the FUS2-GFP reporter gene and analyzed for a functional signaling pathway by measuring green fluo-rescence after induction with pheromone. The assay system will be evaluated, optimized and adapted to the scale used in microtier plates.
EXAMPLE VIII
Fluorescence resonance energy transfer (FRET) as probe for protein-protein interactions The engineering of different GFP mutants with altered fluorescence characteristics allows the use of fluorescence resonance energy transfer (FRET) to probe protein-protein interactions (Heim and Tsien (1996) Curr. Biol. 6, 178-182). The FRET phenomenon consists in a fluorescence transfer between a donor and a recep-tor fluorochrome. If excitation and emission wave-lengths are compatible, the FRET is easily measurable.
The main parameter of the reaction is the distance between donor and receptor, which must be in the range of nanometers. This is precisely the kind of values in protein-protein interactions.
We propose to develop a novel yeast assay system which uses FRET to measure the in vi vo interaction between CaCdc42p and Cacla4p. The CaCDC42 gene will be fused to a GFP mutant that acts as donor, and the CaCLA4 gene will be fused to a mutant that acts as receptor. The yeast strain used as an expression sys tem will be humanized as described in Example VII.
Inhibitors of the interaction are expected to reduce energy transfer, and this reduction can be easily meas-ured spectroscopically. The interaction of unrelated proteins known to interact will be used as a reference to exclude non-specific inhibitors of the assay system.
Compounds inhibiting the interaction of the human homologs or of general toxicity will be excluded by inhibition of growth and therefore reduced fluorescence in both screens.
The CaCDC42 gene will be fused to the gene encoding the GFPY6sx mutant as donor, and the CaCLA4 gene will be fused to the gene encoding the GFPss5T
mutant as receptor (Heim and Tsien (1996), Curr. Biol.

6, 178-182). The constructs will then be transformed into the humanized yeast strain described in Example VII, and the FRET phenomenon will be analyzed in yeast cultures using fluorescence spectroscopy. The condi-tions for the assay will be worked out and optimized.
We will adapt the assay conditions to the scale used in microtiter plates for automated screening.
While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the-.present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

SEQUENCE LISTING
(1) GENERAL INFORMATION
{i) APPLICANT: National Research Council of Canada (ii) TITLE OF THE INVENTION: CANDIDA ALBICANS PROTEINS
ASSOCIATED WITH VIRULENCE AND HYPHAL FORMATION AND USES
THEREOF
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B} TYPE: nucleic acid (C) STRANDEDNESS: single {D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CGGGATCCAG ACCAACCACT CGAACTACT 2g (2) INFORMATION FOR SEQ ID N0:2:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
CGGGATCCGA AGGTGAACCA CCATATTTG 2g (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GAAGATCTTG TAATCAATGT TCCCGTGGA 2g (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
__ _ (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

(2) INFORMATION FOR SEQ ID N0:5:
' (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4492 base pairs (B) TYPE: nucleic acid . (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic RNA

(ix) FEATURE:

(A) NAME/KEY: Coding Sequence (B) LOCATION: 355...4049 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

TACAATCACT
TACAAGTCAA
ATAATTACAA
CTTGACAATC
CTCACTTTAA

TATACGCGTA
CACCATCTTA
TACTCCACAT
ACATATTGGA
TTCAATTTTT

TTAGTTTATA
TCCAACCACT
GACAATTACC
AATAGTTTTC
AATTAATATT

CTATTTGTTT
GACAGCTGAA
AAGAGATAAA
AAAAGAATCA
AGTGCTATAA

CTAGAAATAA
GTTTGCAAAA
AACAAGTTTT
AAAAATAGTA
ACTGCACTTT

TTCACCTCCC
CATTGAATTT
AACTGAACAC
AAATAAAGCC
TATC

Met ACA GAT

SerIleLeu Ser Glu Asn Asn Pro Thr Pro Thr Ser Ile Pro Thr Asp GGA ACG

AsnGluSer Ser His Leu His Asn Pro Glu Leu Asn Ser Arg Gly Thr ACA CCA

ValAlaSer Gly Pro Gly Pro Gly Pro Glu Val Glu Ser Leu Thr Pro ACT TCT

AlaProPro Thr Glu Val Met Asn Thr Thr Ser Ala Asn Ser Thr Ser TTT GAT

LeuSerLeu Gly Ser Pro Met His Glu Lys Ile Lys Gln Gln Phe Asp GAA TCT

AspGluVal Asp Thr Gly Glu Thr Asn Asp Arg Thr Ile Gly Glu Ser AAC AAC

SerSerAsp Ile Asp Asp Ser Gln Gln Ser His Asn Asn Asn Asn Asn GAA GGC

AsnAsnAsn Asn Asn Asn Glu Ser Asn Pro Glu Ser Ser Asp Glu Gly ACA TTC

AspGluLys Thr Gln Gly Met Pro Pro Arg Met Pro Gly Asn Thr Phe AAA CAG

ValLysGly Leu His Gln Gly Asp Asp Ser Asp Asn Glu Tyr Lys Gln GAT TCG

ThrGluLeu Thr Lys Ser Ile Rsn Lys Arg Thr Ser Lys Tyr Asp Ser Ser Pro Gly Thr Leu Glu Ser Pro Gly Thr Leu Asn .Ala Leu Glu Thr Asn Asn Val Ser Pro Ala Val Ile Glu Glu Glu Gln His Thr Leu Ser Leu GluAspLeu SerLeuSer LeuGln HisGlnAsn Glu Arg Asn Ala Leu SerAlaPro ArgSerAla ProPro GlnValPro ThrSerLys Thr Ser SerPheHis AspMetSer LeuVal IleSerSer SerThrSer Val His LysIlePro SerAsnPro ThrSer ThrArgGly SerHisLeu Ser Ser TyrLysSer ThrLeuAsp ProGly LysProAla GlnAlaAla Ala Pro ProProPro GluIleAsp IleAsp AsnLeuLeu ThrLysSer Glu Leu AspLeuGlu ThrAspThr LeuSer SerAlaThr AsnSerPro Asn Leu LeuArgAsn AspThrLeu GlnGly IleProThr ArgAspAsp Glu Asn IleAspAsp LeuProArg GlnLeu SerGlnAsn ThrSerAla Thr Ser ArgAsnThr SerGlyThr SerThr SerThrVal ValLysAsn Ser Arg SerGlyThr SerLysSer ThrSer ThrSerThr AlaHisAsn Gln Thr AlaAlaIle ThrProIle IlePro SerHisAsn LysPheHis Gln Gln ValIleAsn ThrA_snAla ThrAsn SerSerSer SerLeuGlu Pro _ 405 410 415 Leu Gly Val Gly Ile Asn Ser Asn Leu Ser Pro Lys Ser Gly Lys Lys AAA GTG ATG
AGT
GGA
AGT
AAA

ArgLys Ser SerLys ValArg Gly Phe SerSer PheGly Gly Val Met TCA TCT GGT

LysAsn Lys ThrSer SerSer Ser Ser AsnSer LeuAsn Ser Ser Gly CAG ATC TTC

SerHis Ser GluVal AsnIle Lys Ser ThrPro AsnAla Gln Ile Phe GCC GAT TAC

LysHis Leu HisVal GlyIle Asp Asn GlySer ThrGly Ala Asp Tyr GAG TCT ATT

LeuPro Ile TrpGlu ArgLeu Leu Ala SerGly ThrLys Glu Ser Ile CAA GTG GTG

LysGlu Gln GlnHis ProGln Ala Met AspIle AlaPhe Gln Val Val ACA GAC AAA

TyrGln Asp SerGlu AsnPro Asp Ala AlaPhe LysPhe Thr Asp Lys AAT AGT AAT

HisPhe Asp AsnLys SerSer Ser Gly TrpSer GluAsn Asn Ser Asn GCA AAC GGC

ThrPro Pro ThrPro GlyGly Ser Ser GlySer SerGly Ala Asn Gly GCT CGT TCA

Gly..Gly Gly ProSer SerPro His Thr ProPro SerIle Ala Arg Ser AAC GTG TCT

IleGlu Lys AsnVal GluGln Lys Ile ThrPro GlnSer Asn Val Ser AAG CTG CAC

MetPro Thr ThrGlu SerLys Gln Glu AsnGln ProHis Lys Leu His GCT AGA TCC

GluAsp Asn ThrGln TyrThr Pro Thr ProThr HisVal Ala Arg Ser Gln Glu GlyGln PheIlePro SerArgPro ProLys ProPro Ser Ala AAA

Thr Pro LeuSer SerMetSer ValSerHis ThrPro SerSer Gln Lys ATT

Ser Leu ProArg SerAspSer GlnSerAsp Arg5er SerThr Pro Ile ATC

Lys Ser HisGln AspValSer ProSerLys LysIle ArgSer Ile Ile AGA

Ser Ser LysSer LeuLysSer MetArgSer LysSer GlyAsp Lys Arg CCA

Phe Thr HisIle AlaProAla ProProPro SerLeu ProSer Ile Pro TCA

Pro Lys SerLys SerHisSer AlaSerLeu SerGln LeuArg Pro Ser Ala Thr GlySer ThrThr AlaProIle AlaSer AlaAlaPhe Asn Pro AGA

Gly Gly GluAsnAsn AlaLeu ProLysGln IleAsn GluPheLys Arg GCA

Ala His ArgAlaPro ProPro ProProLeu ProPro AlaProPro Ala TCG

Val Pro ProAlaPro ProAla AsnLeuLeu GluGln ThrSerGlu Ser ATA CCT CAACAACGT ACTGCT CCTCTGCAA TTAGCT.-GATGTTACT 2853 GCA

Ile Pro GlnGlnArg ThrAla ProLeuGln LeuAla AspValThr Ala ACT

Ala Pro ThrAsnIle TyrGlu IleGlnGln LysTyr GlnGluAla Thr ' CAA CAG AAATTACGT GAGAAG AAGGCTAGA CTTGAA GAAATACAA 2949 GAA

Gln Gln LysLeuArg GluLys LysAlaArg LeuGlu GluIleGln Glu Arg Leu Arg Glu Lys Asn Glu Arg Gln Asn Arg Gln Gln Glu Thr Gly Gln Asn Asn Ala Asp Thr Ala Ser Gly Gly Ser Asn Ile Ala Pro Pro Val Pro ValPro Asn Lys Pro Pro Gly Ser GlyGly Arg Lys Ser Gly _ GCT CAA CGA

Asp Ala LysGln Ala Leu Ile Ala Lys Lys GluGlu Lys Ala Gln Arg CAA AAA ACA

Lys Arg LysAsn Leu Ile Ile Ala Leu Lys IleCys Asn Gln Lys Thr GAA GAT AAA

Pro Gly AspPro Asn Leu Tyr Val Leu Val IleGly Gln Glu Asp Lys GTT CAT CGT

Gly Ala SerGly Gly Phe Leu Ala Asp Val AspLys Ser Val His Arg AAA TTA CAA

Asn Ile ValAla Ile Gln Met Asn Glu Gln ProLys Lys Lys Leu Gln GAA ATG AGT

Glu Leu IleIle Asn Ile Leu Val Lys Gly LeuHis Pro Glu Met Ser ATT CTT GGT

Asn Ile ValAsn Phe Asp Ser Tyr Leu Lys AspLeu Trp Ile Leu Gly ATG TCC GAT

Val Ile MetGlu Tyr Glu Gly Gly Leu Thr IleVal Thr Met Ser Asp GAA GGA TGT

His Ser ValMet Thr Gly Gln Ile Val Val ArgGlu Thr Glu Gly Cys TTT AAA ATC

Leu Lys GlyLeu Lys Leu His Ser Gly Val HisArg Asp Phe Lys Ile ATT ATG AAC

Ile Lys SerAsp Asn Leu Leu Asn Asp Gly IleLys Ile Ile Met Asn TGT AAT AAT

Thr Asp PheGly Phe Ala Gln Ile Glu Ile LeuLys Arg Cys Asn Asn Ile Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Ile Val Ser ' CGT AAA GAG TAT GGT CCA AAA GTT GAT GTT TGG TCA TTA GGT ATC ATG 3765 Arg Lys Glu Tyr Gly Pro Lys Val Asp Val Trp Ser Leu Gly Ile Met Ile Ile Glu Met Leu Glu Gly Glu Pro Pro Tyr Leu Asn Glu Thr Pro Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro Lys Leu Lys Asp Pro Glu Ser Leu Ser Tyr Asp Ile Arg Lys Phe Leu Ala Trp Cys Leu Gln Val Rsp Phe ~ n Lys Arg Ala Asp Ala Asp Glu Leu Leu His Asp Asn Phe Ile Thr Glu Cys Asp Asp Val Ser Ser Leu Ser Pro Leu TG

Val Lys Ile Ala Arg Leu Lys Lys Met Ser Glu Ser Asp {2) INFORMATION FOR SEQ ID N0:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1230 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

. Met Ser Ile Leu Ser Glu Asn Asn Pro Thr Pro Thr Ser Ile Thr Asp -Pro Asn Glu Ser Ser His Leu His Asn Pro Glu Leu Asn Ser Gly Thr Arg Val Ala Ser Gly Pro Gly Pro Gly Pro Glu Val Glu Ser Thr Pro Leu Ala Pro Pro Thr Glu Val Met Asn Thr Thr Ser Ala Asn Thr Ser Ser Leu Ser Leu Gly 5er Pro Met His Glu Lys Ile Lys Gln Phe Asp ' 65 70 75 g0 Gln Asp Glu Val Asp Thr Gly Glu Thr Asn Asp Arg Thr Ile Glu 5er Gly Ser Ser Asp Ile Asp Asp Ser Gln Gln Ser His Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Glu Ser Asn Pro Glu Ser Ser Glu Gly Asp Asp Glu Lys Thr Gln Gly Met Pro Pro Arg Met Pro Gly Thr Phe Asn Val Lys Gly Leu His Gln Gly Asp Asp Ser Asp Asn Glu Lys Gln Tyr Thr Glu Leu Thr Lys Ser Ile Asn Lys Arg Thr Ser Lys Asp Ser Tyr Ser Pro Gly Thr Leu Glu Ser Pro Gly Thr Leu Asn Ala Leu Glu Thr Asn Asn Val Ser Pro Ala Val Ile Glu Glu Glu Gln His Thr Leu Ser Leu Glu Asp Leu Ser Leu Ser Leu Gln His Gln Asn Glu Asn Ala Arg Leu Ser Ala Pro Arg Ser Ala Pro Pro Gln Val Pro Thr Ser Lys Thr Ser Ser Phe His Asp Met Ser Leu Val Ile Ser Ser Ser Thr Ser Val His Lys Ile Pro Ser Asn Pro Thr 5er Thr Arg Gly Ser His Leu Ser Ser Tyr Lys Ser Thr Leu Asp Pro Gly Lys Pro Ala Gln Ala Ala Ala Pro Pro Pro Pro Glu Ile Asp Ile Asp Asn Leu Leu Thr Lys Ser Glu Leu Asp Leu Glu Thr Asp Thr Leu Ser Ser Ala Thr Asn Ser Pro Asn Leu Leu Arg Asn Asp Thr Leu Gln Gly Ile Pro Thr Arg Asp Asp G_iu Asn Ile Asp Asp Leu Pro Arg Gln Leu Ser Gln Asn Thr Ser Ala Thr Ser Arg Asn Thr Ser Gly Thr Ser Thr Ser Thr Val Val Lys Asn Ser Arg Ser Gly Thr Ser Lys Ser Thr Ser Thr Ser Thr Ala His Asn Gln Thr Ala Ala Ile Thr Pro Ile Ile Pro Ser His Asn Lys Phe His Gln Gln Val Ile Asn Thr Asn Ala Thr Asn Ser Ser Ser Ser Leu Glu Pro Leu Gly Val Gly Ile Asn Ser Asn Leu Ser Pro Lys Ser Gly Lys Lys Arg Lys Ser Gly Ser Lys Val Arg Gly Val Phe Ser Ser Met Phe Gly Lys Asn Lys Ser Thr Ser Ser Ser Ser Ser Ser Asn Ser Gly Leu ' Asn Ser His Ser Gln Glu Val Asn Ile Lys Ile Ser Thr Pro Phe Asn Ala Lys His Leu Ala His Val Gly Ile Asp Asp Asn Gly Ser Tyr Thr Gly Leu Pro Ile Glu Trp Glu Arg Leu Leu Ser Ala Ser Gly Ile Thr Lys Lys Glu Gln Gln Gln His Pro Gln Ala Val Met Asp Ile Val Ala Phe Tyr Gln Asp Thr Ser Glu Asn Pro Asp Asp Ala Ala Phe Lys Lys Phe His Phe Asp Asn Asn Lys Ser Ser Ser Ser Gly Trp Ser Asn Glu Asn Thr Pro Pro Ala Thr Pro Gly Gly Ser Asn Ser Gly Ser Gly Ser Gly Gly Gly Gly Ala Pro Ser Ser Pro His Arg Thr Pro Pro Ser Ser Ile Ile Glu Lys Asn Asn Val Glu Gln Lys Val Ile Thr Pro Ser Gln Ser Met Pro Thr Lys Thr Glu Ser Lys Gln Leu Glu Asn Gln His Pro His Glu Asp Asn Ala Thr Gln Tyr Thr Pro Arg Thr Pro Thr Ser His Val Gln Glu Gly Gln Phe Ile Pro Ser Arg Pro Ala Pro Lys Pro Pro Ser Thr Pro Leu Ser Ser Met Ser Val Ser His Lys Thr Pro Ser Ser Gln Ser Leu Pro Arg Ser Asp Ser Gln Ser Asp Ile Arg Ser Ser Thr Pro Lys Ser His Gln Asp Val Ser Pro Ser Lys Ile Lys Ile Arg Ser Ile Ser Ser Lys Ser Leu Lys Ser Met Arg Ser Arg Lys Ser Gly Asp Lys Phe Thr His Ile Ala Pro Ala Pro Pro Pro Pro Ser Leu Pro Ser Ile Pro Lys Ser Lys Ser His Ser Ala Ser Leu Ser Ser Gln Leu Arg Pro Ala Thr Asn Gly Ser Thr Thr Ala Pro Ile Pro Ala Ser Ala Ala Phe Gly Gly Glu Asn Asn Ala Leu Pro Lys Gln Arg Ile Asn Glu Phe Lys Ala His Arg Ala Pro Pro Pro Pro Pro Leu Ala Pro Pro Ala Pro Pro Val Pro Pro Ala Pro Pro Ala Asn Leu Leu Ser Glu Gln Thr Ser Glu Ile Pro Gln Gln Arg Thr Ala Pro Leu Gln Ala Leu Ala Asp Val Thr Ala Pro Thr Asn Ile Tyr Glu Ile Gln Gln Thr Lys Tyr Gln Glu Ala Gln Gln Lys Leu Arg Glu Lys Lys Ala Arg Glu Leu Glu Glu Ile Gln Arg Leu Arg Glu Lys Asn Glu Arg Gln Asn Arg Gln Gln Glu Thr Gly Gln Asn Asn Ala Asp Thr Ala Ser Gly Gly Ser Asn Ile Ala Pro Pro Val Pro Val Pro Asn Lys Lys Pro Pro Ser Gly Ser Gly Gly Gly Arg Asp Ala Lys Gln Ala Ala Leu Ile Ala Gln Lys Lys Arg Glu Glu Lys Lys Arg Lys Asn Leu Gln Ile Ile Ala Lys Leu Lys Thr Ile Cys Asn Pro Gly Asp Pro Asn Glu Leu Tyr Val Asp Leu Val Lys Ile Gly Gln Gly Ala Ser Gly Gly Val Phe Leu Ala His Asp Val Arg Asp Lys Ser Asn Ile Val Ala Ile Lys Gln Met Asn Leu Glu Gln Gln Pro Lys Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys Gly Ser Leu His Pro Asn Ile Val Asn Phe Ile Asp Ser Tyr Leu Leu Lys Gly Asp Leu Trp Val Ile Met Glu Tyr Met Glu Gly Gly Ser Leu Thr Asp Ile Val Thr His Ser Val Met Thr Glu Gly Gln Ile'Gly Val Val Cys Arg Glu 1045. 1050 1055 Thr Leu Lys Gly Leu Lys Phe Leu His Ser Lys Gly Val Ile His Arg Asp Ile Lys Ser Asp Asn Ile Leu Leu Asn Met Asp Gly Asn Ile Lys Ile Thr Asp Phe Gly Phe Cys Ala Gln Ile Asn Glu Ile Asn Leu Lys Arg Ile Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Ile Val Ser Arg Lys Glu Tyr Gly Pro Lys Val Asp Val Trp Ser Leu Gly Ile Met Ile Ile Glu Met Leu Glu Gly Glu Pro Pro Tyr Leu Asn Glu Thr Pro Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro Lys Leu Lys Asp Pro Glu Ser Leu Ser Tyr Asp Ile Arg Lys Phe Leu Ala Trp Cys Leu Gln Val Asp Phe Asn Lys Arg Ala Asp Ala Asp Glu Leu Leu His Asp Asn Phe Ile Thr Glu Cys Asp Asp Val Ser Ser Leu Ser Pro Leu Val Lys Ile Ala Arg Leu Lys Lys Met Ser Glu Ser Asp (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3496 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 432...3344 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

AGAAAAAATT TTTAATTCCA

ATCCTAAATTTTTTTTTATATAGCTAGTTTTTGGTTGAAA p~AAAHAAAATAGGGGGAAGG 300 Met Thr Ser Ile Tyr Thr Ser Asp Leu Lys Asn His Arg Arg AlaPro ProProPro AsnGly AlaA1aGly SerGlySer GlySer Gly SerGly SerGlySer GlySer GlySerLeu AlaAsnIle ValThr Ser SerAsn SerLeuGly ValThr AlaAsnGln ThrLysPro IleGln Leu AsnIle AsnSerSer LysArg GlnSerGly TrpValHis ValLys Asp AspGly IlePheThr SerPhe ArgTrpAsn LysArgPhe MetVal Ile AsnAsp LysThrLeu AsnPhe TyrLysGln GluProTyr SerSer Asp GlyAsn SerAsnSer AsnThr ProAspLeu SerPhePro LeuTyr Leu IleAsn AsnIleAsn LeuLys ProAsn5er GlyTyrSer LysThr Ser GlnSer PheGluIle ValPro LysAsnAsn AsnLysSer IleLeu Ile SerVal LysThrAsn AsnAsp TyrLeuAsp TrpLeuAsp AlaPhe Thr ThrLys CysProLeu ValGln IleGlyGlu AsnAsnSer GlyVal Ser SerSer HisProHis LeuGln IleGlnHis LeuThrAsn GlySer Leu AsnGly AsnSerSer Ser5er ProThrSer GlyLeuLeu SerSer WO 98/18927 PCTlCA97/00809 ACT GTT GGT ATT
GGA AAT
GGT
AAT

Ser ValLeu Thr Gly AsnSer Gly Ser Pro AsnPhe Gly Val Gly Ile GTG CCT AGT AAT

Thr HisLys Val His GlyPhe Rsp Ala Gly PheThr Val Pro Ser Asn TGG TTA CAT AAA

Gly LeuPro Asp Thr LysSer Leu Gln Ser IleThr Trp Leu His Lys AAA GCT ATT GTT

Asn GluAsp Trp Lys AspPro Val Val Glu LeuGlu Lys Ala Ile Val AAT TCA GCT ACT

Phe TyrSer Asp Ile GlyGly Asn Ala Gly ProIle Asn Ser Ala Thr AAT AAC AAT AAT

Gly SerPro Met Ile SerLys Thr Asn Asn AspPro Asn Asn Asn Asn ACC GTC GAG AAT

_ Asn AsnTyr Ser Ser LysAsn Asn Gln Ala LeuGln Thr Val Glu Asn TGG AAA AAA CAA
CCT TCT TTC
ACT AAA

GluTrp ValLysPro ProAlaLys SerThrVal SerGln Phe Pro Lys CAA

SerArg AlaAlaPro LysProPro ThrProTyr HisLeu Thr Leu Gln CCT

AsnGly SerSerHis GlnHisThr SerSerSer GlySer Leu Ser Pro 370 375, 380 ACT

SerGly AsnAsnAsn AsnAsnAsn SerThrAsn AsnAsn Asn Lys Thr AACGTT TCACCATTG AATAATTTG ATGAATAAA TCTGAA..-CTT CCT 1670 ATT

AsnVal SerProLeu AsnAsnLeu MetAsnLys SerGlu Leu Pro Ile GAT

AlaArg ArgAlaPro Pro.ProPro ThrSerGly ThrSer Ser Thr Asp CAA

TyrSer AsnLysAsn HisGlnAsp ArgSerGly TyrGlu Gln Arg Gln CAA

GlnGln ArgThrAsp SerSerGln GlnGlnGln GlnGln Lys His Gln Gln TyrGln GlnLys SerGlnGln GlnGlnGln GlnPro GlnGlnPro Leu SerLeu HisGln GlyGlyThr SerHisIle ProLys GlnValPro Pro ThrLeu ProSer SerGlyPro ProThrGln AlaAla SerGlyLys Ser MetPro SerLys IleHisPro AspLeuLys IleGln GlnGlyThr Asn AsnTyr IleLys SerSerGly ThrAspAla AsnGln ValAspGly Asp AlaLys GlnPhe IleLysPro PheAsnLeu GlnLeu LysLysSer Gln GlnGln LeuAla SerLysGln ProSerPro ProSer SerGlnGln Gln GlnGln LysPro MetThrSer HisGlyLeu MetGly ThrSerHis Ser ValThr LysPro LeuAsnPro ValAsnAsp ProIle LysProLeu Asn LeuLys SerSer LysSerLys GluAlaLeu AsnGlu ThrLeuGly Val LeuLys ThrPro SerProThr AspLysSer AsnLys ProThrAla Pro AlaSer GlyPro AlaValThr LysThrAla LysGln LeuLysLys Glu ArgGlu ArgLeu AsnAspLeu GlnIleIle AlaLys LeuLysThr Val ValAsn AsnGln AspProLys ProLeuPhe ArgIle ValGluLys CAA AGT AAT ATG
GGT ATC
AAA

AlaGly Gly Ala Gly Val TyrLeu AlaGluMet IleLys Gln Ser Asn AAT ATT ATT

AspAsn Arg Lys Ala Lys GlnMet AspLeuAsp AlaGln Asn Ile Ile AAA ATA AAT

ProArg Glu Leu Ile Glu IleLeu ValMetLys AspSer Lys Ile Asn 720 725 _ 730 AAA GTT TTT

GlnHis Asn Ile Asn Leu AspSer TyrLeuIle GlyAsp Lys Val Phe TTA ATT GAA

AsnGlu Trp Val Met Tyr MetGln GlyGlySer LeuThr Leu Ile Glu ATT AAT TTT

GluIle Glu Asn Asp Lys LeuAsn GluLysGln IleAla Ile Asn Phe TGT ACC AAG

ThrIle Phe Glu Leu Gly LeuGln HisLeuHis LysLys Cys Thr Lys ATT GAT AAA

HisIle His Arg Ile Ser AspAsn ValLeuLeu AspAla Ile Asp Lys AAT ATC GAT

TyrGly Val Lys Thr Phe GlyPhe CysAlaLys LeuThr Asn Ile Asp AGA CGT ACA

AspGln Asn Lys Ala Met ValGly ThrProTyr TrpMet Arg Arg Thr GAA AAA AAG

AlaPro Val Val Gln Glu TyrAsp GluLysVal AspVal Glu Lys Lys TTG ATG ATT

TrpSer Gly Ile Thr Glu MetIle GluGlyGlu ProPro Leu Met Ile AAT CCA AAA

TyrLeu Glu Glu Leu Ala LeuTyr LeuIleAla ThrAsn Asn Pro Lys eso 885 s9o CCA AAA CCC

GlyThr Lys Leu Lys Glu LeuLeu SerAsnSer IleLys Pro Lys Pro TTA TGT TGT

LysPhe Ser Ile Leu Val AspVal ArgTyrArg AlaSer Leu Cys Cys TCA ATT AAA

ThrAsp Glu Leu Leu Glu His Phe Gln His Lys Ser Gly Ser Ile Lys TTA TGG AAG

IleGlu Glu Leu Ala Pro Leu Glu Lys Lys Gln Gln Gln Leu Trp Lys ACA GAT

HisGln Gln His Lys Gln Glu Leu Thr Gly Phe Ala Thr Asp TAGAAAACTG TATATTTATG
GTACTTTGGT TGATGTGTTG

TAGTTTAGAT AGTTTAGTTT
TTGGATTTTT TTCAATTTTT

ACAATATTCT
AGA

(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 971 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Thr Ser Ile Tyr Thr Ser Asp Leu Lys Asn His Arg Arg Ala Pro Pro Pro Pro Asn Gly Ala Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Leu Ala Rsn Ile Val Thr Ser Ser Asn Ser Leu Gly Val Thr Ala Asn Gln Thr Lys Pro Ile Gln Leu Asn Ile Asn Ser Ser Lys Arg Gln Ser Gly Trp Val His Val Lys Asp Asp Gly Ile Phe Thr Ser Phe Arg Trp Asn Lys Arg Phe Met Val Ile Asn Asp Lys Thr Leu Asn Phe Tyr Lys Gln Glu Pro Tyr Ser Ser Asp Gly Asn Ser Asn Ser Asn Thr Pro Asp Leu Ser Phe Pro Leu Tyr Leu Ile Asn Asn Ile Asn Leu Lys Pro Asn Ser Gly Tyr Ser Lys Thr Ser Gln Ser Phe Glu Ile Val Pro Lys Asn Asn Asn Lys 5er Ile Leu Ile Ser Val Lys Thr Asn Asn Asp Tyr Leu Asp Trp Leu Asp Ala Phe Thr Thr Lys Cys Pro Leu Val Gln Ile Gly Glu Asn Asn Ser Gly Val Ser Ser Ser His Pro His Leu Gln Ile Gln His Leu Thr Asn Gly Ser Leu Asn Gly Asn Ser Ser Ser Ser Pro Thr Ser Gly Leu Leu Ser Ser Ser Val Leu Thr Gly Gly Asn Ser Gly Val Ser Gly Pro Ile Asn Phe Thr His Lys Val His Val Gly Phe Asp Pro Ala Ser Gly Asn Phe Thr Gly Leu Pro Asp Thr Trp Lys Ser Leu Leu Gln His Ser Lys Ile Thr Asn Glu Asp Trp Lys Lys Asp Pro Val Ala Val Ile Glu Val Leu Glu Phe Tyr Ser Asp Ile Asn Gly Gly Asn Ser Ala Ala Gly Thr Pro Ile Gly Ser Pro Met Ile Asn Ser Lys Thr Asn Asn Asn Asn Asn Asp Pro Asn Asn Tyr Ser Ser Thr Lys Asn Asn Val Gln Glu Ala Asn Leu Gln Glu Trp Val Lys Pro Pro Ala Lys Ser Thr Val Ser Gln Phe Lys Pro Ser Arg Ala Ala Pro Lys Pro Pro Thr Pro Tyr His Leu Thr Gln Leu Asn Gly Ser Ser His Gln His Thr Ser Ser Ser Gly Ser Leu Pro Ser Ser Gly Asn Asn Asn Asn Asn Asn Ser Thr Asn Asn Asn Asn Thr Lys Asn Val Ser Pro Leu Asn Asn Leu Met Asn Lys Ser Glu Leu Ile Pro Ala Arg Arg Ala Pro Pro Pro Pro Thr Ser Gly Thr Ser Ser Asp Thr Tyr Ser Asn Lys Asn His Gln Asp Arg Ser Gly Tyr Glu Gln Gln Arg Gln Gln Arg Thr Asp Ser Ser Gln Gln Gln Gln Gln Gln Lys Gln His Gln Tyr Gln Gln Lys Ser Gln Gln Gln Gln Gln Gln Pro Gln Gln Pro Leu Ser Leu His Gln Gly Gly Thr Ser His Ile Pro Lys Gln Val Pro Pro Thr Leu Pro Ser Ser Gly Pro Pro Thr Gln Ala Ata Ser Gly Lys Ser Met Pro Ser Lys Ile His Pro Asp Leu Lys Ile Gln Gln Gly Thr Asn Asn Tyr Ile Lys Ser Ser Gly Thr Asp Ala Asn Gln Val Asp Gly Asp Ala Lys Gln Phe Ile Lys Pro Phe Asn Leu Gln Leu Lys Lys Ser Gln Gln Gln Leu Ala Ser Lys Gln Pro Ser Pro Pro Ser Ser Gln Gln Gln Gln Gln Lys Pro Met Thr Ser His Gly Leu Met Gly Thr Ser His Ser Val Thr Lys Pro Leu Asn Pro Val Asn Asp Pro Ile Lys Pro Leu Asn Leu Lys Ser Ser Lys Ser Lys Glu Ala Leu Asn Glu Thr Leu Gly Val Leu Lys Thr Pro Ser Pro Thr Asp Lys Ser Asn Lys Pro Thr Ala Pro Ala Ser Gly Pro Ala Val Thr Lys Thr Ala Lys Gln Leu Lys Lys Glu Arg Glu Arg Leu Asn Asp Leu Gln Ile Ile Ala Lys Leu Lys Thr Val Val Asn Asn Gln Asp Pro Lys Pro Leu Phe Arg Ile Val Glu Lys A1a Gly Gln Gly Ala Ser Gly TyrLeu GluMet IleLysAsp Asn Ala Asn Val Asn Arg LysIle AlaIleLys GlnMet AspLeuAsp AlaGlnPro ArgLys Glu LeuIle IleAsnGlu IleLeu ValMetLys AspSerGln HisLys Asn IleVal AsnPheLeu AspSer TyrLeuIle GlyAspAsn GluLeu Trp ValIle MetGluTyr MetGln GlyGlySer LeuThrGlu IleIle Glu AsnAsn AspPheLys LeuAsn GluLysGln IleAlaThr IleCys Phe GluThr LeuLysGly LeuGln HisLeuHis LysLysHis IleIle 7as 790 7ss 800 His ArgAsp IleLysSer AspAsn ValLeuLeu AspAlaTyr GlyAsn Val LysIle ThrAspPhe GlyPhe CysAlaLys LeuThrAsp GlnArg Asn LysArg AlaThrMet ValGly ThrProTyr TrpMetAla ProGlu Val ValLys GlnLysGlu TyrAsp GluLysVal AspValTrp SerLeu Gly IleMet ThrIleGlu MetIle GluGlyGlu ProProTyr LeuAsn Glu GluPro LeuLysAla LeuTyr LeuIleAla ThrAsnGly ThrPro Lys LeuLys LysProGlu LeuLeu SerAsnSer TleLysLys PheLeu Ser IleCys LeuCysVal AspVal ArgTyrArg AlaSerThr AspGlu Leu LeuGlu HisSerPhe IleGln HisLysSer GlyLysIle GluGlu Leu AlaPro LeuLeuGlu TrpLys LysGlnGln GlnLysHis GlnGln His LysGln GluThrLeu AspThr GlyPheAla (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1031 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 271...843 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

_ TTTTAGTTCC TATAATCATA CAGATTTCTC GTCCTAAATC TATTTTTATT GTTATTTTTA 180 w Met Gln Thr Ile Lys Cys Val Val GAT TGC TTA ACC

ValGly Gly Ala Val Gly Lys Thr Leu Ile Ser Tyr Asp Cys Leu Thr AAA CCT ACT TAT

ThrSer Phe Pro Ala Asp Tyr Val Val Phe Asp Asn Lys Pro Thr Tyr 25 . 30 35 q0 ACC CCA TTT TTT

AlaVal Val Met Ile Gly Asp Glu Thr Leu Gly Leu Thr Pro Phe Phe GCT AGA TTA TAT

AspThr Gly Gln Glu Asp Tyr Asp Arg Pro Leu Ser Ala Arg Leu Tyr ACT TTT TCC GCT

ProSer Asp Val Phe Leu Val Cys Val Ile Ser Pro Thr Phe Ser Ala GAA TTC CCA CPrT

SerPhe Asn Val Lys Glu Lys Trp Glu Val His His Glu Phe Pro His GGT GGT ACC CGA

CysPro Val Pro Ile Ile Ile Val Gln Thr Asp Leu Gly Gly Thr Arg GAT CAC AGA CCA

AsnAsp Val Ile Leu Gln Arg Leu Gln Lys Leu Ser Asp His Arg Pro CAG GCT AAG GTC

IleThr Glu Gln Gly Glu Lys Leu Glu Leu Arg Ala Gln Ala Lys Val GTT CAA AGA GTG

LysTyr Glu Cys Ser Ala Leu Thr Gly Leu Lys Thr Val Gln Arg Val GAG GAA CCT AAA

PheAsp Ala Ile Val Ala Ala Leu Pro Val Ile Lys Glu Glu Pro Lys AAG ATACTAGAAG ATAGAGGATA TTGG

SerLys Cys Thr Ile Leu Lys TACATGAGAT
ATTGAATATC
TATCATTAAA

TTTAGGTTTG
ATCTCGTTTG
ATGTGTTGGG

AAAAGATGTG
TGTAAGACTC
TAGA

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 191 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
MetGln ThrIleLys CysValVal ValGly Gly ValGlyLys Asp Ala ThrCys LeuLeuIle SerTyrThr ThrSerLys PhePro AlaAspTyr ValPro ThrValPhe AspAsnTyr AlaValThr ValMet IleGlyAsp GluPro PheThrLeu GlyLeuPhe AspThrAla GlyGln GluAspTyr AspArg LeuArgPro LeuSerTyr ProSerThr AspVal PheLeuVal CysPhe SerValIle SerProAla SerPheGlu AsnVal LysGluLys TrpPhe ProGluVal HisHisHis CysProGly ValPro IleIleIle ValGly ThrGlnThr AspLeuArg AsnAspAsp ValIle LeuGlnArg LeuHis ArgGlnLys LeuSerPro IleThrGln GluGln GlyGluLys LeuAla LysGluLeu ArgAlaVal LysTyrVal GluCys SerAlaLeu ThrGln ArgGlyLeu LysThrVal PheAspGlu AlaIle ValAlaAla LeuGlu ProProVal IleLysLys SerLysLys CysThr IleLeu (2) INFORMATION FOR SEQ ID NO:11:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2231 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 291...2195 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:

AGTCTATCAG
TTATATCTCC
CTCCCCTTTT

Met Ile AGT AGT AGT

Lys ThrPheArg Lys Lys Arg Leu Ser Asn Ser Ser Pro Ser Ser Ser CGA TCA CAA

Lys LysThrIle Ser Val Ser Ser Thr Ser Asn Thr Ser Arg Ser Gln CAA GTC GCT

His AspGlyIle Leu Ser Pro Lys Lys Ile Arg Leu Tyr Gln Val Ala GGT AAA AAA

Asp TyrGluPro Gln Pro Gly Glu Leu Phe Phe Gly Asp Gly Lys Lys AAT GAA AAA

Phe PheHisVal Leu Asp Val Asp Asp Leu His Glu Ala Asn Glu Lys ATA CCA CAA

Glu ArgAsnGly Trp Glu Ala Thr Asn Met Thr Leu Lys Ile Pro Gln AGT TTT TCT

Gly MetValPro Ile Tyr Phe Glu Ile Asp Arg Arg Pro Ser Phe Ser TCA AAT GAT

Thr ValThrAla Ser Asn Ser Phe Thr Ser Ile Ile Gln Ser Asn Asp GGA ACA CGA

His GlnHisGln Gln Ile His Asri Gly Gly Asn Asn Leu Gly Thr Arg GCT GAA GCT

Asn GlnThrLeu Tyr Val Thr Leu Tyr Phe Lys Glu Arg Ala Glu Ala ATA AAT ATT

Asp AspGluLeu Asp Met Pro Asn Glu Leu Ile Cys Ala Ile Asn Ile TGG CCA CGA

His HisAspTyr Glu Phe Ile Ala Lys Ile Asn Leu Gly Trp Pro Arg CCT AAA GAT

Gly ProGlyLeu Val Val Ser Tyr Val Ile Ile Leu Leu Pro Lys Asp AAT TAT ACA CAA
ACA

Asn Pro Ser His Thr SerIleAsp ThrSerArg ArgSer Gln Asn Tyr CAA AAT

Val Ile Val Ile Gly PheAsnIle ProThrVal GluGln Trp Gln Asn CAA AAA

Lys Asn Thr Ala-LysTyr GlnAlaSer ThrIlePro LeuGly Ser Gln GGA ACT

Ile Ser Ser Gly Pro ProThrSer AlaAsnSer GlnTyr Phe Gly Thr CAT ACT

Asp Asn Thr Met Ser AsnArgSer SerLeuGly SerSer Ile His Thr ATT AGT

Ser Ile Glu Ala Val AspSerTyr GlnLeuAsp HisGly Arg Ile Ser TAT ACT

Tyr Gln Ser Ile Ala ArgLeuAsn AsnGlyArg IleArg Tyr Tyr Thr CGA CAA

Leu Tyr Tyr Tyr Asp PheTyrAsp LeuGlnVal LysLeu Leu Arg Gln TTT GAA

Glu Leu Pro Tyr Ala GlyArgIle GluAsnSer LysArg Ile Phe Glu TCT GGA

Ile Pro Ile Pro Pro LeuIleAsn ValAsnAsp SerIle Ser Ser Gly CGA AAA

Lys Leu Arg Glu Leu AspTyrTyr LeuSerAsn LeuIle Ala Arg Lys AGT TCT

Leu Pro His Ile Arg SerGluGlu ValLeuLys LeuPhe Asp Ser Ser GAT TTT

Val Leu Asn Gly Asp ArgGluThr AspAlaIle AsnLys Arg Asp Phe ARA AGT

Phe Ser Pro Ile Gln LysSerAsn SerHisGln AspArg Leu Lys Ser Ser Gln Tyr Ser Asn Phe Asn Val Leu Gln Gln Gln Gln Gln Gln Gln CAA TAT GGT GAT
CAG

Gln GlnGln TyrAla HisHis SerArgGly Ser Asn SerPro Gln Asp AAT TCT

Thr GluSer SerGly SerAsn LeuIleAsn Ser Ser HisAsn Asn Ser TCA CCA

Asp SerLeu SerSer SerPro ProProPro Pro Gln ThrVal Ser Pro 485 . 490 495 ACC GAC

Thr ThrAsn ThrThr AsnThr ThrIleThr Thr Ser SerSer Thr Asp CAA GAT

Lys ProLys AlaLys ValLys PheTyrPhe Asp Asp IlePhe Gln Asp TTA TTA

Val LeuIle ProThr AsnLeu ArgLeuGln Asp Lys SerLys Leu Leu TTT TAT

Leu LysArg LeuGlu LeuAsp IleThrTyr Lys Glu LysPro Phe Tyr CAA TTA

Asp GlnGln LysPro ThrSer GluSerIle His Phe LeuLys Gln Leu GAT AGC

Asn PheGlu AspPhe LeuIle GluAsnGlu Thr Asn AsnAsn Asp Ser CTG GAA

Asn GluIle AspPhe GluAsn GluIleIle Lys Lys LeuGly Leu Glu TTT ATT

Glu GluVal AsnAsp AspGlu LysPheGln Ser Leu PheAsp Phe Ile TGT TCAATAAGAG
AGAGAGA

Lys LysLeu MetVal LeuVal Tyr Cys ~~~~T 2231 (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 635 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Ile Lys Thr Phe Arg Lys Ser Lys Arg Leu Ser Ser Asn Ser Ser Ser Pro Lys Lys Thr Ile Ser Arg Val Ser Ser Thr Ser Ser Asn Gln Thr Ser His Asp Gly Ile Leu Gln Ser Pro Lys Lys Val Ile Arg Ala Leu Tyr Asp Tyr Glu Pro Gln Gly Pro Gly Glu Leu Lys Phe Phe Lys Gly Asp Phe Phe His Val Leu Asn Asp Val Asp Asp Glu Leu His Lys Glu Ala Glu Arg Asn Gly Trp Ile Glu Ala Thr Asn Pro Met Thr Gln Leu Lys Gly Met Val Pro Ile Ser Tyr Phe Glu Ile Phe Asp Arg Ser Arg Pro Thr Val Thr Ala Ser Ser Asn Ser Phe Thr Asn Ser Ile Asp Ile Gln His Gln His Gln Gln Gly Ile His Asn Gly Thr Gly Asn Arg Asn Leu Asn Gln Thr Leu Tyr Ala Val Thr Leu Tyr Glu Phe Lys Ala Glu Arg Asp Asp Glu Leu Asp Ile Met Pro Asn Glu Asn Leu Ile Ile Cys Ala His His Asp Tyr Glu Trp Phe Ile Ala Lys Pro Ile Asn Arg Leu Gly Gly Pro Gly Leu Val Pro Val Ser Tyr Val Lys Ile Ile Asp Leu Leu Asn Pro Asn Ser His Tyr Thr Ser Ile Asp Thr Ser Arg Arg Ser Gln Val Ile Gln Val Ile Asn Gly Phe Asn Ile Pro Thr Val Glu Gln Trp Lys Asn Gln Thr Ala Lys Tyr Gln Ala Ser Thr ile Pro Leu Gly Ser Ile Ser Gly Ser Gly Thr Pro Pro Thr Ser Ala Asn Ser Gln Tyr Phe Asp Asn His Thr Met Thr Ser Asn Arg Ser Ser Leu Gly Ser Ser Ile Ser Ile Ile Glu Ala Ser Val Asp Ser Tyr Gln Leu Asp His Gly Arg Tyr Gln Tyr Ser Ile Thr Ala Arg Leu Asn Asn-Gly Arg Ile 305 31.0 315 320 Arg Tyr Leu Tyr Arg Tyr Tyr Gln Asp Phe Tyr Asp Leu Gln Val Lys Leu Leu Glu Leu Phe Pro Tyr Glu Ala Gly Arg Ile Glu Rsn Ser Lys Arg Ile Ile Pro Ser Ile Pro Gly Pro Leu Ile Asn Val Asn Asp Ser Ile Ser Lys Leu Arg Arg Glu Lys Leu Asp Tyr Tyr Leu Ser Asn Leu Ile Ala Leu Pro Ser His Ile Ser Arg Ser Glu Glu Val Leu Lys Leu Phe Asp Val Leu Asp Asn Gly Phe Asp Arg Glu Thr Asp Ala Ile Asn Lys Arg Phe Ser Lys Pro Ile Ser Gln Lys Ser Asn Ser His Gln Asp Arg Leu Ser Gln Tyr Ser Asn Phe Asn Val Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Tyr Ala His His Ser Arg Gly Ser Asp Asn Ser Pro Thr Asn Glu Ser Ser Gly Ser Asn Leu Ile Asn Ser Ser Ser _ His Asn Asp Ser Ser Leu Ser Ser Ser Pro Pro Pro Pro Pro Pro Gln Thr Val Thr Thr Thr Asn Thr Thr Asn Thr Thr Ile Thr Thr Asp Ser Ser Ser Lys Gln Pro Lys Ala Lys Val Lys Phe Tyr Phe Asp Asp Asp Ile Phe Val Leu Leu Ile Pro Thr Asn Leu Arg Leu Gln Asp Leu Lys Ser Lys Leu Phe Lys Arg Leu Glu Leu Asp Ile Thr Tyr Lys Tyr Glu Lys Pro Asp Gln Gln Gln Lys Pro Thr Ser Glu Ser Ile His Leu Phe Leu Lys Asn Asp Phe Glu Asp Phe Leu Ile Glu Asn Glu Thr Ser Asn Asn Asn Asn Leu Glu Ile Asp Phe Glu Asn Glu Ile Ile Lys Glu Lys Leu Gly Glu Phe Glu Val Asn Asp Asp Glu Lys Phe Gln Ser Ile Leu Phe Asp Lys Cys Lys Leu Met Val Leu Val Tyr

Claims (6)

WE CLAIM:

1. An in vitro screening test for compounds to inhibit the activity of at least one protein selected from the group consisting of CaCla4p (SEQ ID NO:8), Cst20p (SEQ ID NO:6), CaCdc42p (SEQ ID NO:10) and CaBem1p (SEQ ID NO:12), which comprises:
a) provinding a mixture comprising a tested compound and at least one of said proteins; and b) means to monitor the activity of said at least one protein;
thereby compounds are tested for their inhibiting potential.

2. The screening test of claim 1, wherein the inhibition of the interactions between CaCla4p (SEQ ID NO:8) and CaCdc42p (SEQ ID NO:10) is determined.

3. The screening test of claim 1, wherein she inhibition of the interactions between Cst20p (SEQ ID NO:6) and CaCdc42p (SEQ ID NO:10) is determined.

4. The screening test of claim 1, wherein the inhibition of the interactions between CaCla4p (SEQ ID NO:8) and CaBem1p (SEQ ID NO:12) is determined.

5. The screening test of claim 1, wherein the inhibition of the interactions between Cst20p (SEQ =D NO:6) and CaBem1p (SEQ ID NO:12) is determined.

6. A method for determining at least one gene involved in filamentous growth associated with virulence, which comprises disrupting and analyzing one gene selected from the group consisting of CaCla4p (SEQ ID NO:8), Cst20p (SEQ
ID NO:6), CaCdc42p (SEQ ID NO:10) and CaBem1p (SEQ ID
NO:12), in Candida albicans, thereby determining the role in virulence of said gene.