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WO1998018927A1 - Proteines de candida albicans, associees a la virulence et a la formation d'hyphes, et leurs utilisations - Google Patents

Proteines de candida albicans, associees a la virulence et a la formation d'hyphes, et leurs utilisations Download PDF

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WO1998018927A1
WO1998018927A1 PCT/CA1997/000809 CA9700809W WO9818927A1 WO 1998018927 A1 WO1998018927 A1 WO 1998018927A1 CA 9700809 W CA9700809 W CA 9700809W WO 9818927 A1 WO9818927 A1 WO 9818927A1
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Ekkehard Leberer
David Y. Thomas
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National Research Council Of Canada
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Priority to US10/093,524 priority patent/US20030166886A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi

Definitions

  • the 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 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 sexual 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.
  • 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) .
  • S. cerevisiae 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.
  • morphological transitions are controlled by signaling components 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 cascade including Ste7 ⁇ (a homolog of MAP kinase kinase or MEK), Stellp (a MEK kinase homolog) and Ste20p (a MEK kinase kinase) (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al . (1993) Science 262, 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).
  • Ste20p serine/threonine protein kinases are thought to be involved in triggering 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) EMBO 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.
  • Cst20p a C. albicans homolog of the Ste20p protein kinase
  • Cst20p a C. albicans homolog of the Ste20p protein kinase
  • CaCla4p a C. albicans homolog of the Cla4p protein kinase
  • CaCla4p a C. albicans homolog of the Cla4p protein kinase
  • 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 provide screening tests for inhibitors of CaCla4p, Cst20p, CaCdc42p and CaBemlp or of their interactions.
  • 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, Cladosporiu , 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.
  • an in vi tro 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.
  • 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.
  • FIG. IA to ID illustrate photomicrographs which show that C. albicans CST20 gene complements defects in pseudohyphal growth of ste20/ste20 S. cerevisiae diploid cells.
  • Figs. 2A to 2C show the morphology of S. cere- visiae MAT ⁇ cells (strain YEL306-1A) deleted for STE20 and CLA4, and transformed with plasmids expressing CLA4 (Fig. 2A), STE20 (Fig. 2B) and C. albicans CST20 (Fig. 2C).
  • Figs. 3A to 3C show the nucleotide (SEQ ID NO: 5) and predicted amino acid sequences of CST20 (SEQ ID NO : 6 ) .
  • 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 cst20 ⁇ /cst20 ⁇ : : URA3 strain CDH22
  • B ura3/ura3 cst20 ⁇ /cst20 ⁇ : : CST20 : : URA3 strain CDH36 (obtained by reintegration of CST20 into strain CDH25 by homologous recombination using linearized plasmid PDH190)
  • C ura3/ura3 cst20 ⁇ /cst20 ⁇ strain CDH25 transformed with plasmids pYPBl-ADHpt
  • E pYPBl- ADH ⁇ t-HST7
  • E ura3/ura3 hst l ⁇ /hst l ⁇ strain CDH12 transformed with plasmids pVEC
  • Figs. 7A to 7B illustrate the nucleotide (SEQ ID NO: 7) and predicted amino acid (SEQ ID NO: 8) sequences of CaCLA4.
  • Fig. 8A illustrates the deletion of CaCLA4 in C. albicans .
  • Fig. 8C illustrates the Northern blot analysis with the CaCLA4 fragment as a probe.
  • PCR with the divergent oligodeoxynucleotides OEL109 and OELllO was used to delete the coding sequence of CaCLA4.
  • 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 NO: 9) and predicted amino acid (SEQ ID NO: 10) sequences of CaCdc42p.
  • Figs. 12A to 12B illustrate the nucleotide (SEQ ID NO: 11) and predicted amino acid (SEQ ID NO: 12) sequences of CaBemlp.
  • 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 mycelial formation in asexual C. albicans .
  • C. albicans cells into kidneys after infection into mice and completely suppressed virulence in the mouse model.
  • hyphal formation of C. albicans mediated by the CaCla4p protein kinase may contribute to the pathogenicity of this dimorphic fungus.
  • 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) K 2 HP0 4 , 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 stationary 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.
  • the CST20 gene was isolated from a genomic C. albicans library constructed in plasmid YEp352 from genomic DNA of the clinical isolate W01 (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 FUSl : :HIS3 expression and mating in S. cerevisiae strain YEL64 which was disrupted in STE20.
  • the S. cerevisiae MAT ⁇ strain YEL257-1A-2 deleted for STE20 and CLA4 and carrying plasmid pDH129 with CLA4 under control of the GAL1 promoter was transformed 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.
  • a plasmid that contained CST20- lanking 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 0DH68 (5 1 - CGGGATCCAGACCAACCACTCGAACTACT-3' (SEQ ID N0:1) and ODH69 ( 5 ' -CGGGATCCGAAGGTGAACCACCATATTTG-3 ' ( SEQ ID N0:2); newly introduced BamHI sites are underlined) and plasmid pDH119 as a template.
  • a plasmid that contained CaCLA4 flanking sequences joined with Bglll sites was then created by PCR using the divergent oligodeoxynucleotide primers OEL109 (5 1 - GAAGATCTTGTAATCAATGTTCCCGTGGA-3' (SEQ ID NO : 3 ) and OELllO ( 5 ' -GAAGATCTCATCGTGATATTAAATCCGAT-3 ' ( SEQ ID NO:4); newly introduced Bglll sites are underlined) and plasmid pDH205 as template.
  • the amplified DNA was cleaved with Bglll and ligated with a 4 kb BamHI-Bglll 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 transformed into the Ura " C. albicans strain CAI4 (Fonzi, W. A. & Irwin, M. Y.
  • the C. albicans integration plasmid pDH190 was constructed by subcloning a Kpnl to PstI fragment of CST20 into pBS-cC7RA3 (pBluescript KS( + ) into which the C. albicans URA3 gene was cloned between the NotI and Xbal sites of the polylinker). The integration plasmid was then linearized with Nsil and transformed into C. albicans to target integration into the Nsil site of the CST20 ⁇ : :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 Kpnl to SacI insert of YEp 352-CaCLA4 into plasmid pVEC.
  • 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.
  • 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
  • the diploid strain YEL306 heterozygous for ste20 ⁇ : : TRP1 /STE20 cla4 ⁇ : : 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 ste20 ⁇ cla4 ⁇ spores were obtained from transformants with the plasmid without insert, but were obtained from transformants with plasmids carrying CLA4 (Fig. 2A), STE20 (Fig. 2B) or CST20 (Fig. 2C).
  • the open reading frame of CST20 is capable of encoding a protein of 1,229 amino acids with a pre- dieted molecular weight of 133 kDa and a domain structure characteristic of the Ste20p/p65- PAK 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) EMBO 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 present at similar levels in yeast cells grown in YPD at room temperature and germ tubes induced by a temperature shift to 37°C. Isolation and characterization of CaCLA4
  • 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 CLA4 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 protein 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 p ⁇ - ⁇ (Fig. 7).
  • the amino terminal, noncatalytic region contains a sequence from amino acid residues 69 to 180 with similarity to pleckstrin homology (PH) domains and a sequence from amino acid residues 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 noncatalytic sequences are less conserved. Chromosomal deletion of CST20
  • Homologous recombination was used in a multistep procedure to partially delete CST20 in a URA ⁇ C. albi - cans strain (Fig. 4A) .
  • PCR with the divergent oligodeoxynucleotides 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 Xhol were from following strains: Lane #1, CAI4 ( ura3/ura3 CST20/CST20 ) ; lane 2, CDH15 ⁇ ura3/ura3 CST20/cst20 ⁇ : :hisG-URA3-hisG) ; lane 3, CDH18 ( ura3/ura3 CST20/cst20 ⁇ : :hisG) ; lane 4, CDH22 ( ura3/ura3 cst20 ⁇ : :hisG-URA3-hisG/cst20 ⁇ : :hisG) ; lane 5, CDH25 ⁇ ura3/ura3 cst20 ⁇ : :hisG/cst20 ⁇ : :hisG) .
  • Northern blots showed that the CST20 transcript was absent in the corresponding homozygous deletion strains.
  • 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 reintegrated into the genome by targeted homologous recombination (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 glucosamine. Normal hyphae formation was also observed on rice agar and on agar containing Lee ' s medium or 10% serum.
  • Fig. 8A shows the restriction endonu- clease map of CaCLA4.
  • the coding sequence is indicated by the arrow.
  • PCR with the divergent oligodeoxynucleotides OEL109 and OELllO 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 endonuclease restriction sites are as follows: B, BamHI; Bg, Bglll; E, EcdRl ; H, HindiII; P, PstI; S, Sad ; X, Xbal .
  • the deletions were confirmed by Southern blot analyses (Fig. 8B). Southern blot analysis with a 1.1 kb CaCLA4 fragment from Pstl-Xbal 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/cacla4 ⁇ : :hisG-URA3-hisG) ; 3, CDH88 ( ura3/ura3 CaCLA4/cacla4 ⁇ : :hisG) ; 4, CLJ1 ⁇ ura3/ura3 cacla4 ⁇ : :hisG-URA3- hisG/cacla4 ⁇ : :hisG) ; and 5, CLJ5 ⁇ ura3/ura3 cacla4 ⁇ : :hisG/cacla4 ⁇ : :hisG) .
  • mice were injected intravenously with wild type and mutant strains and monitored for survival and for fun- gal invasion into kidneys.
  • the Ura- strain CAI4 was not pathogenic (Figs. 6A and B).
  • infection with Ura + wild type cells resulted in rapid mortality with a rate that was dependent on the dose of injected cells (1 x 10 ⁇ cells in Fig. 6A, and 1 x 10 ⁇ cells in Fig. 6B).
  • Survival was significantly prolonged, however, in mice infected with Ura + cells deleted for both alleles of CST20 ( cst20 ⁇ /cst20 ⁇ : : URA3 ) .
  • a C. albicans homolog of the CaCDC42 gene was cloned by functional complementation of the temperature-sensitive growth defect of S. cerevisiae cells carrying the cdc42-l ⁇ s mutation.
  • the growth defect was fully complemented by plasmid YEp352-CaCDC42.
  • the open reading frame of the CaCDC42 gene is capable of encoding 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 CaBEMl gene was cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM1 gene.
  • Ste20p fulfills multiple functions during mating (Leberer, E. et al . (1992) EMBO 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.
  • Cst20p has the potential to fulfill similar functions in C. albicans .
  • 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 CPH1 alleles, which encode a homolog of the S. cerevisiae Stel2p transcription factor (Liu, H. et al .
  • 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.
  • CaCla4p and CaCdc42p 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 vi tro assay will be used to test compounds that inhibit the interaction between Cst20p and CaBemlp.
  • the CaCDC42 gene will be fused to the DNA binding domain of GAL4
  • 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 interaction system will be excluded by performing a parallel screen with unrelated fusion proteins known to inter- act. Compounds of general toxicity or inhibitors of 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 GAL4-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.
  • an integrating plasmid will be constructed carrying the HsCDC42 gene fused to a URA3 blaster gene and CDC42 flanking sequences. After linearization, 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 CaCLA4 gene fused to the transcriptional activation domain of GAL4 will be constructed by standard procedures.
  • sequences of either one of the genes CaCLA4, CST20, CaCDC42 and CaBEMl may be used to derive probes for the detection of C. albicans using PCR techniques or hybridization assays.
  • CaCDC42 and CaBEMl may be used to identify and clone homologues from other fungi.
  • the STE20 gene will be replaced in a supersensi- tive sstl yeast strain by the human PAK gene using homologous recombination.
  • an integrating plasmid will be constructed carrying the PAK gene fused to a URA3 blaster gene and STE20 flanking sequences. The construct will be linearized and transformed 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.
  • Fluorescence resonance energy transfer 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).
  • FRET fluorescence resonance energy transfer
  • the FRET phenomenon consists in a fluorescence transfer between a donor and a receptor 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.
  • 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 measured 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 GFP Y66H mutant as donor, and the CaCLA4 gene will be fused to the gene encoding the GFP 3657 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 conditions 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.
  • MOLECULE TYPE cDNA
  • MOLECULE TYPE cDNA
  • cDNA cDNA
  • MOLECULE TYPE Genomic RNA
  • FEATURE :
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • AGT TCT AAT AGT CTT GGC GTA ACA GCA AAT CAA ACC AAA CCT ATT CAA 614 Ser Ser Asn Ser Leu Gly Val Thr Ala Asn Gin Thr Lys Pro He Gin 50 55 60
  • ACT CAT AAA GTA CAC GTG GGA TTT GAT CCT GCC AGT GGT AAT TTT ACT 1190 Thr His Lys Val His Val Gly Phe Asp Pro Ala Ser Gly Asn Phe Thr 240 245 250
  • AAC AAT TAC TCA TCA ACC AAA AAC AAT GTC CAA GAG GCA AAT TTA CAA 1430 Asn Asn Tyr Ser Ser Thr Lys Asn Asn Val Gin Glu Ala Asn Leu Gin 320 325 330
  • CAA CAT AAA AAT ATT GTT AAT TTT TTG GAT TCT TAT TTA ATT GGT GAT 2678 Gin His Lys Asn He Val Asn Phe Leu Asp Ser Tyr Leu He Gly Asp 735 740 745
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • AAA AAT CAA ACT GCC AAA TAT CAA GCT TCA ACA ATC CCC CTT GGT TCA 1064 Lys Asn Gin Thr Ala Lys Tyr Gin Ala Ser Thr He Pro Leu Gly Ser 245 250 255
  • AAA TTA CGA AGA GAA AAA TTG GAT TAT TAT TTA TCA AAT TTA ATT GCA 1448 Lys Leu Arg Arg Glu Lys Leu Asp Tyr Tyr Leu Ser Asn Leu He Ala 375 380 385
  • TTT TCT AAA CCA ATA AGT CAA AAA TCA AAT TCT CAT CAA GAT AGA TTA 1592 Phe Ser Lys Pro He Ser Gin Lys Ser Asn Ser His Gin Asp Arg Leu 420 425 430

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Abstract

L'invention porte sur des protéines de Candida albicans, telles que les CaCla4p, Cst20p, CaCdc42p et CaBemlp associées à la virulence et à la formation d'hyphes, et sur leurs utilisations notamment pour l'élaboration d'essais de criblage d'inhibiteurs destinés au traitement d'infections et/ou inflammations dues à des champignons pathogènes. L'invention porte également sur un essai de criblage in vitro des composés inhibiteurs de l'activité biologique d'au moins une protéine choisie dans un groupe consistant en CaCla4p, Cst20p, CaCdc42p et CaBemlp, et comprenant: a) au moins une desdites protéines; et b) des moyens de suivre l'activité biologique d'au moins l'une de ces protéines. On peut ainsi vérifier le potentiel inhibiteur des composés.
PCT/CA1997/000809 1996-10-30 1997-10-29 Proteines de candida albicans, associees a la virulence et a la formation d'hyphes, et leurs utilisations WO1998018927A1 (fr)

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AU48585/97A AU4858597A (en) 1996-10-30 1997-10-29 (candida albicans) proteins associated with virulence and hyphal formation and uses thereof
CA002269633A CA2269633A1 (fr) 1996-10-30 1997-10-29 Proteines de candida albicans, associees a la virulence et a la formation d'hyphes, et leurs utilisations
US10/093,524 US20030166886A1 (en) 1996-10-30 2002-03-11 Candida albicans proteins associated with virulence and hyphal formation and uses thereof

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US60/029,458 1996-10-30

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DE10023130A1 (de) * 2000-05-11 2001-11-22 Fraunhofer Ges Forschung Hyphenspezifische Faktoren aus Candida albicans
US6639064B1 (en) * 1999-09-17 2003-10-28 New York University NRIF3, novel co-activator for nuclear hormone receptors
US7033621B1 (en) 1997-04-28 2006-04-25 Novogen, Inc. Isoflavone compositions produced from legumes
US7763660B2 (en) * 2003-02-05 2010-07-27 The University Of Vermont And State Agricultural College Inhibitors of Candida albicans
CN113502345A (zh) * 2021-04-23 2021-10-15 江苏师范大学 黄曲霉致病基因cdc48在筛选防治黄曲霉污染药物中的应用

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LEBERER E ET AL: "SIGNAL-TRANSDUCTION THROUGH HOMOLOGS OF THE STE20P AND STE7P PROTEIN-KINASES CAN TRIGGER HYPHAL FORMATION IN THE PATHOGENIC FUNGUS CANDIDA-ALBICANS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93, 13217-13222, XP002057299 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7033621B1 (en) 1997-04-28 2006-04-25 Novogen, Inc. Isoflavone compositions produced from legumes
US6639064B1 (en) * 1999-09-17 2003-10-28 New York University NRIF3, novel co-activator for nuclear hormone receptors
DE10023130A1 (de) * 2000-05-11 2001-11-22 Fraunhofer Ges Forschung Hyphenspezifische Faktoren aus Candida albicans
WO2001085989A3 (fr) * 2000-05-11 2003-06-05 Fraunhofer Ges Forschung Facteurs specifiques aux filaments mycelliens extraits de candida albicans
US7220574B2 (en) 2000-05-11 2007-05-22 Fraunhafer-Gasellschaft Zur Forderung Der Angewandten Forschung E.V. Hyphen-specific factors from Candida albicans
DE10023130B4 (de) * 2000-05-11 2007-10-18 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Hyphenspezifische Faktoren aus Candida albicans
US7763660B2 (en) * 2003-02-05 2010-07-27 The University Of Vermont And State Agricultural College Inhibitors of Candida albicans
CN113502345A (zh) * 2021-04-23 2021-10-15 江苏师范大学 黄曲霉致病基因cdc48在筛选防治黄曲霉污染药物中的应用

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