CA2314611A1 - New candida albicans kre9 and uses thereof - Google Patents

Abstract

The present invention relates to an isolated DNA which codes for a gene essential for cell wall glucan synthesis of Candida albicans, wherein the gene is referred to as CaKRE9, wherein the sequence of the DNA is as set forth in Fig. 1. The present invention relates to antifungal in vitro and in vivo screening assays for identifying compounds which inhibit the synthesis, assembly and/or regulation of .beta.1,6-glucan. There is also disclosed an in vitro method for the diagnosis of diseases caused by fungal infection in a patient.

Description

- 1 - _ BACFCGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to a novel gene, CaKRE9, isolated in the yeast pathogen, Candida albicans, that is a functional homolog of the S. cerevisiae KRE9 gene and which is essential for cell wall glucan synthesis, and to novel antifungal screening assays.
to (b) Description of Prior Art Fungi constitute a vital part of our ecosystem but once they penetrate the human body and start spreading they cause infections or "mycosis" and they can pose a serious threat to human health. Fungal is infections have dramatically increased in the last 2 decades with the development of more sophisticated medical interventions and are becoming a significant cause of morbidity and mortality. Infections due to pathogenic fungi are frequently acquired by debilitated 2o patients with depressed cell-mediated immunity such as those with human immunodeficiency virus (HIV) and now also constitute a common complication of many medical and surgical therapies. Risk factors that predispose individuals to the development of mycosis include neu-25 tropenia, use of immunosuppressive agents at the time of organ transplants, intensive chemotherapy and irra-diation for hematopoietic malignancies or solid tumors, use of corticosteroids, extensive surgery and pros-thetic devices, indwelling venous catheters, hyperali-3o mentation and intravenous drug use, and when the deli-cate balance of the normal flora is altered through antimicrobial therapy.
The yeast genus Candida constitutes one of the major groups that cause systemic fungal infections and 35 the five medically relevant species which are most _ Z _ _ often recovered from patients are C. albicans, C.
tropicalis, C. glabrata, C. parapsilosis and C. krusei.
Much of the structure of fungal and animal cells along with their physiology and metabolism is highly s conserved. This conservation in cellular function has made it difficult to find agents that selectively dis-criminate between pathogenic fungi and their human hosts, in the way that antibiotics do between bacteria and man. Because of this, the common antifungal drugs, io like amphotericin B and the azole-based compounds are often of limited efficacy and are frequently highly toxic. In spite of these drawbacks, early initiation of antifungal therapy is crucial in increasing the sur-vival rate of patients with disseminated candidiasis.
15 Moreover, resistance to antifungal drugs is becoming more and more prominent. For example, 6 years after the introduction of fluconazole, an alarming proportion of Candida strains isolated from infected patients have been found to be resistant to this drug and this is 2o especially the case with vaginal infections. There is thus, a real and urgent need for specific antifungal drugs to treat mycosis.
The fungal cell wall: a resource for new antifungal targets z5 Tn recent years, we have focused our attention on the fungal extracellular matrix, where the cell wall constitutes an essential, fungi-specific organelle that is absent from human/mammalian cells, and hence offers an excellent potential target for specific antifungal 3o antibiotics. The cell wall of fungi is essential not only in maintaining the osmotic integrity of the fungal cell but also in cell growth, division and morphology.
The cell wall contains a range of polysaccharide poly-mers, including chitin, (3-glucans and O- and N-linked 35 mannose sidechains of glycoproteins. (3-glucans, homo-polymers of glucose, are the main structural component component of the yeast cell wall, and constitute up to 60% of the dry weight of the cell wall. Based on their chemical linkage, two different types of polymers can be f ound : (31, 3 -glucan and (31, 6 -glucan . The (31, 3 -glucan s is the most abundant component of the cell wall and it contains on average 1500 glucose residues per molecule.
It is mainly a linear molecule but contains some 1,6-linked branchpoints. The (31,6-glucan is a smaller and highly branched molecule comprised largely of 1,6-lo linked glucose residues with a small proportion of 1,3-linked residues. The average size of X31,6-glucan is approximately 400 residues per molecule. The (31,6-glucan polymer is essential for cell viability as it acts as the "glue" covalently linking glycoproteins and 15 the cell wall polymers (31,3-glucan and chitin together in a crosslinked extracellular matrix.
In United States Patent No. 5, 194, 600 issued on March 16, 1993 in the names of Bussey et al . , there is disclosed the screening of specific yeast strains 2o defective in certain mutants of genes which participate in ~3-glucan assembly.
It would be highly desirable to be provided with the identification and subsequent validation of new cell wall related targets that can be used in specific 2s enzymatic and cellular assays leading to the discovery of new clinically useful antifungal compounds.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide 3o the identification and subsequent validation of a new target that can be used in specific enzymatic and cellular assays leading to the discovery of new clinically useful antifungal compounds.
~,;,M lDfcD SHEE'~' - 3a -Although a gene involved in the cellular growth of S. cerevisiae was identified, there are no certainties that there would be a homolog in Candida albicans or if present that it would have the same s function.
In accordance with the present invention a gene was isolated, CaKRE9, in the yeast pathogen, Candida albicans, that is a functional homolog of the S.
A~,~-~'~°L~E~ ~~~~-~'~

glucan synthesis . The gene is not found in humans and when it is inactivated in C. albicans, the cell cannot survive when grown on glucose, thus, validating it as a wholly new target for antifungal drug discovery.
s Using the gene of the present invention, we intend to utilize novel drug screening assays for which we possess all the genetic tools.
In accordance with the present invention there is provided an isolated DNA which codes for a gene io essential for cell wall glucan synthesis of Candida albicans, wherein the gene is referred to as CaKRE9, wherein the sequence of the DNA is as set forth in Fig. 1.
In accordance with the present invention there i5 is also provided an antifungal screening assay for identifying a compound which inhibits the synthesis, assembly and/or regulation of X31,6-glucan, which com-prises the steps of:
a) synthesizing (31,6-glucans in vitro from acti 2o vated sugar monomer/polymer and specific (31,6 glucan synthetic proteins;
b) subjecting step a) to a high throughput compound screen determining absence or presence of (31,6-glucan, wherein absence of (31,6-glucan is 2s indicative of an antifungal compound.
In accordance with the present invention there is also provided an in vivo antifungal screening assay for identifying compounds which inhibit the synthesis, assembly and/or regulation of (31,6-glucan, which com so prises the steps of:
a) separately cultivating a mutant yeast strain lacking one gene for synthesis of (31,6-glucans and a wild type yeast strain with activated sugar monomer/polymer UDP-glucose;

b) subjecting both yeast strains of step a) to the screened compound and determining if the com pound selectively inhibits growth of wild type strain which is indicative of an antifungal com pound.
In accordance with the present invention there is also provided an in vitro method for the diagnosis of diseases caused by fungal infection in a patient, which comprises the steps of:
io a) obtaining a biological sample from the patient;
b) subjecting the sample to PCR using a primer pair specific for CaKRE9 gene, wherein a presence of the gene is indicative of the presence of fungal infection.
u5 In accordance with the present invention, the gene is CaKRE9.
In accordance with the present invention there is also provided an in vitro method for the diagnosis of diseases caused by fungal infection in a patient, 2o which comprises the steps of:
a) obtaining a biological sample from the patient;
b) subjecting the sample to an antibody specific for CaKre9p antigen, wherein a presence of the antigen is indicative of the presence of fungal 25 infection.
In accordance with~one embodiment of the present invention, the fungal infection may be caused by Can-dida.
In accordance with the present invention there 3o is also provided the use of at least one of KRE9 and CaKre9 nucleic acid sequences and fragments thereof as a probe for the isolation of KRE9 homologs in all fungi .
For the purpose of the present invention the 35 following terms are defined below.

- 6 - _ The term a "mutant yeast strain" is intended to mean any yeast strain lacking one gene for synthesis of (31,6-glucan, such as KRE9 and homologs thereof.
The term a "wild type yeast strain" is intended s to mean any yeast strain containing the KRE9 gene or a homolog thereof or a plasmid overexpressing the KRE9 gene or a homolog thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
to Fig. 1 illustrates the complete nucleotide and predicted amino acid sequence of CaKRE9 (SEQ ID NO:1-2) .
Fig. 2 illustrates the comparison of the sequence of Kre9p from Candida albicans (SEQ ID N0:2) 15 and Kre9p (SEQ ID N0:3) and Knhlp (SEQ ID N0:4) from Saccharomyces cerevisiae;
Fig. 3 illustrates the CaKRE9-dependent effect on the growth (A) and Killer phenotype (B) of kre9A
null mutants;
2o Fig. 4A illustrates the schematic representation of the strategy for disruption of the Candida albicans KRE9 gene;
Fig. 4B illustrates the Southern blot verifica tion of the correct integration of the hisG-URA3-hisG
z5 disruption module into the CaKRE9 gene and proper CaURA3 excision after 5-FOA treatment; and Fig. 5 illustrates the quantification of (31,6-Glucan levels of different Candida albicans strains.
3o DETAINED DESCRIPTION OF THE INVENTION
In accordance with the present invention, the synthesis and the assembly of the cell wall polymer (31,6 glucan which plays a central role in the organiza-tion of the yeast cell wall and which is indispensable 35 for cell viability were extensively studied. Although _ 7 _ _ the biochemistry of ~i1,6 glucosylation is incompletely understood, a genetic analysis of genes required for 1,6 synthesis has been performed in Saccharomyces cere-visiae, and has identified many genes required for this s process. These encode products acting in the endoplas-mic reticulum, the Golgi complex and at the cell sur-f ace .
In accordance with the present invention a novel gene was identified, KRE9, whose product is required io for the synthesis of (31, 6 linked glucans (Brown JL. et al. (1993) Molecular & Cellular Biology 13:6346-6356).
KRE9 appears to be a fungal specific gene, as it is absent from animal lineages based on data base searches of the Caernorhabditis elegans, mouse and Homo Sapiens i5 genomes and it also appears to be absent from the plant, bacterial and archaebacterial lineages.
KRE9 and its homolog KNX1 KRE9 encodes a 30-kDa secretory pathway protein involved in the synthesis of cell wall X31,6 glucan 20 (Brown JL. et aI. (1993) Molecular & Cellular Biology 13:6346-6356). Disruption of KRE9 in S. cerevisiae leads to serious growth impairment and an altered cell wall containing less than 20~ of the wild-type amount of (31,6 glucan. Analysis of the glucan material 2s remaining in a kre9 null mutant indicated a polymer with a reduced average molecular mass (Brown JL. et al.
(1993) Molecular & Cellular Biology 13:6346-6356). The kre9 null mutants also displayed several additional cell-wall-related phenotypes, including an aberrant 3o multiple budded morphology, a mating defect, and a failure to form projections in the presence of alpha-factor. Antibodies generated against Kre9p detected an O-glycoprotein of approximately 55 to 60 kDa found in the extracellular medium of a strain overproducing - 8 - _ Kre9p, indicating it is normally localized at the cell surface .
In the yeast genome a KRE9 homolog was recently found, KNH1, whose product, Knhlp, shares 46~ overall identity with Kre9p (Dijkgraaf GJ. et al. (1996) Yeast 12:683-692). Disruption of the KNH1 locus has no effect on growth, killer toxin sensitivity or (31,6-glu-can levels. Overexpression of KNH1 suppressed the severe growth defect of a kre9 null mutant and restored io the level of alkali-insoluble (31,6-glucan to almost wild type levels. When overproduced, Knhlp, like Kre9p, can be found in the extracellular culture medium as an O-glycoprotein, and is likely also a cell surface protein under conditions of normal expression. The dis-cs ruption of both KNH1 and KRE9 is lethal. Transcription of KNH1 is carbon-source and KRE9 dependent. The severe growth defect of a kre9d null mutant observed on glucose can be partially restored when galactose becomes the major carbon source. Transcription of the 2o KNHI gene is normally low in wild type cells grown on glucose but increases approximately five fold in galac-tose grown cells, where it partially compensates for the loss of Kre9p and allows partial suppression of the slow growth phenotype of kre9d cells. These results 2s suggest that KRE9 and KNH1 are specialized in vivo to function under different environmental conditions (Dijkgraaf GJ. et al. (2996) Yeast 12:683-692).
The essential nature of the KRE9/KNH1 gene pair, and the putative extracellular location of their gene 3o products make these proteins a new and potentially valuable target for antifungal compounds that need not enter the fungal cell.
~i1,6-glucan in pathogenic fungi The yeast Saccharomyces cerevisiae, although not 35 a pathogen, is a proven model organism for pathogenic - 9 - _ fungi as it is closely related taxonomically to oppor-tunistic pathogens like the dimorphic yeast Candida albicans. The composition of the cell wall of C. albi-cans resembles that of S. cerevisiae in containing s (31, 3- and (31, 6-glucans, chitin, and mannoproteins (Mio, T. et al., J. Bacteriol. 179:2363-2372 Analyses of the Candida albicans genes involved in extracellular matrix assembly are limited but indicate that the proteins responsible for synthesis of the polymers often resem-to ble those found in the more extensively studied yeast, Saccharomyces cerevisiae. The ~i1,6 glucosylation of proteins appears to be widespread among fungal groups, and the polymer varies in abundance between fungal spe-cies. In C. albicans this polymer is particularly i5 abundant, comprising approximately half of the alkali insoluble glucan. Comparative studies with C. albicans have so far identified three genes involved in (31,6 glucosylation based on their relatedness to those in S.
cerevisiae, indicating that synthesis of this polymer 2o is functionally conserved and essential for the growth of Candida albicans.
Isolation of the CaKRE9 gene In order to validate KRE9 as a possible new antifungal target, we have examined if genes related to 25 S. cerevisiae KRE9 were present in C. albicans. Using complementation of the S. cerevisiae kre9 mutant pheno-type as a screen, we have isolated a C. albicans gene that encodes a protein similar to the S. cerevisiae KRE9 gene product.
3o CaKRE9 was identified by a plasmid shuffle approach as a gene being able to restore the slow growth of a Saccharomyces cerevisiae kre9::HIS3 dis-rupted strain. A diploid strain heterozygous for a kre9::HIS3 deletion was transformed with a centromeric 35 LYS2-based pRS317 vector containing a wild type copy of - 10 _ the S. cerevisiae KRE9 gene. Transformants were selected by prototrophic growth on minimal media, sporulated and a haploid kre9::HIS3 strain containing a plasmid-based copy of KRE9 was obtained by tetrad dis-section and spore progeny analysis. This strain was shown to possess wild type growth and killer toxin sen-sitivity and was subsequently transformed with a Can-dida albicans genomic library contained within the mul-ticopy YEp352-plasmid harboring the URA3 gene as a io selectable marker. In order to screen for plasmids that could restore growth to a kre9::HIS3 mutant, about 20,000 His3+ Lys2+ Ura3+ cells were replica plated on minimal medium containing a-aminoadipate as a primary nitrogen source to select for cells that have lost the i5 LYS2 plasmid-based copy of KRE9 but are still able to grow, indicating that a copy of the complementing CaKRE9 gene could be present in such growing cells.
These cells were further tested for loss of the pRS317-KRE9 plasmid by failure to grow on medium lacking 20 lysine. YEp352-based Candida albicans genomic DNA was recovered from cells that grew in the presence of lysine but did not grow in its absence. Upon retransformation in yeast, only 2 different genomic inserts were able to partially restore growth of the 2s kre9::HIS3 haploid strain. DNA from both inserts were sequenced.
The CaKRE9 gene was contained in only one of the C. albicans clones. Complete sequencing of the 8-kb fragment containing the CaKRE9 gene revealed an open 3o reading frame of 813 by encoding a 29-kDA secretory protein of 271 amino acid residues (see Fig. 1). As is the case with Kre9p and Knhlp (Brown JL. et al. (1993) Molecular & Cellular Biology 13:6346-6356; Dijkgraaf GJ. et al. (1996) Yeast 12:683-692), the hydrophobic N-35 terminal region of CaKre9p comprises an eukaryotic sig-- 11 - _ nal sequence, with the most likely cleavage site occur-ring between amino acid residues 21 and 22. CaKre9p shares 43~ overall identity with Kre9p and 32~ with Knhlp (see Fig. 2). The amino acid residues are shown in single-letter amino acid code. Sequences were aligned with gaps to maximize homology. Dots represent a perfect match between all sequences while a vertical slash indicates conservative substitution at a given position. The most conserved region between the 3 pro-io teins encompasses a large part of the central region and most of the C-terminal portion, with the N-terminal part being largely unique to each protein. Kre9p, Knhlp and CaKre9p share a high proportion of serine and threonine residues (26~), potential sites for O-glyco-i5 sylation, a modification known to occur on Kre9p and Knhlp, and characteristic of many yeast cell surface proteins. In addition, all 3 proteins have lysine and arginine rich C-termini and lack potential N-linked glycosylation sites.
2o The functional capacity of CaKre9p was assessed in Saccharomyces cerevisiae by measuring its ability to restore the growth and killer toxin sensitivity of a kre9 null mutant. Firstly, the YEp352-based Candida albicans genomic DNA containing the CaKRE9 gene was 25 transformed into a diploid strain of S. cerevisiae heterozygous for a kre9::HIS3 deletion, sporulated and a haploid kre9::HIS3 strain containing a plasmid-based copy of CaKRE9 was obtained from spore progeny follow-ing tetrad dissection. As can be seen in Fig. 3A, a so strain harboring the CaKRE9 gene grows at a slower rate than a wild type strain or the mutant strain harboring a copy of KRE9 but significantly faster than the kre9 null mutant which has a severe growth phenotype. Sec-ondly, the haploid kre9 strain carrying the CaKRE9 was 35 submitted to a killer toxin sensitivity assay (Fig.

3B). K1 killer yeast strains secrete a small pore-forming toxin that requires an intact cell wall recep-tor for function. KRE9 null mutations lead to a con-siderable decrease in the level of (31,6-glucan disrupt-s ing the toxin receptor (Brown JL. et al. (1993) Molecu-lar & Cellular Biology 13:6346-6356), leading to killer resistance and showing no killing zone in the assay.
The killer phenotype of the kre9 mutant allowed a test of possible suppression by CaKre9p. Overexpression of to CaKRE9 in the S. cerevisiae haploid strain carrying a disrupted copy of KRE9 partially suppressed the killer resistance phenotype (Fig. 3B).
These results imply that Kre9p and CaKre9p both play very similar roles in (31,6-glucan assembly in S.
15 cerevisiae and C. albicans.
Disruption of the CaKRE9 gene Experimental strategy:
The gene disruption was performed by the URA
blaster protocol using the hisG-CaURA3-hisG module. A
20 1.6-kb DraI DNA fragment containing the CaKRE9 gene was subcloned from the original insert into the SmaI site and the blunted XbaI site (treated with the Klenow fragment of DNA polymerase I) of YEp352 (see Fig. 4A).
Extracted genomic DNAs are from . CAI4 wild type cells 2s (lane 1), CaKRE9/Cakre9::hisG-URA-hisG heterozygous mutant (lane 2), CaKRE9/Cakre9::hisG heterozygous mutant obtained after 5-FOA treatment (lane 3) and Cakre9/Cakre9::hisG-URA-hisG homozygous null mutant which is able to grow only when galactose is used as 3o the sole source of carbon.
The CaKRE9 gene was disrupted by deleting a 485 by BstxI-BamHI fragment of the open reading frame and replacing it by a 4.0 kb BglII/BamHI fragment carrying the hisG-URA3-hisG module from plasmid pCUB-6 (see 35 Fig. 4A). The sticky ends were enzymatically treated to accommodate the ligation. This disruption plasmid was digested by HindIII and KpnI, precipitated with ethanol and sodium acetate and 100 ~g of the 5.2 kb-disruption fragment was transformed into CAI4 Candida albicans s cells by the lithium acetate method.
Putative heterozygous disruptants were selected on minimal medium carrying glucose or galactose as car-bon sources but lacking uracil. In preparation for a second round of gene disruption, the CaURA gene was to excised using a 5-FOA selection. The second round of transformation was performed in the same way as the primary one.
The accurate integration of the hisG-CaURA3-hisG
cassette into the CaKRE9 gene and its excision from i5 genomic DNA was verified by Southern hybridization using 3 different probes:
(1) a 405-by fragment from. C. albicans genomic DNA con-taining coding and 3' flanking sequences of CaKRE9;
(2) a 783 by DNA fragment obtained by PCR and covering 2o the entire CaURA3 coding region; and (3) a 898 by fragment amplified by PCR that encompasses the whole of the Salmonella typhimurium hisG gene (see Fig. 4B) .
All genomic DNAs were digested with the BamHI
2s and SalI restriction enzymes.
Results:
In the first round of transformation where transformants were selected on glucose containing plates, the Southern blotting results revealed that the 3o hisG-CaURA3-hisG module correctly integrated into the Candida albicans KRE9 gene (see Fig. 4). When genomic DNA of putative heterozygous CaKRE9 disruptions was digested with the SalI and BamHI restriction enzymes and probed with the CaKRE9 405-by SalI-BstXI DNA frag-35 ment along with the hisG and the CaURA3 probes, 2 Candida albicans KRE9 gene (see Fig. 4). When genomic DNA of putative heterozygous CaKRE9 disruptions was digested with the SalI and BamHI restriction enzymes and probed with the CaKRE9 405-by SalI-BstXI DNA
s fragment along with the hisG and the Ca URA3 probes, 2 expected bands could be detected (see Fig. 4B, land 2, for representative result): a 773 by band corresponding to the wild type gene that could only be detected by the CaKRE9 probe and a 4318 by diagnostic band, to revealed by all 3 probes, indicating successful disruption of one copy of the CaKRE9 gene. After removal of the CaURA3 using 5-FOA (5-fluoroorotic acid), the 773 by wild type band could still be visualized but the disrupted band from which the CaURA3 is was excised shifted to an anticipated 1428 by when probed with the CaKRE9 and hisG probes but not with the CaURA3 probe (see Fig. 4B, lane 3).
In order to assess if the CaKRE9 gene is essential in C. albicans, a second round of disruptions 2o was undertaken in the heterozygous strain where the CaURA3 gene was eliminated. However, in view of the nature of the carbon source regulation of the KRE9/KNH1 pair in S. cerevisiae, the second round of transformation was executed using both glucose and 2s galactose as carbon sources. 32 Ura+ colonies from the glucose plated transformation were analyzed by Southern blot hybridization using the 3 different probes and only yeast cells heterozygous at the CaKRE9 locus could be found. The absence of the expected homozygous 3o double disruption among the transformants is consistent with the fact that CaKRE9 is an essential gene in C.
albicans when glucose is the sole carbon source.
Demonstration of CaKRE9 as an essential gene under these conditions validates the CaKRE9 gene product as a 35 therapeutical target in Candida albicans.
~n.~~ ~!_ ~r~
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Besting that they could be homozygous disruptants.
Southern blot hybridizations were performed on these 8 transformants and they were shown to be homozygous dis-ruptants for the CaKRE9 locus: one copy corresponded to the disrupted gene in which CaURA3 has been removed (1428 bp) and the second one represented the inactiva-tion of the remaining wild type copy by the hisG-caURA3-hisG module (4318 bp; Fig. 4B, lane 4) . Thus a homozygous disruption of kre9 in C. albicans is lethal to when glucose constitutes the exclusive carbon source.
Further, it should be appreciated that glucose is the main source of carbon of human beings.
~i1,6-glucan analysis of C. albicaas CaKRE9 mutants Experimental strategy:
is Yeast total-cell protein extracts were prepared from exponentially growing cultures by cell lysis with glass beads. Cellular extracts were standardized for total cellular protein and equivalent amounts of pro-tein were alkali extracted (0.75M NaOH final lh, 75°C) .
ao The alkali soluble fractions were then spotted onto nitrocellulose and immunoblots were carried out.
Briefly, blots were treated in TBST buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.05 TweenT"" 20, containing 5~ non fat dried milk powder) and subsequently incubated with 25 affinity purified rabbit anti-(31,6-glucans antibodies (prepared as described Montijn, R.C. et al. (1994) J.
Biol. Chem. 296:19338-19342) in the same buffer. After antibody binding, membranes were washed in TBST and a second antibody directed against rabbit immunoglobulins so and conjugated with horseradish peroxidase, was then added. The blots were again washed and whole cell ~i1,6 glucans detected using an enhanced chemiluminescence procedure.
Results In order to directly measure the effect of inac-tivating CaKRE9 on X31,6-glucan synthesis and assembly, a specific rabbit anti-(31, 6-glucan antiserum was raised against BSA-coupled pustulan (a commercially available s (31,6 glucan), affinity purified, and used to detect antigen-antibody complexes by Western blotting of total cell protein extracts of different yeast strains grown on galactose. As expected, wild type cells yielded a strong (31,6-glucan signal (see Fig. 5). The affinity to purified Ab detected about a quarter of the glucan in the C. albicans heterozygous dcakre9 whereas no (31,6-glucan could be detected from a C. albicans homozygous dcakre9 disruptant grown on galactose (Fig. 5).
Discussion 15 The essential nature of the KRE9 gene in C.
albicans, and the possible extracellular location of its gene product make this protein a new and poten-tially valuable target for antifungal compounds that need not enter the fungal cell. The precise role of 2o Kre9p in ~i-glucan synthesis remains to be precisely determined but does not prevent the establishment of a antifungal drug screening assay The present invention will be more readily un derstood by referring to the following examples which 2s are given to illustrate the invention rather than to limit its scope.
EXAMPLE I
In vitro screening method for specific antifungal agents (enzymatic-based assay) 30 The primary objective is to identify novel com-pounds inhibiting the synthesis, assembly and/or regu-lation of X31,6-glucans. This enzymatic assay would utilize some of the gene products (KRE) involved in (31,6-glucan synthesis, including using an in vitro 35 assay for CaKre9p. Using specific reagents such as an antibody to (31,6-glucan, and a specific glucanase for the polymer, the approach is to synthesize the polymer in vitro from the activated sugar monomer UDP-glucose.
This task can be accomplished by existing methodologies such as the production of large amounts of each protein s and by the availability of genetic tools, such as the ability to delete or overexpress gene products that are involved in synthesis of this and the other major poly-mers. Once the assay has been established it will per-mit the screening of possible compounds that inhibit io steps in the synthesis of this essential polymer. When such inhibitors will be found, they will then be evalu-ated as candidates for specific antifungal agents.
The effects of such compounds on (31,6-glucan levels may be directly measured using the anti-(31,6 i5 glucan antibody. This approach can be used on all type of fungi and can be adapted to a high throughput immu-noassay to find ~i1,6-glucan inhibitors.
EXAMPhE II
2o In vfvo screening method for specific antifungal agents (cellular-based assay) Yeast strains possessing or lacking (31,6-glucans permit a differential screen for compounds inhibiting synthesis of this cell wall polymer. Specifically, an 2s antifungal drug screen can be devised based on a whole-cell assay in which the fungal-specific CaKre9p would be targeted.
The strains that may be used in accordance with the present invention include, without limitation, any 3o yeast strain mutant for CaKRE9 and homologs thereof disrupted strain, conditional mutants, overexpression strains and suppressed disrupted strains.
Compounds can be tested for their ability to inhibit growth or kill a wild type C. albicans strain 3s while having no effect on a Cakre9 suppressor strain.
In addition, compounds leading to hypersensitivity in a CaKRE9 deletion will also be of value as candidate antifungal drugs. The finding of new antifungal com-pounds will be greatly simplified by these types of screens. The direct scoring on cells of the level of s efficacy of a particular compound (natural product extracts, pure chemicals...) alleviates the costly and labor intensive establishment of an in vitro enzymatic assay. The availability of genetic tools, such as the ability to delete or overexpress gene products that are to involved in synthesis of this and the other major poly-mers will permit the establishment of this new screen-ing method. When such inhibitors will be found, they will then be evaluated as candidates for specific anti-fungal agents.
EXAMPLE III
The use of CaKRE9 in the diagnosis of fungal infection Detection based on PCR
Candida spp. and other pathogenic fungi are tradi 2o tionally identified by morphological and metabolic characteristics and often this require days to weeks to isolate on culture from a patient's sample. Identifi cation is time-consuming and often unreliable and this impedes the selection of antimicrobial agents in cases in which species identification of the organism is nec-essary. Moreover, culture-based diagnostic methods are not within the scope of many routine microbiology labo-ratories and are frequently limited to detection of pathogenic organisms in patients at an advanced stage of disease or even at autopsy. The detection of dis-seminated Candida mycosis is an area where there is an urgency for new sophisticated techniques of identifica-tion. Polymerase Chain Reaction (PCR) based tests to establish the presence of a fungal infection are at this point highly desirable for laboratory diagnosis and management of patients with serious fungal dis-eases. The CaKRE9 gene is fungi specific and could be used to develop new diagnostic procedures of mycosis based on the PCR. Such diagnostic tests would be pre-dicted to be highly sensitive and specific. Ulti-mately, simple kits permitting the diagnosis of fungal infections will be sold to hospitals and specialized clinics. Current trends in the hospital microbiology laboratories indicate that there will be a considerable future increase in use of the PCR as a diagnostic tool.
to Detection based on anti-CaKre9p antibodies CaKre9p is thought to be localized at the cell surface and as such could be detected as a circulating candidal antigen by an enzyme-linked immunoabsorbent assay (ELISA) detection kit based on antibodies i5 directed against CaKre9p. Antibodies directed against CaKre9p could allow levels of specificity and sensitiv-ity high enough to permit commercialization of a diag-nostic kit.
20 EXAMPhE IV
The use of Kre9p in all fuagi Isolation and use of functional homologs of KRE9/CaKRE9 from all fungi. Most fungi have (31,6-glu-cans and likely have KRE9 homologs in their genome.
25 The kre9 mutant can allow isolation of similar genes by functional complementation from other pathogenic fungi as what was done to isolate CaKRE9. KRE9 could also serve as a probe to isolate by homology KRE9 homologs from other yeasts. In addition, Kre9p allows isolation 30 of homologs in other species by the techniques of reverse genetics where antibodies raised against Kre9p could be used to screen expression libraries of patho-genic fungi for expression of KRE9 homologs that would immunologically cross react with antibodies raised 35 against S. cerevisiae KRE9 and C. albicans CaKRE9.

- 2~ -The kre9 mutant can allow isolation of similar genes by functional complementation from other pathogenic fungi as what was done to isolate CaKRE9. KRE9 could also serve as a probe to isolate by homology KRE9 homologs s from other yeasts. In addition, Kre9p allows isolation of homologs in other species by the techniques of reverse genetics where antibodies raised against Kre9p could be used to screen expression librairies of pathogenic fungi for expression of of KRE9 homologs io that would immunologically cross react with antibodies raised against S. cerevisiae KRE9 and C. albicans CaKRE9. These putative KRE9 homologs in these pathogenic fungi could serve as targets for potential new antifungals.
15 Other methods are used to find proteins which interact with Kre9p and homologs thereof, such as two-hybrid, co-immunoprecipitation and chromatography using an activated Kre9p matrix.
A~~'~DED ~~E~

SEQUENCE LISTING
<110> McGILL UNIVERSITY
BUSSEY, Howard LUSSIER, Marc SDICU, Anne-Marie SHAHINIAN, Sarkis, Serge <120> NEW CANDIDA ALBICANS KRE9 AND USES
THEREOF
<130> 1770-195PCT FC/ld <150> CA 2,218,446 <151> 1997-12-12 <160> 4 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 816 <212> DNA
<213> Artificial Sequence <400>

atgagacaatttcaaatcatattaatttcccttgttgtttccataataagatgtgttgtt60 gcagatgttgacatcacatcaccaaagagtggagaaactttttctggtagttctggatca120 gcaagtatcaagattacctgggatgattcagacgattcagactcaccgaaatctttggat180 aatgccaaagggtacacaatttctttatgtactggacctacttcagatggggatatccag240 tgtttggatccattagtcaagaacgaagctattgcaggtaaatctaaaacagtttctatt300 ccccagaactcagtacctaatggttattactatttccaaatttacgttactttcactaat360 ggaggtaccactattcattattcaccacgtttcaaattgactggtatgtctggtccaact420 gccactttagatgtcaccgaaacaggatcggtgccagcggatcaagcttcaggatttgat480 actgcaactactgccgactccaaatctttcacagttccatataccctacaaacagggaag540 accagatacgcaccaatgcaaatgcaaccaggtaccaaagtgactgctacaacctggagt600 atgaagttcccaactagtgctgttacttactactcaacaaaggctggcacaccaaatgtg660 gcctctactattaccccaggttggagttatactgctgaatctgccgttaactatgctagt720 gttgctccatatccaacatactggtatcctgccagtgaacgagtgagtaaggctacaatt780 agtgctgctacaaagagaagaagatggttggattga 816 <210> 2 <211> 271 <212> PRT
<213> Artificial Sequence <400> 2 Met Arg Gln Phe Gln Ile Ile Leu Ile Ser Leu Val Val Ser Ile Ile Arg Cys Val Val Ala Asp Val Asp Ile Thr Ser Pro Lys Ser Gly Glu Thr Phe Ser Gly Ser Ser Gly Ser Ala Ser Ile Lys Ile Thr Trp Asp Asp Ser Asp Asp Ser Asp Ser Pro Lys Ser Leu Asp Asn Ala Lys Gly Tyr Thr Ile Ser Leu Cys Thr Gly Pro Thr Ser Asp Gly Asp Ile Gln Cys Leu Asp Pro Leu Val Lys Asn Glu Ala Ile Ala Gly Lys Ser Lys Thr Val Ser Ile Pro Gln Asn Ser Val Pro Asn Gly Tyr Tyr Tyr Phe Gln Ile Tyr Val Thr Phe Thr Asn Gly Gly Thr Thr Ile His Tyr Ser Pro Arg Phe Lys Leu Thr Gly Met Ser Gly Pro Thr Ala Thr Leu Asp Val Thr Glu Thr Gly Ser Val Pro Ala Asp Gln Ala Ser Gly Phe Asp Thr Ala Thr Thr Ala Asp Ser Lys Ser Phe Thr Val Pro Tyr Thr Leu Gln Thr Gly Lys Thr Arg Tyr Ala Pro Met Gln Met Gln Pro Gly Thr Lys Val Thr Ala Thr Thr Trp Ser Met Lys Phe Pro Thr Ser Ala Val Thr Tyr Tyr Ser Thr Lys Ala Gly Thr Pro Asn Val Ala Ser Thr Ile Thr Pro Gly Trp Ser Tyr Thr Ala Glu Ser Ala Val Asn Tyr Ala Ser Val Ala Pro Tyr Pro Thr Tyr Trp Tyr Pro Ala Ser Glu Arg Val Ser Lys Ala Thr Ile Ser Ala Ala Thr Lys Arg Arg Arg Trp Leu Asp <210> 3 <211> 276 <212> PRT
<213> Artificial Sequence <400> 3 Met Arg Leu Gln Arg Asn Ser Ile Ile Cys Ala Leu Val Phe Leu Val Ser Phe Val Leu Gly Asp Val Asn Ile Val Ser Pro Ser Ser Lys Ala Thr Phe Ser Pro Ser Gly Gly Thr Val Ser Val Pro Val Glu Trp Met Asp Asn Gly Ala Tyr Pro Ser Leu Ser Lys Ile Ser Thr Phe Thr Phe Ser Leu Cys Thr Gly Pro Asn Asn Asn Ile Asp Cys Val Ala Val Leu Ala Ser Lys Ile Thr Pro Ser Glu Leu Thr Gln Asp Asp Lys Val Tyr Ser Tyr Thr Ala Glu Phe Ala Ser Thr Leu Thr Gly Asn Gly Gln Tyr Tyr Ile Gln Val Phe Ala Gln Val Asp Gly Gln Gly Tyr Thr Ile His Tyr Thr Pro Arg Phe Gln Leu Thr Ser Met Gly Gly Val Thr Ala Tyr Thr Tyr Ser Ala Thr Thr Glu Pro Thr Pro Gln Thr Ser Ile Gln Thr Thr Thr Thr Asn Asn Ala Gln Ala Thr Thr Ile Asp Ser Arg Ser Phe Thr Val Pro Tyr Thr Lys Gln Thr Gly Thr Ser Arg Phe Ala Pro Met WO 99/31269 PCT/CA9$/01151 Gln Met Gln Pro Asn Thr Lye Val Thr Ala Thr Thr Trp Thr Arg Lys Phe Ala Thr Ser Ala Val Thr Tyr Tyr Ser Thr Phe Gly Ser Leu Pro Glu Gln Ala Thr Thr Ile Thr Pro Gly Trp Ser Tyr Thr Ile Ser Ser Gly Val Asn Tyr Ala Thr Pro Ala Ser Met Pro Ser Asp Asn Gly Gly Trp Tyr Lys Pro Ser Lys Arg Leu Sex Leu Ser Ala Arg Lys Ile Asn Met Arg Lys Val <210> 4 <211> 267 <212> PRT
<213> Artificial Sequence <400> 4 Met Leu Ile Val Leu Phe Leu Thr Leu Phe Cys Ser Val Val Phe Arg Thr Ala Tyr Cys Asp Val Ala Ile Val Ala Pro Glu Pro Asn Ser Val Tyr Asp Leu Ser Gly Thr Ser Gln Ala Val Val Lys Val Lys Trp Met His Thr Asp Asn Thr Pro Gln Glu Lys Asp Phe Val Arg Tyr Thr Phe Thr Leu Cys Ser Gly Thr Asn Ala Met Ile Glu Ala Met Ala Thr Leu 65 70 75 g0 Gln Thr Leu Ser Ala Ser Asp Leu Thr Asp Asn Glu Phe Aen Ala Ile Ile Glu Asn Thr Val Gly Thr Aap Gly Val Tyr Phe Ile Gln Val Phe Ala Gln Thr Ala Ile Gly Tyr Thr Ile His Tyr Thr Asn Arg Phe Lys Leu Lys Gly Met Ile Gly Thr Lys Ala Ala Asn Pro Ser Met Ile Thr Ile Ala Pro Glu Ala Gln Thr Arg Ile Thr Thr Gly Asp Val Gly Ala Thr Ile Asp Ser Lys Ser Phe Thr Val Pro Tyr Asn Leu Gln Thr Gly Val Val Lys Tyr Ala Pro Met Gln Leu Gln Pro Ala Thr Lys Val Thr Ala Lys Thr Trp Lys Arg Lys Tyr Ala Thr Ser Glu Val Thr Tyr Tyr Tyr Thr Leu Arg Asn Ser Val Asp Gln His Thr Thr Val Thr Pro Gly Trp Ser Tyr Ile Ile Thr Ala Asp Ser Asn Tyr Ala Thr Ala Pro Met Pro Ala Asp Asn Gly Gly Trp Tyr Asn Pro Arg Lys Arg Leu Ser Leu Thr Ala Arg Lys Val Asn Ala Leu Arg His Arg

Claims (8)

WHAT IS CLAIMED IS:

1. An isolated DNA which codes for a gene essential for cell wall glucan synthesis of Candida albicans, wherein said gene is referred to as CaKRE9, wherein the sequence of said DNA is as set forth in Fig. 1.

2. An antifungal screening assay for identifying a compound which inhibits the synthesis, assembly and/or regulation of .beta.1,6-glucan through CaKRE9 gene of claim 1, which comprises the steps of:
a) synthesizing .beta.1,6-glucan in vitro from activated sugar monomer/polymer and specific .beta.1,6-glucan synthetic proteins containing at least CaKre9;
b) subjecting step a) to a high throughput compound screen determining concentration of .beta.1,6-glucan, wherein reduction in .beta.1,6-glucan is indicative of an antifungal compound.

3. An in vivo antifungal screening assay for identifying compounds which inhibit the synthesis, assembly and/or regulation of .beta.1,6-glucans, which comprises the steps of:
a) separately cultivating a mutant yeast strain lacking CaKRE9 gene for synthesis of .beta.1,6-glucans and a wild type yeast strain with activated sugar monomer/polymer UDP-glucose;
b) subjecting said both yeast strains of step a) to the screened compound and determining if said compound selectively inhibits growth of wild type strain which is indicative of an antifungal compound.

4. An in vitro method for the diagnosis of diseases caused by fungal infection in a patient, which comprises the steps of:
a) obtaining a biological sample;
b) subjecting said sample to PCR using a primer pair specific for CaKRE9 gene, wherein a presence of said gene is indicative of the presence of fungal infection.

5. The method of claim 6, wherein said fungal infection is caused by Candida.

6. An in vitro method for the diagnosis of diseases caused by fungal infection in a patient, which comprises the steps of:
a) obtaining a biological sample;
b) subjecting said sample to an antibody specific for CaKre9p antigen, wherein a presence of said antigen is indicative of the presence of fungal infection.

7. The method of claim 6, wherein said fungal infection is caused by Candida.

8. The use of CaKRE9 nucleic acid sequences and fragments thereof as a probe for the isolation of CaKRE9 homologs.