AN ARO1 DEHYDROQUINATE SYNTHASE OF CANDIDA ALBICANS This invention relates. in pan. to newly identified polynucleotides and polypeptides; variants of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants; agonists and antagonists of the polyeptidcs : and uses of these polynucleotides, polycptidcs. variants. agonists and antagonists, In particular. in these and in other regards. die invention relates to polynucleotides and polypeptides of arom, hereinafter referred to as "ARO1" and "ARO1" BACKGROUND OF THE INVENTION It is particularly preferred to emplov candidal genes and gene products as targets for the development of antifungals. Candida spp. make up spp. medically important genus of microbes. They are known to produce two main types of infection. superficial infections of the mucosa and skin. and systemic infections, particularly in immuno-compromised patients, characterized by dissemination to the bod ! tissucs ith poor prognosis without intervention. C. albicans is the principal species causing candida infections The frequency of Candida albicans infections has risen dramatically in the past 2 years. This has been attributed to an incrcasing population of people with weakened immune systems and the use of broad spectrum antibacterials and more interventional surgical practices. The amoury of good antifungal agents is small and resistance to existing agents is an emerging problem. This has created a demand for agents new antifungal agents and diagnostic tests for this organism.

Fungi like bacteria and plants. but unlike mammals, are ablc to ssntl1esizc the aromatic amino acids phcnyiaianine. tyrosine and tryptophan. In fungi, a single polypeptide, the arom multifucntional enzyme, catalyses five consecutive reactions of the central aromatic biosynthetic pathway (Lambert et al. (1985). The 3-dehydroquinate synthase activity of the pentafunctionai auom enzyme complcx of AM/Y.'.s'/j'u/Y?c/'t7.y.y<isZn'-dependent.;c'c!/c'/./!< ;/'fr?/226 : 817-29. Duncan et al. (1987). The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.

Biochemical Journal 246 : 375-86.). IWibition of any step catalyzed by this enzyme will prevent the synthesis of aromatic amino acids. p-aminobenzoic acid (precursor for folate) and ubiquinone. These essential metabolites are in limiting concentrations in mammalian tissues and thus inhibition of this enzyme is a valid antifungal strategy.

Clearly, there is a need for factors that may be used to screen compounds for antifungai activity and which may also bc used to determine their roles in pathogenesis of infection, dysfunction and disease.

There is a need, therefore, for identification and characterization of such factors which can plav a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.

The potypeptide of the present invention has the conserved residues, and have ammo acid sequence homology to known arom multifunctional protein protein.

SUMMARY OF THE INVENTION Toward these ends. and others. it is an object of the present invention to provide poly eptides. inter alia. that have been identified as novel AROI peptides by homology between the amino acid sequence set out in Table 2 and known aiTimo acid sequences of other proteins such as Saccharomyces cerevisiae ARO1 protein.

It is a further object of the invention, morcover, to provide polynucleotides that encode ARO1 polypeptides, particularly polynucleotides that encode the polypeptide herein designated ARO1.

In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding ARO) pohpeptides in the sequence set out in Table 1 ISEQ ID NO : 1]. or a variant thereof.

In another particularly preferred embodiment of the present invention there is a novel arom multifunctional protein protein from Candida albicans comprising the amino acid seqeunce of Table 2 SEQ 1D NO : 2). or a variant thereof.

In accordance with this aspect of the present invcntion tthcrc is provided an isolated nucteic acid molecule encoding a mature polypeptide expressible by the strain Candida albicans SC5314 contained in ATCC Deposit No. PTA-567.

In accordancc with this aspcct of the invention there are provided isolated nucteic acid molccules encoding ARO1, particularly Candida ARO1, including mRNAs, cDNAs. genomic DNAs and, in further embodiments of this aspect of the invention include biologicallv. diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising same.

In accordance with another aspect of the present invention, there is provided the use of a potynucteotide of the invention for therapeutic or prophylactic purposes, in particutar genetic immunization.

Among the particularly preferred embodiments of this aspect of me invention are naturall occurring allclic varints of ARO1 and polypeptides encoded therefrom.

In accordance wdth uus aspect of the invention there are provided novel polypeptides of Candida referred to herein as ARO1 as well as biologically, diagnosticaliy, prophylactically, clinically or therapeutically useful fragments. variants thereof. and compositions comprising same.

Among the particularly preferred embodiments of this aspect of the invention are vanants of ARO I polypeptide encoded by naturally occurring alleles of the AROI gene.

lii a preferred embodiment of this aspect of the invention there are provded methods for producing the aforementioned ARO1 polypeptides.

In accordance with yet another aspect of the present invention. there are provided inhibitors to such polypeptides. useful as antifungal agents. including, for example, antibodies.

In accordance with certain preferred embodiments of the aspect of the invention, there are provided products. compositions and methods, inter alia: assessing ARO1 expression : to treat, for example, candidosis (candidiasis). for cxnple. superficial candidosis : candidosis of the oropharynx, such as, oral thrush (acute pseudomembraneous candodisis). denture stomatitis. angular choilitis (per) eche), Candida Icukoplakia (chronic hyperplastic candidosis), midline glossitis (median rbomboid glossitis, glossal central papillary atrophy, "antibiotic sore tongue", acute atrophic candidosis), and miscellaneous forms of oral candidosis : candidosis of the genitalia, such as. vulvovaginal candidosis (vaginal thrush, Candida colpitis), and candidosis of the penis (Candida balanitis, balanoposthitis and urethritis); candidosis of the skin. nails and other external sites, such as. Candida intertrigo, napkin (diaper) dermatitis, Candida onychia and paronychia. candidosis of die external car. and miscellaneous cutaneous fbmis of candidosis: chronic mucocutaneous candidosis; systemic candidosis; candidosis of the gastrointcstinal tract. such as. esophageal candidosis, gastric candidosis, candidosis of die intestine. Candida cholecystitis, and die"autobre\very syndrome" ("meitei-sho") : candidosis of the Urinas Tract, such as. renal candidosis (Candida pyelonephritis), candidosis of die urinary bladder (Candida cystitis), Candida urethritis and prostatitis; cardiovascular Candida infections, such as. candida endocarditis, and myocarditis : Candida endocariditis, Candida myocarditis and pericarditis. Candida phlebitis and thrombophlebiits; Candida infections of the eye. such as. Candida endophthalmitis, Candida infections of die comea and conjuctiva. and candidosis of die lacrimal sacs (dacryocystitis) : candidosis of the central nervous system : Candida meningitis, cerebral candidosis. and candidosis of the inner ear : Candida infections of bones and joints : Candida peritonitis. hepatitis and miscellaneous odher fonns of systemic candidosis. such as, Candida peritonitis. Candida hepatitis and spleutis. Candida pancreatitis, Congenital and intrauterine candidosis (Candida chorioamnionitis and funisitis); disseminated candidosis, such as, in neonates. young infants, and heroin addicts : Candida allergy such as, cutaneous Candida allergy and "candidids", Respiratory tract allergy to Candida. Candida allergy in other bodily sites, "chronic candidosis" and "the yeast connection"; assaying genetic variation: and administering a ARO1 polypeptide or polvnucleotide to an organism to raise ale immunological response against a fungi, especially a C'andida.

In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polyucleotides that hybridize to ARO1 polvnucleotide sequences.

In certain additional preferred embodiments of this aspect of dhe invention there are provided antibodies against ARO1 polypeptides.

In accordance with yet another aspect of the present invention. there are provided ARO1 antagonists which are also preferably rungistatic or fungicidal.

In a further aspect of the invention there are provided compositions comprising a ARO1 polwnucleotide or a ARO 1 polypeptide for administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

Table 1 AROI cloned DNA sequence SEQ ID NO: 11 1 ATGTCTATTG AAAAGGTGCC AATTTTGGGT AAGGAAACTA TCCATGTTGG 51 TTATGGTATT GCCGACCATA TTGTCAGGGA AGTGATAGCC AACTTGGCTT 101 CTTCAACTTA TGTTATAGTG ACTGACACAA ACATGGCAAG AACTCCCCAA 151 TATTCCAAAT TGACTGATGA TTTCAAAACT AATTTGTCTG AAAAACGTCC 201 TGAATCCAGA TTATTGACTT ATTGTGTATC ACCGGGTGAA AACAACAAAA 251 ACAGAGCTAC CAAAGCTGCA GTGGAAGATT TTCTTTTACA ACAAGGTTGT 301 ACCAGAGACA CCGTCATATT GGCTGTTGGT GGGGGTGTTA TTGGTGACAT 351 GATTGGGTTT GTTGCCGCCA CATTTATGAG AGGTGTCAGA GTTGTTCAAG 401 TTCCAACTAC ATTGTTAGCC ATGGTAGATT CATCTGTTGG TGGGAAAACC 451 GCCATTGATA CTCCATTAGG TAAGAATTTC ATTGGTGCTT TCCATCAACC 501 CGAATACGTT TTCTGTGATG TATCCTTTTT AGAGACTTTA CCTGCTAGAC 551 AATTTATTAA TGGTATGGCT GAAGTTGTGA AAACTGCGGC CATTTGGAAT 601 GAAGARGAAT TCACCAGATT AGAAAACTTT TCCAAGAAGT TTCTTTCTGT 651 CGTTACCTCC AAAAAACCAG ATTTACAGTC AATTAAGGCT GAATTAGTGA 701 AAACCGTTTT GGAATCTGTT AGAGTGAAAG CTGGTGTTGT GTCATCTGAT 751 GAAAAGGAAG CTGGTCTTAG AAATTTACTT AATTTTGGCC ATACTATTGG 801 TCATGCAATT GAAGCGGTTT TAACTCCAGA AGCATTACAT GGAGAATGTG 851 TTTCTATTGG TATGATTAAG GAAGCTGAGT TGTCTAGATA TTTGGGTATA 901 CTACCACCTG TTGCAGTTGC CAGATTGTCG AAATGTCTCG TTGCTTACGG 951 CTTACCTGTG TCTATTGACG ATAAAGAATT CTTGAAAAAA GTGGGACCAA 1001 AACGTCATTA TGTTGAAATC GATATTTTGT TGAAAAAAAT GGCCATTGAC 1051 AAGAAAAATG ATGGTAGTAA GATTAGATGT GTCTTGTTAG AGAAAATTGG 1101 TAAATGTTAC CAATTGAAAG CACATCAAGT ATCCAAACAA GATTTAAGTT 1151 TTGTGTTGAC TGATGAAGTG TTGGTTCACC CATTCACCAA TCCACCTAAA 1201 GAAAACATAA TTGTTCCACC TGGTTCCAAA TCTATCTCCA ATAGAGCATT 1251 GATTTTAGCT GCTTTGGGTA ATGGTACTGT TCGTGTGAAA AATTTGTTAC 1301 ATTCAGACGA TACTAAGCAT ATGTTAGATG CTGTTGCTTC TTTAAAGGGA 1351 GCTGAAATAT CCACTGAAGA TAATGGTGAA ACCATTGTAG TTAAAGGAAA 1401 TGGTGGCAAC TTGGTCACAT CTGGCGAAGA ATTGTACTTG GGTAATGCTG 1451 GTACTGCTTC CAGATTTTTG ACTACTGTAG CTTCATTAGT GGGCAAATCA 1501 CAAGCTAGTG ATGATGTTAT TTTAACTGGT AACGCAAGAA TGCAAGAAAG 1551 ACCTATTGGA CCATTAGTGG ATGCCTTGGG ATCTAATGGT TCTGAGATTG 1601 AGTATTTGAA TAAGCAGGGT TCATTGCCAT TGAAAATCAG TGCTGGCAAT 1651 GGATTGAAAG GTGGAAGAAT AGAATTGGCT GCAACAATCT CTTCGCAGTA 1701 TGTTTCTTCA ATTTTAATGT GTGCACCATA TGCTAAGGAG CCAGTTACTT 1751 TGGCTTTAGT CGGAGGTAAA CCAATTTCTC AATTATACAT AGATATGACA 1801 TGTGCAATGA TGAAATCATT TGGTATTGAA GTTACCAAAT CAACTACTAA 1851 GGAATACACC TATCATATTC CAAAGGGGAC ATACAAGAAT CCATCTGAAT 1901 ATGTCATTGA ATCAGATGCG TCATCTGCAA CTTATCCATT AGCATTTGCT 1951 GCTATGACTG GAACATCCTG TACTATTCCG AACATAGGTT CTTCCTCATT 2001 ACAAGGAGAT GCCAAATTTG CTGTTGATGT GTTGAAACCA ATGGGGTGTA 2051 AAGTTGAACA AACCACAACT TCAACAACTG TAACTGGTCC GCCGAGAGGT 2101 CACTTGAAAC CATTACCTCA TGTTGATATG GAGCCAATGA CTGATGCATT 2151 TTTGACTGCT TCTGTTGTTG CTGCTGTTGC TAAAGGCGGT TCTTCTACTT 2201 CTATAACTGG TATTGCTAAC CAAAGAGTTA AAGAATGTAA TAGAATTGAA 2251 GCTATGGTTA CTGAATTGGC AAAATTTGGT GTGCCAGCAA ATGAATTACC 2301 CGATGGAATT GAAATACATG GTATTGATAT TGAAGATTTG AAAACACCAG 2351 AAATTTCTAA AAGAGGTGTT TCCTCCTATG ATGATCATAG AGTGGGTATG 2401 TCATTTTCAT TATTGGCAGG TTTGTGTAAA GAACCCGTTT TGATTTTGGA 2451 AAGATCAACC ACTGGTAAGA CTTGGCCAGG TTGGTGGGAT ATCTTACATT 2501 CCAAtTTTAA GATTGAGCTT GATGGTTATG AACCACCATT CAATACTGAT 2551 AAACATGTGG ATAAATCTAG TGATAAAAGT ATCATTGTCA TTGGTATGAG 2601 AGGCACTGGA AAATCTACTT TATCTGAATG GTTGGCTTCC TTTTTGGGGT 2651 TCAAGATGTT AGATATGGAC AAATATCTTG AAGAGAAATT GGGCACTGGT 2701 ATCAAATCAT TGATTAAAGC CAAAGGTTGG GAATATTTCC GTCAAGAAGA 2751 AGCAATTGTT GCTAAAGAAT GTTTCACCAA GTTTTCCAAG GGGTATGTAC 2801 TTTCCACTGG TGGAGGAATT GTTGAAGGTG AAGATGCCAG ACAGCAATTG 2851 AAATCTTATG CTGATAATGG AGGGATTGTT TTGCACTTGC ATCGTGATTT 2901 AGATGAAACT GTCACTTTCT TGGCTGCTGA TACAACCAGA CCTGCTTATA 2951 GCAGTGAAGT TCAAGAAGTA TGGTTAAGAA GAGAAAAATG GTACCATGAA 3001 TGTTCTAACT ACCACTTCTA TTCCAGTCAC TGTAGTACTG AGGATGAATT 3051 CAACCATTTG AGAAGATCCT TTGTAAACTA CATTAAACTT ATCACTGGTG 3101 CTGAAAGACC TGTTGTGCCA GCTGGTAGAT CAGCTGCTGT TGTCTTGACA 3151 TCACCGGATT TGAATGAAGT TGTTGGAGAT TTGGAATCTA TTACAATTGG 3201 TGCTGATGCA GTTGAATTAA GAGTTGATTT ATTTAAGGAT ACTTCTGCTG 3251 AATTTGTTGC TGCCCAAATT GCTGTAATAA GAAAGCATGC CGATTTACCA 3301 ATTATTTACA CTGTGAGAAC TGTGTCGCAA GGTGGTAAAT TCCCAGATGA 3351 AAATGTTGAC GAATTAAAGA GTTTGTTGTT ACTTGGTATC AGATTAGGTG 3401 TTGCATACGT TGATCTTCAA TTGACTGCTC CGAATGAACT CATTGAAGAG 3451 ATTAGCAGTA AGAAGGGTTT CACTAGAGTC ATTGGTACTT ATCAAGATAT 3501 AAATGGTGAA TTGAAATGGA ACAATGTTGA ATGGAAAAAC AAATATAATC 3551 AAGGTGTTTC CATGAATGCT GATATTGTGA GACTAGTCGG TAAAGCAAAC 3601 TCAATTCAAG ATAATTTAGA TTTGGAAAAC TTTAAAAAGC AGAATACTTT 3651 AAAACCATTG ATTGCTTTCA ATTTAGGTTC TCAAGGTAAG TTGTCACAAG 3701 TATTGAATGG AACATTCACC CCTATTTCCC ACAAATTACT TCCAAATGAT 3751 GAAGAGTTCT TAACAATCGG CGAATTAAAT CAAACTTATT TTGATATTGG 3801 AGGTTTCACT GCTAAAAAAT TTTGGGTCAT TGGTTCACCC ATTGAACATT 3851 CAAGATCACC AAACTTGCAC AATGCAGGGT ATAAAGCATT GAATTTGCCT 3901 TACCAATTTG GCAGATTTGA AGCTACAGAT GTTGATGTTG TTTATGATAA 3951 CTTGATCAAT AAACCAGACT TTGGTGGTTT AGCCATCACT ATGCCATTGA 4001 AATTGGATAT CATGAAGTTT GCCACTAAAC TATCTGATGC AGCAGAGACC 4051 ATTGGAGCCG TCAACACATT GATTCCAATT GAAGGCGGAT ATTTTGGTGA 4101 TAACACTGAT TGGGTGGGTA TTAGTAATTC TTTCATAAGA GCAGGTGTTC 4151 CACCTAAGCT GAGCTCGAAT GGGTTAGTTG TTGGTGCAGG CGGCACTTCT 4201 AGAGCTGCCA TTTATGCCTT ACATCAAATG GGATGTGCAA AGATTTACTT 4251 GGTGAATCGT ACTGCTGCTA AATTGGAGGA ACTTGTCAAG TCATTCCCTA 4301 AAGACTATAA TCTTGAGATT GTCGAAACCG AACAACAAGC CGATAAAGCA 4351 AGTAAAGTCC TGTTGGCAGT GTCTTGTATT CCTGCTGATA AACCATTAGA 4401 TGGAGAAGTG TTGAAGAAAA TTGAGCGAAT TTTATCTAAT GGAAGTGAAC 4451 AATCTGCTGG TTTCAAGCCA ACCTTATTGG AAGCTAGTTA CAAACCAAGA 4501 GTGACACCAA TTATGAAGCT TGCTGAAGAA CAATATAAAT GGAAAGTTAT 4551 CCCAGGTGTT GAGATGTTGG TAAATCAAGG TGATAGACAG TTTAAACTTC 4601 ATACTGGTTT TACTGCACCA TATGAGATTA TTCATCGTGC TGTTGTTGAG 4651 GAATAA Table 2 ARO1 deduced amino acid sequence [SEQ ID NO : 2 1 MSIEKVPILG KETIHVGYGI ADHIVREVIA NLASSTYVIV TDTNMARTPQ 51 YSKLTDDFKT T1LSEKRPESR LLTYCVSPGE 2dIJRKNRATKAA VEDFLLQQGC 101 TRDTVILAVG GGVIGDMIGF VAATFMRGVR WQVPTTLLA MVDSSVGGKT 151 AIDTPLGKNF IGAFHQPEYV FCDVSFLETL PARQFINGMA EWKTAAIWN 201 EEEFTRLENF SKKFLSWTS KKPDLQSIKA ELVKTVLESV RVKAGVVSSD 251 EKEAGLRNLL NFGHTIGHAI EAVLTPEALH GECVSIGMIK EAELSRYLGI 301 LPPVAVARLS KCLVAYGLPV SIDDKEFLKK VGPKRHYVEI DILLKKMAID 351 KKNDGSKIRC VLLEKIGKCY QLKAHQVSKQ DLSFVLTDEV LVHPFTNPPK 401 ENIIVPPGSK SISNRALILA ALGNGTVRVK NLLHSDDTKH MLDAVASLKG 451 AEISTEDNGE TIWKGNGGN LVTSGEELYL GNAGTASRFL TTVASLVGKS 501 QASDDVILTG NARMQERPIG PLVDALGSNG SEIEYLNKQG SLPLKISAGN 551 GLKGGRIELA ATISSQYVSS ILMCAPYAKE PVTLALVGGK PISQLYIDMT 601 CAMMKSFGIE VTKSTTKEYT YHIPKGTYKN PSEYVIESDA SSATYPLAFA 651 AMTGTSCTIP NIGSSSLQGD AKFAVDVLKP MGCKVEQTTT STTVTGPPRG 701 HLKPLPHVDM EPMTDAFLTA SWAAVAKGG SSTSITGIAN QRVKECNRIE 751 AMVTELAKFG VPANELPDGI EIHGIDIEDL KTPEISKRGV SSYDDHRVGM 801 SFSLLAGLCK EPVLILERST TGKTWPGWWD ILHSKFKIEL DGYEPPFNTD 851 KHVDKSSDKS IIVIGMRGTG KSTLSEWLAS FLGFKMLDMD KYLEEKLGTG 901 IKSLIKAKGW EYFRQEERIV AKECFTKFSK GYVLSTGGGI VEGEDARQQL 951 KSYADNGGIV LHLHRDLDET VTFLAADTTR PAYSSEVQEV WLRREKWYHE 1001 CSNYHFYSSH CSTEDEFNHL RRSFVNYIKL ITGAERPWP AGRSAAWLT 1051 SPDLNEWGD LESITIGADA VELRVDLFKD TSAEFVAAQI AVIRKHADLP 1101 IIYTVRTVSQ GGKFPDENVD ELKSLLLLGI RLGVAYVDLQ LTAPNELIEE 1151 ISSKKGFTRV IGTYQDINGE LKWNNVEWKN KYNQGVSMNA DIVRLVGKAN

1201 SIQDNLDLEN FKKQNTLKPL IAFNLGSQGK LSQVLNGTFT PISHKLLPND 1251 EEFLTIGELN QTYFDIGGFT AKKFWVIGSP IEHSRSPNLH NAGYKALNLP 1301 YQFGRFEATD VDWYDNLIN KPDFGGLAIT MPLKLDIMKF ATKLSDAAET 1351 IGAVNTLIPI EGGYFGDNTD WVGISNSFIR AGVPPKLSSN GLWGAGGTS 1401 RAAIYALHQM GCAKIYLVNR TAAKLEELVK SFPKDYNLEI VETEQQADKA 1451 SKVLLAVSCI PADKPLDGEV LKKIERILSN GSEQSAGFKP TLLEASYKPR 1501 VTPIMKLAEE QYKWKVIPGV EMLVNQGDRQ FKLHTGFTAP YEIIHRAWE 1551 E BRIEF DESCRIPTION OF THE DRAWINGS The following drawings depict certain embodiments of the invention. Tliev are illustrative oniv and do not limit the invention otherwise disclosed herein.

Table 1 shows the polyucleotide sequence of Candida albicans ARO1 [SEQ ID NO : 1].

Table 2 shows me amino acid sequence of Canclicla alhicans Ald SEQ ID NO:2] deduced from dte polynuclcotide sequence of Table 1.

GLOSSARY The following illustrativc explanations arc providcd to facilitate understanding of certain terms used frequency herein, particularlv ui die Exnples. The explanations are provided as a convenience and are not limitative of the invention.

ARO I-BINDING MOLECULE. as used herein. refers to molecules or ions which bind or interact specifically widi AROI polyeptides or polynucleotides of the present invention-including, tor examp) e enzyme substrates. cell membrane components and classical receptors. Binding between polypeptidcs of the invention and such molecules. including binding or binding or interaction molecules may be exclusive to polypeptides of the invention, which is preferred. or it may be highly specific for polyeptidcs of me invention, which is also preferred. or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred. or it mav be specific to several groups of proteins at least one of which includes a polypeptide of the'invention. Binding molecules also include antibodies and antibody-derived reagents that bind specifically to polypeptides of the invention.

GENETIC ELEMENT generally means a polynucleotide comprising a region that encodes a polypeptide or a poiynucieotide region that regulates replication, transcription or translation or other processes important to expression of the polypeptide in a host cell, or a poiynucieotide comprising bom a region that encodes a polypeptide and a region operably linked thereto that regulates expression. Genetic elements may be comprised within a vector that replicates as an episomal element : mat is, as a molecule physically independent of the host cell genome. They may be comprised within plasmids. Genetic elements also may be comprised within a host cell genome not in their natural state but, rather, following

manipulation such as isolation. cloning and introduction into a host cell in the form of purifie DNA or in a vector, among others.

HOST CELL is a cell which has been transfonmed or transfected, or is capable of transformation or transfection by an exogenous polvnucleotide sequence.

IDENTITY or SIMILARITY, as known in the art, are relationships between two or more polypeptide sequences or two or more polwucleotide sequences, as determined by comparing the sequences.

In the art. identity also means the degree of sequence relatedness between polvpeptide or polvnucleotide sequences. as the case may be. as determined by the match between strings of such sequences. Both identity and similaritv can be readily calculated (Computational Molecular Biology, Lesk. A. M.. ed.

Oxford Univcrsity Press. New York. 1988 : Biocomputing: Informatics and Genome Projects, Smith.

D. W.. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin.

A. M.. and Griffin. H. G.. eds., Humana Press, New Jersey, 1994: Sequence Analysi, s in Molecular Biology, von Heinje, G., Academic Press. 1987; and Sequence Analysis Primer, Gribskov, M. and Devercux. J.. eds.. M Stockton Press. New York. 1991). While there exist a number of methods to measure identity and similarity between two polynucleotide or two polypeptide sequences, both terms are well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devercux. J.. cds.. M Stockton Press. New York, 1991 : and Carillo, H., and Lipman. D., SIAM. J Applied Math., 48: 1073 (1988) Methods commonly employed to détermine identity or similarity between sequences include. but are not limited to those disclosed in Carillo, H.. and Lipman. D., SIAM. J. Applied Math., 48:1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identitv and similarity are codified in computer programs.

Preferred computer program methods to détermine identity and similarity between two sequences include. but are not limited to. GCG program package (Devereux, J., et al.. Nucleic Acicl. s Research 12 (1) : 387 (1984)). BLASTP. BLASTN. and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403 (1990)).

ISOLATED means altered"by the hand of man"from its natural state : i. e., that. if it occurs in nature. it has been changed or removed from its original environment. or both. For example a naturally occurring polvnucleotide or a polypeptide naturally present in a living organism in its natural state is not isolated.'but the same polvnucleotide or polypeptide separated from the coexisting materials of its natural state is"isolated". as the term is employed herein. As part of or following isolation, such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance. The isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms

hitroduced into host cells in culture or in whole organisms. such DNAs still would be isolated. as the term is used herein, because they would not be in their naturally occuring form or environment. Similarly, the polyucleotides and polypeptides may occur in a composition. such as a media formulations. solutions for introduction of polynucleotides or polypeptides. for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions. and. therein remain isolated polynucleotides or poiypeptides within me meaning of that term as it is employed herein.

POLYNUCLEOTIDE (S) generally refers to any polyribonucleotide or polvdeoxribonucleotide. which mav be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to. among others, single-and double-stranded DNA. DNA that is a mixture of single-and double-stranded regions or single-, double-and triple-stranded regions, single-and double-stranded RNA. and RNA that is mixture of single-and double-stranded regions. hybrid molecules comprising DNA and RNA that may be single-stranded or. more typically, double-stranded, or triple-stranded, or a mixture of single-and double-stranded regions. In addition, polynucleotide as used herein refers to triplc-strvdcd regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the moleucles. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein. the term polvnucleotide uacludes DNAs or RNAs as described above that contain one or more modified bases. Thus. DNAs or RNAs with backbones modified for stabilitv or for other reasons are"polvnucleotides"as that term is intended herein. Moreover. DNAs or RNAs comprising unusual bases. such as inosine, or modified bases. such as tritylated bases. to name just two examples, are polynucleotides as me term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve manv useful purposes known to those of skill in the art. The term polvnucleotide as it is employez herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides. as well as the chemical fomls of DNA and RNA characteristic of viruses and cells, including simple and complex cells. inter alia. Polynucleotides embraces short polynucleotides often referred to as oligonucleotide (s).

POLYPEPTIDES. as used herein, includes all polypeptides as described below. The basic structure of poiypeptides is well known and has been descnbed m innumerable textbooks and other publications in the art. hi this context. the term is used herein to refer to any peptide or protein comprising two or more amino acids jouie to each other in a linear chain by peptide bonds. As used herein. the term refers to both short chauvis, which also commonly are referred to in the art as peptides oligopeptides and oligomers, for example. and to longer chains, which generally are referred to in the art as proteins, of which there are many types. It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that manv amino acids.

Lnduding the terminal amino acids, may be modified in a given polypeptide. eimer by natural processes. such as processing and other post-translational modifications. but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here. but thev are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. and thev are well known to those of skill in the art. Among the known modifications wluch mav be present in polypeptides of the present are. to name an illustrative few. acetylation. acylation. ADP-ribosylation. unidation. covalent attaclunent of flavin. covalent attachment ofaheme moiety, covalent attachment of a nucleotide or nucleotide derivative. covalent attachent of a lipid or lipid derivative. covalent attachment of phosphotidvlulositol. cross-linking, cyclization. disulfide bond formation, demethyiation. formation of covalent cross-links. fondation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor fomiation. hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifcations are well known to those of skill and have been described in great detail in die scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic textes, such as. for instance PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed.. T. E. Creighton, W. H.

Freeman and Company, New York (1993). Many detaiied rcviews are available on diis subject, such as, for example, those provided by Wold. F.. Posttranslational Protein Modifications : Pcrspcctives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.

Johnson, Ed.. Acadcmic Press. New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) and Rattan et al../'/e//?'//!c.s'/.')'.'/.y/-?.s'/<:7o/Ayoa'///cof.t'7f :/g;/?.Ann. N. Y. Acad. Sci. 663 : 48-62 (1992). It will be appreciated, as is well known and as noted above. that pohpeptidcs are not always cntircly linear. For instance. polypeptides may be generally as a result of posttranslational events, including natural processing event and events brought about bv human manipulation which do not occur naturally.

Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods. as Modifications can occur anywhere ul a polypeptide, including the peptide backbone. the ammo acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group i a polypeptide. or both. by a covalent modification. is common in naturally occurring and synthetic polypeptides and such modifications mav be present in polxpeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E coli or other cells, prior to proteolytic processing, almost invariably will be N-formylmethionine. During post- translational modification of the peptide. a methionine residue at the NH,-terminus may be deleted.

Accordingly, this invention contemplates the use of both the methionine-containing and the methionineless amino terminal variants of the protein of the invention. The modifications that occur in a polypeptide often will be a function of how it is made. For polypeptides made bv expressing a cloned gene in a host. for instance. the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present m the polypeptide aitinio acid sequence. For instance, as is well known, glycosyiation often does not occur in fungal hosts such as, for example. E. coli. Accordingly, when glycosylation is desired. a polypeptide should be expressed in a glycosvtating host, generally a eukaryotic cell. Insect cell often carry out the same posttranslational glycosylations as mammalian cells and. for this reason. insect cell expression systems have been developed to express efficiently mammalian proteins having native patterns of glycosylation, inter ulia. Similar considcrations apply to other modifications. It will bc appreciated that me saxe type of modification may be present in the same or varying degree at several sites in a given polyeptide. Also, a given polypeptide may contain many types of modifications. In gcneral. as used herein. the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized recombinantly by expressing a polynucleotide in a host cell.

VARIANT (S), as the term is used herein, are polynucleotides or polypeptides that differ from a reference polvnucleotide or polypeptidc respectively. Variants in this sense are described bclow and elsewhere in me present disclosure in greater detail. (1) A polynucleotide that differs in nucleotide sequence from another. reference polynucleotide. Generally. differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and. in many regions, identical.

Changes in me nucleotide sequence of the variant may be silent, i. e., they may not alter the amino acids encoded by the polxnucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with me same ammo acid sequence as me reference polypeptide. Changes in die nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substituions, additions, deletions, fusions and truncations in die polypeptide encoded by the reference sequence. as discussed below.

(2) A polypeptide mat differs in amino acid sequence from another, reference polypeptide. Generally. differences are limited so that the sequences of the reference and the variant are closely similar overall and, in many region. identical. A variant and reference polypeptide may differ in amino acid sequence bv one or more substitutions. additions, deletions. fusions and truncations, which may be present in any combination.

(3) A variant may also be a fragment of a polynucleotide or polypeptide of the uivention that differs from a reference polvnucleotide or polypeptide sequence by being shorter than the reference sequence, such as by a terminal or internal deletion. A variant of a polypeptide of the invention also includes a polypeptide which retains essentially the same biological function or activitv as such polypeptide, e. g., proproteins which can

be activated by cleavage of the proprotein portion to produce an active mature polyeptide. (4) A variant may also be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded bv the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the poiypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is emploved for purification of the mature poiypeptide or a proprotein sequence. (5) A variant of the polynucleotide or polypeptide may be a naturally occurring variant such as a naturally occurring allelic variant. or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides. cells or organisms. or may be made by recombinant mcans. Among polynucleotide variants in this regard are variants that differ from the aforementioned polwnucleotides by nucleotide substitutions, deletions or additions. The substitutions. deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both.

Alterations in the coding regions may produce conservative or non-conservative ammo acid substitutions, deletions or additions. Among g polypeptid variants in this regard are variants that differ from thc aforementioned polypeptides by amino acid substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more amino acids. Alterations in the of the ammo acids may be conservative or non-conscrvativc an o acid substitutions, deletions or additions. All such variants defined above are deemed to be w idiin die scope of those skilled in die art from the teachings herein and from die art.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel AROI polypeptides and polynucleotides, among other things, as described in greater detail below. In particular, the uivention relates to polypeptides and polynucleotides of a novel AROI gene of Candida albicans, which is related by ammo acid sequence homology to Saccharomyces cerevisiae AROI polypeptide. The invention relates especially to AROI having the nucleotide and encoded amino acid sequences set out in Table 1 and Table 2 respectively, and to the AROI nucleotide and amino acid sequences of the DNA in ATCC Deposit No. PTA-567, which is herein referred to as"the deposited strain"or as the"DNA of the deposited strai 1. It w ill be appreciated that the nucleotide and amino acid sequences set out in Figures 1 [SEQ ID NO : 1] and 2 [SEQ ID NO: 2] were obtained by sequencing the DNA of the deposited strain. Hence, the sequence of AROI of the

deposited strain is controlling as to any discrepancies between it (and the sequence it encodes) and the sequences of Table 1 [SEQ ID NO : 1] and Table 2 [SEQ ID NO : 2].

Techniques are available to evaluate temporal gene expression in fungi, particularly as it applies to viability under laboratory and host infection conditions. A number of methods can be used to identify genes which are essential to survival per se, or essential to the establishment/maintenance of an infection. Identification of expression of a sequence by one of these methods yields additional information about its function and permits the selection of such sequence for further development as a screening target. Briefty. these approches include: 1) Signature Tagged Mutagenesis (STM) This technique is described by Hensel et al.. Science 269: 400-403 (199-). the contents of which is incorporated by reference for background purposes.

Signature tagged mutagenesis identifies genes necessary for the establishment/maintenance of infection in a given infection model.

The basis of the technique is the random mutagenesis of target organism by various means (c. g.. transposons) such that unique DNA sequence tags are inserted in close proximity to the site of mutation. The tags from a mixed population of fungal mutants and fungi recovered from an infected hosts are detected by amplification, radiolabeiing and hvbridization analvsis. Mutants attenuated in virulence are revealed bv absence of the tag from the pool of fungi recovered from infected hosts.

In Canclicla albicans. because the transposon system is not developed. a more efficient way of creating the tagged mutants is to use the insertion-duplication mutagenesis technique as described by Morrison el al., J. Bacteriol, 159: 870 (1984) the contents of which is incorporated by reference for background purposes.

2) In Vivo Expression Technology (IVET) This technique is described by Camilli et al., Proc. Nat'l. Acad. Sci. USA. 91: 2634-2638 (1994). the contents of which is incorporated by reference for background purposes. IVET identifies genes up-regulated during infection when compared to laboratorv cultivation. implying an important role in infection. Sequences identified by this technique are implied to have a significant role in infection establishment/maintenance.

In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less reporter gene in a plasmid vector. The pool is introduced into a host and at various times after infection fungi may be recovered and assessed for the presence of reporter gene expression. The chromosomal fragment carried upstream of an expressed reporter gene should carry a promoter or portion of a gene normallv upregulated during infection. Sequencing upstream of the reporter gene allows identification of the up regulated gene.

3) Differential display This technique is described by Chuang et al., J. Bacteriol. 175 : 2026-2036 (1993) and Zhao et al.. Microb. Pathog. 25 : 121-9 (1998). the contents of which are incorporated bs reference for background purposes. This method identifies those genes which are expressed in an organism bv identifying mRNA present using randomly-primed RT-PCR. By comparing pre-infection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to librarv sequences.

4) Generation of conditional lethal mutants by transposon mutagenesis.

This technique described bx Chun and Goebl, Genetic. 142-. 39-50 (1996). the contents of which is incorporated by reference for background purposes. identifies genes whose expression are essential for ccll viability.

In this technique transposons carrving controllable promoters. which provide transcription outward from the transposon in one or both directions. are generated. Random insertion of these transposons into target organisms and subsequent isolation of insertion mutants in the presence of inducer of promoter activity ensures that insertions which separate promoter from coding region of a gene whose expression is essential for cell viability will be recovered. Subsequent replica plating in the absence of inducer identifies such insertions. since they tail to survive. Sequencing of the flanking regions of the transposon allows identification of site of insertion and identification of the gene disrupted. Close monitoring of the changes in cellular processes/morphology during growth in the absence of inducer yields information on likelv function of the gene. Such monitoring could include floxv cytometrv (cell division. lysis. rcdox potential DNA replication). incorporation of radiochemicallv labeled precursors into DNA. RNA, protein. lipid. ccll wall polysaccharides. monitoring reporter enzyme gene fusions which respond to known cellular stresses.

5) Generation of conditional lethal mutants by chemical mutagenesis.

This technique is described bv Beckwith. J., Method. s in Enzvmology 204 : 3-18 (1991), the contents of which are incorporated herein bN, reference for background purposes. In this technique random chemical mutagenesis of target organism, growth at temperature other than physiological temperature (permissive temperature) and subsequent replica plating and growth at different temperature (e. g., 42°C to identify ts, 25°C to identify cs) are used to identify those isolates which now fail to grow (conditional mutants). As above close monitoring of the changes upon growth at the non-permissive temperature vields information on the function of the mutated gene.

Complementation of conditional lethal mutation by library from target organism and sequencing of complementing gene allows matching with library sequences.

Each of these techniques mav have advantages or disadvantage depending on the particular application. The skilled artisan would choose the approach that is the most relevant with the particular end use in mind. For example, some genes might be recognised as essential for infection but in reality are only necessary for the initiation of infection and so their products would represent relatively unattractive targets for antifungals developed to cure established and chronic infections.

6) RT-PCR Fungal messenger RNA, preferably that of C'anclida alhicans. is isolated from fungal infected tissue e. g. 48 hour murine lung infections. and the amount of each mRNA species assessed bv reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs. The detennination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the fungal genes which are transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in fungal pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for novel antifungals. Because of the gene specific nature of the PCR primers cmployed it should be understood that the fungal mRNA preparation need not be free of mammalian RNA. This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain fungal mRNA species which are very short lived in the bacterium (in the order of 2 minute halflives).

Optimally the fungal mRNA is prepared from infected murine lung tissue bv mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time. subsequent processing according to the manufacturers ofTRIzole reagent and DNAase treatment to remove contaminating DNA. Preferably the process is optimized by finding those conditions which give a maximum amount of fungal 16S ribosomal RNA, preferablv that of Candida albicans, as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe. Typically a 5 due labelled primer is used in each PCR primer pair in a PCR reaction which is tenninated optimally between 8 and 25 cycles. The PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI).

Use of the of these technologies when applied to the sequences of the present invention enables identification of fungal proteins expressed during infection, inhibitors of which would have utility in anti-fungal therapy.

Polynucleotides In accordance with one aspect of the present invention, there are provided isolated polvnucleotides which encode the ARO 1 polypeptide having the deduced amino acid sequence of Table 2 [SEQ ID NO : 2].

Using the information provided herein, such as the polxnucleotide sequence set out il Table 1 [SEQ ID NO : 11,. a polvnucleotide of the present invention encoding AROI polypeptide may be obtained using standard cloning nid screening procedures, such as those for cloning and sequencing chromosomal DNA fragments from Candida albicans SC5314 cells as starting material. followed by obtaining a full length clone. For example, to obtain a polynucleotide of the invention sequence. such as that sequence given in Table I [SEQ ID NO : 1] typically a librarv of clones of chromosomal DNA of Candida albicans SC5314 in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide. preferably a 17-mer or longer. derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using high stringency washes. By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence. Conveniently such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone.

Suitable techniques are described by Maniatis T.. Fritsch E. F. and Sambrook et al.. MOLECULAR CLONING. A LABORATORY MANUAL, 2nd Ed. : Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Ncw York (1989). (sce Screening By Hybridization 1. 90 and Sequencing Denatured Double- Stranded DNA Templates 13. 70). Illustrative of the invention, the polynucleotide set out in Table 1 [SEQ ID NO : I] was discovered in a DNA library derived from Candida albicans SC5314.

ARO I polvpeptides of the invention is structurally related to other proteuis of the arom fnilv as shown by the results of sequencing die DNA encoding AROI polvpeptide of the deposited strain. The DNA sequence thus obtained is set out in Table I SEQ ID N0 : 1]. It contains an open reading frame encoding a protein of having about the number ofamino acid residues set fordi in Table 2 [SEQ ID NO : 2] widi a deduced molecular weigllt that can be calculated using amino acid residue molecular weight values well known in die art. The protein exhibits greatest homology to Saccharomyces cerevisiae ARO I protein among known proteins. ARO I of Table 2 [SEQ ID NO : 2] has about 59. 5% identity over its entire length and about 68. 8% similarity over its entire length widi die amino acid sequence of Saccharomyces cerevisiae AROI polypeptide.

Polynucleotides of the present invention may be ul the form of RNA. such as mRNA. or in the form of DNA, including, for instance, cDNA and genonuc DNA obtained by cloning or produced bs chemical svnthetic techniques or by a combination thereof. The DNA may be double-stranded or single- stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coing strand, also referred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical to the coding sequence of the polynucleotide shown in Table I [SEQ ID NO : 1]. It also may be a polynucleotide widi a different

sequence, which, as a result of the redundancy (degeneracx) of the genetic code. encodes the polypeptide of Table 2 [SEQ ID NO:2].

Polynucleotides of the present invention which encode the polypeptide of Table 2 [SEQ ID NO : 2] may include. but are not limited to the coding sequence for the mature polypeptide. by itself : die coding sequence for the mature polypeptide and additional coding sequences. such as those encoding a leader or secretory sequence, such as a pre-, or pro-or prepro-proteui sequence : the coding sequence of the mature polypeptide. with or without the aforementioned additional coding sequences, together with additional. non- coding sequences, including tor example, but not limited to non-coding 5'and 3'sequences, such as the transcribed, non-translated sequences that plav a role in transcription (including termination signals, for example,), ribosome binding, mRNA stability elements, and additional coding sequence which encode additional amino acids. such as those which provide additional functionalities. Thus, for instance, die polypeptide may be fused to a marker sequence, such as a peptide. which facilitates purification of the fused polypeptide. In certain embodiments of this aspect of the invention, the marker sequences is a hexa-histidine peptide. such as the tag provided in die pQE vector (Qiagen. hic.). among others, many of which ; commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The HA tag may also be used to create fusion proteins and corresponds to an epitope derived of influenza hemagglutuiin protein, which has been described by Wilson et al., Cell 37: 767 (1984), for instance. Polynucleotides of the invention also include, but are not limited to. polynucleotides comprising a structural gene and its naturally associated genetic elements.

In accordance with the foregoing, the term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides wliich include a sequence encoding a polypeptide of die present invention. particularly fungal, and more particularly the Candida albicans ARO1 having the amino acid sequence set out in Table 2 [SEQ ID NO : 2]. The term encompasses polynucleotides diat include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or insertion sequence or editing) together with additional regions, that also may contais coding and/or non- coding sequences.

The present invention further relates to variants of the herein above described polynucleotides which encode for variants of the polypeptide having the deduced amino acid sequence of Table 2 [SEQ ID NO : 2]. A variant of the poiynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polanucleotide may be made bv mutagenesis techniques, including those applied to polynucleotides, cells or organisms.

Among variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions deletions or additions mav involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

Among the particularly preferred embodiments of me invention in this regard are polvnucleotides encoding polypeptides having the ammo acid sequence of ARO1 set out in Table 2 [SEQ ID NO : 2]; variants thereof.

Further particularly preferred in titis regard are polynucleotides encoding AR01 variants of the fragments which have the amino acid sequence of ARO 1 polypeptide of Table 2 [SEQ ID NO : 2] in which several. a few. 5 to 10. I to 5. 1 to 3, 2. 1 or no amino acid residues are substituted. deleted or added. ui anv combination. Especiallv preferred among these are silent substitutions, additions and deletions, which do not alter the propertics and activitics of ARO1. Also cspcciallv preferred in this regard are conservative substitutions. Most highly preferred are polynucleotides encoding polypeptides having the amino acid sequence of Table 2 [SEQ ID NO : 2], without substitutions.

Further preferred embodiments of die invention are polynucleotides that are at least 70% indentical over their entire length to a polnnucleotide encoding ARO I polypeptide having the amino acid sequence set out in Table 2 [SEQ ID N0 : 2], and polynucleotides which are complementary to such polynucleotides.

Alternativelv, most highly preferred are polwucleotides that comprise a region that is at least 80% identical over their entire length to a polynucleotide encoding ARO1 polypeptide of the Candida albicans DNA of the deposited strain and polynucleotides complementary thereto In this regard. polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polvnucleotides. those with at least 95% are especially preferred. Furthermore, those with at least 97% are highlv preferred among those with at least 95%, and among these those with at least 98% and at least 99% @ are particularly highly preferred, with at least 99% being the more preferred.

Preferred embodiments in this respect, moreover, are polynucleotides wliich encode polypeptides which retain substantiallv the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQIDNO : I].

The present invention further relates to polvnucleotides that hybridize to the herein above-described seqeunces, In this regard the present invention especiallv relates to polynucleotides which hybridize under stringent conditions to the herein above-described polvnucleotides. As herein used, the term"stringent conditions"means hvbridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.

As discussed additionally herein regarding polvnucleotide assays of the inventionN tòr instance polynucleotides of the invention as discussed above. may be used as a hvbridization probe for RNA. cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding ARO 1 and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the ARO1 gene. Such probes generally will comprise at least 15 bases. Preferably, such probes have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less.

For example. the coding region of the AROI gene may be isolated by screening using the known DNA sequence to swthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The polyucleotides and polypeptides of the present invention may be e employed as research reagents and materials for discovery of treatments of and diagnostics for disease. particularly human discase. as further discussed herein relating to polynucleotide assays. inter alia.

The polynucleotides of the invention that are oligonuclcotides. including SEQ ID NOS: 3 and 4, derived from the sequences of SEQ ID NOS: I and 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the Candida albicans genes identified herein in whole or in part are transcribed in infected tissuc. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.

The polynucleotides may encode a polypeptide which is me mature protein plus additional amino or carboxyl-terminal ammo acids, or anuino acids interior to the mature polypeptide (when the mature form has more than one pol ! peptidc chain for ulstancc). Such sequences may plav a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half- life or may facilitate manipulation of a proteii for assay or production. among other timings. As generallv is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having me mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such ulactive precursors generally are activated. Some or all of die prosequences may be removed before activation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein. having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.

Deposited materials The deposit has been made under the temis of the Budapest Treatv on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposit is provided merelv as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S. C. 112.

A deposit containing a Candicla albicans strain has been deposited with me American Type Culture Collection (ATCC), 12301 Park Lawn Drive. Rockville. Maryland 20852. USA. National Collections of Industrial and Marine Bacteria Ltd. (NCIMB). 23 St. Machar Drive. Aberdeen AB2 1 RY. Scotland on 20 August 1999 and assigne ATCC Deposit No. PTA-567. The Canclicla crlbican, v strain deposit is referred to herein as"the deposited strain"or as"me DNA of the deposited strain." The deposited material is a strain that contais the full length AROI DNA. referred to as"C. albicans SC53 4"upon dcposit.

The sequence of the polynucleotides contained in the deposited material. as well as the amino acid sequence of the polypeptide encoded thcreb ! arc controlling in die event of any conflit with any description of sequences herein.

A license may bc required to make. use or sell the deposited materials. and no such license is hereby granted.

Polypeptides The present invention further relates to a AROI polypeptide which lias a deduced amino acid sequence of Glu muno acids in length, as set forth in Table 2 [SEQ ID NO:2], and has a deduced molecular weight of 169 kilodaltons.

Among dic particularly preferred embodvnents of the invention are polypeptides having me amino acid sequence of ARO I set out in Table 2 [SEQ ID NO : 2]. and variants thereof. Alternatively, particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of the ARO 1. and variants thereof.

Among preferred variants are those that vary from a reference bv conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide bv another amino acid of like characteristics. Topically seen as conservative substitutions are the replacements. one for another. among the aliphatic amino acids Ala. Val, Leu and Ile ; interchange of the hydroxyl residues Ser and Thr. exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants thereof having the amino acid sequence of the AROI polypeptide of Table 2 [SEQ ID NO : 2] in which several. a few, 5 to 10. 1 to 5, 1 to 3, 2, 1 or

no ammo acid residues are substituted. deleted or added. in anv combination. Especiallv preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the ARO1, Also especially preferred in this regard are conservative substitutions. Most highly preferred are polypeptides having the amino acid sequence of Table 2 [SEQ ID NO : 2] without substitutions.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form. and preferably are purifie to homogeneity.

The polypeptides of the present invention include the polepeptide of Table 2 [SEQ ID NO:2] (in particular the mature polypeptide) as well as polypeptides which have at leas 70% identity to the polypeptide of Table 2 [SEQ ID NO : 2[, preferably at least 80% identity to me pohpeptide of Table 2 [SEQ ID N0 : 2). and more preferably at least 90% similarity (more preferably at least 90% identity) to the polycptide of Table 2 [SEQ ID NO : 21 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of Table 2 [SEQ ID NO : 2] and also include portions of such polypeptides with such portion of me polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide svnthesis : dierefore. the fragments may be employed as intermediates for producing the fill-lengdi polypeptides. Fragments or portions of me polynucleotides of the present invention may be used to synthesize full-length polynucleotides of die present invention.

Fragments Also among preferred embodiments of this aspect of the present invention are polypeptides comprising fragments of ARO1, most particularly fra Table 2 [SEQ ID NO : 2]. and variants of the AROI of Table 2 [SEQ ID NO: 2].

In this regard a fragment is a polypeptide having an amino acid sequence that entirely is die same as part but not all of the e amino acid sequence of the aforementioned ARO l pcptidcs and variants thereof.

Such fragments may be"free-standing,"i. e., not part of or fused to other amino acids or polypeptides, or they may be comprised widiin a larger polypeptide of winch dieu forum a part or region.

When comprised widiin a larger polypeptide, die presently discussed fragments most preferably form a single continuous region. However. several fragments may be comprised within a single larger polypeptide.

For uistance. certain preferred embodiments relate to a fragment of a ARO1 polypeptide of the present comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro-polypeptide regions fused to the amino terminus of the AROI fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from ARO1.

Representative examples of polypeptide fragments of the invention. uiclude, for example. fragments from amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101-Glu, and any combination of these 20 ammo acid fragments.

In this context "about" herein includes the particularly recited ranges larger or smaller by several. a few, 5. 4. 3. 2 or 1 amino acid at either extreme or at both extremes.

Preferred fragments of the invention include, for example, truncation pohpeptides of AR01.

Truncation potypeptides include ARC I polypeptides having the amino acid sequence of Table 2. or of variants thereof, except for deletion of a continuous series of residues (that is. a continuous region part or portion) that includes me amino terminus, or a continuous series of residues that includes the carboxvl terminus or. as in double truncation mutants, deletion of two continuous series of residues. one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out about also are preferred embodiments of truncation fragments. which are especially preferred among fragments generally. Degradation forms of the polypeptides of the invention in a host cell, particularly a Candida, are also preferred.

Also preferred in this aspect of die invention are fragments charactcrizcd by structural or functional attributes of ARO1. Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forcing regions. beta-sheet and beta-sheet-forming regions. turn and tum-fonning rcgions. coil and coil-forming regions. hydrophilic regions, hydrophobic regions, alpha amphipathic régions. beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region. and high antigenic index regions of ARO I. and combinations of such fragments.

Preferred regions are those that mediate activities of ARO 1. Most highly preferred in this regard are fragments that have a chemical. biological or other activity of ARO 1. including those widf a similar activiez or an unproved active. or widf a decreased undesirable activity. Furthcr preferred polypeptide fragments are those that are antigenic or immunogenic in an animal, especially in a human.

It will be appreciated that the invention also relates to. among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hybridize to polvnucleotides encoding the fragments. particularly those that hybridize under stringent conditions. and polynucleotides. such as PCR primers, for amplifying polwucleotides that encode the fragments. In these regards, preferred poh-nucleotides are those that correspond to the preferred fragments, as discussed above.

Vectors, host cells, expression The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of die present invention, host cells which are genetically engineered with vectors of the invention and the production of polvpeptides of the invention by recombinant techniques.

Host cells can be geneticallv engineered to incorporate polynucleotides and express polypeptides of the present invention. Introduction of a polvnucleotides into the host cell can be affected by calcium phosphate transfection. DEAE-dextran mediated transfection. transvection. microinjection, cationic lipid- mediated transfection. electroporation. transduction, scrape leading, ballistic introduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al.. BASIC METHODSINMOLECULAR BIOLOGY, (1986) and Sambrook et al.. MOLECULAR CLONING: A LABORATORYAzIANUAL. 2nd Ed.. Cold Spring Harbor Laborator Press. Cold Spring Harbor. N. Y.

(1989).

Polvnucelotide constructs in host cells can be used in a conventional mazer to produce the gene product encoded by the recombinant sequence. Alternatively, the poiypeptides of the invention can be svnthetically produced by conventional peptide sniitlicsizcrs.

Mature proteins can be expressed in mammalian cells. yeast. bacteria. or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropnate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al..

MOLECULAR CLONING : A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989).

In accordance with this aspect of the invention the vector may be. for exnple. a plasmid vector, a single or double-stranded phage vector. a single or double-stranded RNA or DNA viral vector. Plasmids generally are designated herein by a lower case p preceded and/or followed by capital Ictters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill il the art.

Starting plasmids disclosed herein are either commercially available, publicly available, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art.

Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operativelv linked to the polynucleotide to be expressed. Appropnate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

In certain preferred embodiments in this regard. the vectors provide for specific expression. Such specific expression may be inducible expression or expression out in certain types of cells or both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.

A vanety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routine by those of skill in the art.

A great vanety of expression vectors can be used to express a polypeptide of the invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e. g., vectors derived from bacterial plasmide. from bactenophage from transposons. from yeast episomes. from insertion elements. from veast chromosomal elements, from viruses such as baculoviruses, papova vimses such as SV40, vaccinia viruses, adenoviruses. fowl pox vimses. pseudorabies vinises and retroviruses, and vectors derived from combinations thereof. such as those derived from plasmid and bacteriophage genetic elements. such as cosmids and phagemids. all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintaul. propagate or express polynucleotides to express a polypeptide in a host may bc used for expression in this regard.

The appropriate DNA sequence ma be inserted into die vector bv any of a variety of well-known and routine techniques, such as. tor example, those set forth in Sambrook ct al.. MOLLCULAR CLONING , A LABORATORY MANUAL, 2nd Ed. ; Cold Spring Harbor Laboratorv Press. Cold Spring Harbor. New York (1989).

The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence (s). including, for instance. a promoter to direct mRNA transcription. Representatives of such promoters include, but are not limited to, the phage lambda PL promoter, the L. coli lac. trp and tac promoters. the SV40 carry and late promoters and promoters of retroviral LTRs.

In general. expression constructs will contain sites for transcription initiation and temunation, and. in the transcribed region. a ribosome binding site for translation. The coding portion of the mature transcripts expressed bv the constructs will include a translation initiatuig codon. for exnplc. AUG or GUG. at the beginning and a temunation codon appropnately positioned at the end of the polypeptide to bc translated.

In addition, die constructs mav contais control regions that regulate as well as engender expression.

Generally, in accordance with many commonly practiced procedures, such regions will operate b\, controlling transcription, such as transcription factors, repressor binding sites and temiination. among others.

Vectors for propagation and expression generally will include selectable markers and amplification regions, such as. for example, those set forth in Sambrook et nl., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed. ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Representative examples of appropriate hosts uiclude bacterial cells. such as streptococci. staphylococci, E. coli. streptomyces and Baciliu. s. subtilis cells: fungal cells. such as yeast cells and Aspergillacs cells; insect cells such as Drosophile S2 and Spocloptena Sf9 cells : animal cells such as CHO, COS. HeLa. C127, 3T3, BHK, 293 and Bowes melanoma cells : and plant cells.

The following vectors, which are commercially available, are provided by way of example.

Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9. available from Qiagen : pBS vectors. Phagescript vectors. Bluescript vectors, pNH8A, pNH16a, pNH18A. pNH46A. available from Stratagene : and ptrc99a. plW223-3. pKK233-3 pDR540. pRIT5 available from Pharmacia. and pBR322 (ATCC 37017). Among preferred eukaryotic vectors are pWLNEO. pSV2CAT. pOG44, pXT1 and pSG available from Stratagene : and pSVK3. pBPV pMSG and pSVL available from Phannacia. These vectors are listed solely bv wav of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of me present invention. It will be appreciated that an other plasmid or vector suitable for. for example, introduction, maintenance, propagation or expression of a polynuclcotide or polypeptide of the invention in a host may be used in this aspect of the invention.

Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region. such as a chloramphenicol acctyl transferase ("CAT") transcription unit. downstream of restriction site or sites tòr i Itroduculg a candidate promoter fragment; i.e., a fragment khat may contain a promoter. As is well known, introduction into the vector of a promoter- containing fragment at me restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available. such as pKK232-8 and pCM7. Promoters for expression of polvnucleotides of me present invention include not only well known and readily available promoters. but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.

Among known prokarvotic promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E coli lacl and lacZ and promoters. the T3 and T7 promoters. the gpt promoter. the lambda PR-PL promoters and the trp promoter.

Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs. such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionei l-l promoter.

Recombinant expression vectors will include, for example, origins of replication. a promoter preferably derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.

Polvnucleotides of the invetion, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site is located appropriately 5'to a ribosome binding site. The ribosome binding site will be 5@ to the codon that initiates translation of the polypeptide to be expressed. for example AUG or GUG. Generally, there will be no other open reading frames that begin with an initiation codon. usually AUG. and lie between the ribosome binding site e and the initiation codon. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal in constructs for use in cukaryotic hosts.

Transcritpion termination signal appropriately disposed at the 3'end of the transcribed region may also be included in die polynucleotide construct.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the pcriplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to me polypeptide or they may bc hctcrologous signals.

The polypcptide may be cxpressed in a modified form, such as a fusion protein, and may include not onlv secretion signals but also additional heterologous functional regions. Thus. for instance, a region of additional amino acids. particularly charged amino acids. may be added to me N-or C-terminus of the polypeptide to improve stability and persistence in the host cell. during purification or during subsequent handling and storage. Also, region also may be added to the polypeptide to facilitate purification. Such regions mav be removed prior to final preparation of me polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion. to improve stability or to facilitate purification, among others, are familiar and routine techniques in me art. A preferred fusion protein comprises a hcterologous region from immunolglobulm that is useful to solubilizc or purify polypeptides. For example. EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another protein or part thereof. In drug discovery, for example, proteins have been fused with antibody Fc portions for the purpose of high- throughput screening assays to identify antagonists. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The. Journal of Biological Chemistry, Vol.

270, No. 16, pp 9459-9471 (1995).

Cells typically then are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by mu convenient method. including freeze-tiaw cyclulg, sonication. mechanical disruption. or use of cell Iysing agents such methods are well know to those skilled in the art.

Mammalian expression vectors may comprise an origin of replication. a suitable promoter and enhancer, and also any necessary ribosome binding sites. polvadenvlation regions. splice donor and acceptor sites. transcriptional termination sequences. and 5'flanking non-transcnbed sequences that are necessary for expression.

ARO1 polxpeptide can be recovered and purifie from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction cliromatography. affiniriy cliromatography. hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid cluomatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the poiypeptide is denatured during isolation and or purification.

Polynucleotide assays This invention is also related to die use of the ARO1 polynucleotides to detect complementary polynucleotides such as, for example as a diagnostic reagent. Detection of ARO1 polynucleotides in a cukayotc, particularly a mammal, and especially a human. will provide a diagnostic method for diagnosis of a discase. Eukaryotes (herein also "individual(s)"), particularly mammals, and especially humans, infected with an organism comprising a ARO1 polynucleotide, particularly the gene sequence may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtauicd from an infected individual's cells and tissues, such as bone, blood. muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al.. Nature 324.- 163-166 (1986) prior to analysais. RNA or cDNA may also be used in die same ways. As an cxnple.

PCR primera complementary to the nucleic acid encoding ARO1 polypeptide can be used to identify and analyze ARUI presence and/or cxpression. Usug PCR. characterization of the strain of prokawote present in a cukaryote. particularly a mammal, and especially a human, may be made by an analysais of the genotype of the prokaryote gene. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to radiolabeled AROI RNA or altematively, radiolabeled ARO1 antisense DNA sequences. Perfectlv matched sequences can be distinguished from mismatched duplexes by RNase A digestion or bv differences in melting temperatures.

Sequence differences between a reference gene and genes having mutations also mav be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced bv appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence

detennination is performed bv conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.

Genetic characterization based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e. g., Myers et at.. Science. 230 : 1242 (1985)).

Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S I protection or me chemical cleavage method (e. g.. Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985)).

TIILIS, the detection of a specific DNA sequence may be achieved by methods such as hvbridization. RNasc protection. chemical cleavage. direct DNA sequencing or the use of restriction enzymes, e.g., restriction fragment length polymorphisms (RFLP) and Souther blotting of genomic DNA.

In addition to more conventional gel-clectrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.

Cells carrying mutations or polymorphisms in the gene of the present invention may also be detected at me DNA level by a variety of techniques, to allow for serotsping, for example. For example, RT-PCR can be used to detect mutations. It is particularly preferred to used RT-PCR il conjunction with automated detection systems, such as. for example. GeneScan, RNA or cDNA may also be used for the same purpose, PCR or RT-PCR. As an example PCR primers complementary to the nucleic acid encoding AROI pohpeptide can be used to identify and analyze mutations. The primers may be used to amplify the gene isolated from the individual such that the gene may then be subject to various techniques for elucidation of the DNA sequence. In tlus way, mutations in the DNA sequence may be diagnosed.

The invention provides a process for diagnosing, disease, preferably fungal infections, more preferably Candida albicans, and most preferably candidosis (candidiasis), for example, superficial candidosis. candidosis of the oropharynx, such as, oral thrush (acute pseudomembraneous candodisis). denture stomatitis. angular choilitis (perlèche), Candida leukoplakia (chronic hyperplastic candidosis), midline glossitis (median rbomboid glossitis, glossal central papillary atrophy,"antibiotic sore tongue", acute atrophic candidosis), and miscellaneous forms of oral candidosis : candidosis of the genitalia, such as. vulvovaginal candidosis (vaginal thrush, Candida colpitis), and candidosis of the penis (Candida balanitis, balanoposthitis and urethritis) ; candidosis of the skin, nails and other external sites, such as, Candida intertrigo, napkin (diaper) dermatitis, Candida onychia and paronyclua, candidosis of the extennal ear, and miscellaneous cutaneous forms of candidosis; chronic mucocutaneous candidosisg systemic candidosis

candidosis of the gastrointestinal tract, such as. esophageal candidosis, gastric candidosis. candidosis of the intestine, Candida cholecystitis, and the"autobrewery syndrome" ("meitei-sho") : candidosis of the Urinary Tract, such as. renal candidosis (Candida pyelonephritis), candidosis of the urinary bladder (Candida cystitis), Candida urethritis and prostatitis: cardiovascular Candida infections, such as, candida endocarditis, and myocarditis; Candida endocariditis. Candida myocarditis and pericarditis, Candida phlebitis and thrombophlebitis : Candida infections of the eye, such as. Candida endophthalmitis, Candida infections of the comea and conjuctiva. and candidosis of the lacrimal sacs (dacryocvstitis) : candidosis of the central nervous system : Candida meningitis, cerebral candidosis, and candidosis of die inner eau : Candida infections of bones and joints ; Candida peritoutis, hepatitis and miscellaneous other forms of systemic candidosis. such as. Candida peritonitis. Candida hepatitis and splenitis Candida pancreatitis.

Congenital and intrauterine candidosis (Candida chorioamnionitis and fuusitis) : disseminated candidosis. such as. in neonates, young infants. and heroin addicts ; Candida allergy, such as. cutaneous Candida allergy and"candidids". Respiratory tract allergy to"Candida". Candida allergy in other bodily sites."chrouc candidosis" and "the yeast connection", comprising determining from a sample derived from an individual a increased level of expression of polynuclcotidc having the sequence of Table 1 [SEQ ID NO: 1]. Increased expression of ARO1 polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotide, such as. for example. PCR. RT-PCR, RNase protection, Northern blotting and other hvbridization methods.

Polypeptide assays The present invention also relates to a diagnostic assays such as quantitative and diagnositc assays for detecting levels of ARO1 protein in cells and tissues, including determination of normal and anormal levels. Thus, for instance, a diagnostic assay in accordance with me invention for detecting over-expression of AROI protein compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a ARC I protein, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these ELISAs frequently are preferred. An ELISA assay initially comprises preparing an antibody specific to ARO1, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds to the monoclonal antibody. The reporter antibody is attached a detectable reagent such as radioactive, fluorescent or enznmatic reagent. in this example horseradish peroxidase enzyme.

Antibodies The polypeptides. and variants thereof or cells expressing them can be used as an unmunogen to produce antibodies thereto. The present invention includes, for examples monoclonal and polyclonal

antibodies, chimeric. single chain, and humanized antibodies. as well as Fab fragments. or the product of an Fab expression library.

Antibodies generated against the polypeptides corresponding to a sequence of the invention can be obtained by direct injection of the polypeptides into ar. animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will men bind me polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding me whole native polypeptides. Such antibodies can then be used to isolate the polyeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies. any technique known in me art winch provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein. C.. Nature 256: 495-497 (1975); Kozbor et al., Immunology 7Oclav 4 72 (1983). Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques described for me production of single chain antibodies (U. S. Patent No. 4.946.778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice. or other organisms such as other mammals, may be used to express humanized antibodies to immunog enic polypeptide products of this invention.

Alternativel phagc display tcchnology could be utilized to select antibody genes w ith binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Fbp or from naivc librarics (McCaffertv. J. et al.. (1990).

Nature 348. 552-554 Marks. J. et al.. (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved bn chain shuffling (Clackson. T. et al.. (1991) Nature 352.624-628).

If two antigen binding domains are present each domain may bc directed against a different epitope-termed'bispecific'antibodies The above-described antibodies may be employed to isolate or to identify clones expressing me polypeptide or purify the polypeptide of the present invention bv attachment of the antibody to a solid support for isolation and/or purification by afnnity chromatography.

Thus, among others. antibodies against ARO1 may be employed to inhibait and/or treat infections. particularly fungal infections and especially candidosis (candidiasis), for example, superficial candidosis ; candidosis of the oropharynx. such as. oral thrush (acute pseudomembraneous candodisis), denture stomatitis, angular choilitis (perleche), Candida leukoplakia (chronic hnperplastic candidosis). nudline glossitis (median rbomboid glossitis, glossal central papillary atrophy,"antibiotic sore tondue". acute atropluc candidosis). and miscellaneous forms of oral candidosis : candidosis of the genitalia, such as. vulvovaginal candidosis (vaginal thrush. Candida colpitis), and candidosis of the penis (Candida balanitis.

balanoposthitis and urethritis) : candidosis of the skin. nails and other external sites. such as. Candida intertrigo, napkin (diaper) dermatitis. Candida on-,, and paronvchia. candidosis of the external ear, and miscellaneous cutaneous forms of candidosis : chronic mucocutaneous candidosis; systemic candidosis; candidosis of the gastrointestinal tract, such as, esophageal candidosis, gastric candidosis. candidosis of the intestine, Candida cholecvstitis. and the"autobrewery syndrome" ("meitei-sho"): candidosis of the Urinary Tract. such as, renal candidosis (Candida pyelonephritis). candidosis of the urinarv bladder (Candida cystitis), Candida urethritis and prostatitis : cardiovascular Candida infections, such as. candida endocarditis, and myocarditis : Candida cndocariditis. Candida myocarditis and pericarditis, Candida phlebitis and thrombophlebitis : Candida infections of the eye, such as, Candida endophthalmitis, Candida infections of the cornea and conjuctiva, and candidosis of the lacrimal sacs (dacryocvstitis) : candidosis of the central nervous system : Candida meningitis, cerebral candidosis, and candidosis of me inner ear; Candida infections of bones and joints : Candida peritonitis, hepatitis and miscellaneous other forms of systemic candidosis, such as, Candida peritonitis, Candida hepatitis and splcnitis. Candida pancrcatitis.

Congénital and intrauterine candidosis (Candida chroioamnionitis and funisitis) : disseminatd candidosis, such as, in neonates, young infants, and heroin addicts: Candida allergy, such as. cutaneous Candida allcrgy and"candidids". Respiratory tract allergy to"Candida". Candida allergy in other bodily sites, "chronic candidosis"and"die yeast connection".

Polypeptide derivatives and variants include antigenically, epitopically or tmmunologically equivalent derivatives which form a particular aspect of this invention. The term "antigenically equivalent derivative" as used herein encompasses a polypeptide or its equivalent which will be specificallv recognised by certain antibodies which. when raised to the protein or polypeptide according to the present invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term "immunologically equivalent derivative" as used herein encompasses a peptide or its equivalent w hich when used in a suitable fonmulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.

The polypeptide. such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stabilitv to the polypeptide. The antigen may be associated, for example bv conjugation. with an immunogenic carrier protein for example bovine serum albumin (BSA) or kevhole limpet haemocvanin (KLH). Alternativelv a multiple antigenic peptide comprising multiple copies of the protein or polypeptide. or an antigenically or immunologically equivalent polypeptide thereof mav be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.

Preferably the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized" ; where the complimentarity determining region (s) of the hvbridoma-derived antibody has been transplanted into a human monoclonal antibody. for example as described in Jones, P. et al.

(1986), Nature 321. 522-525 or Tempest et al., (1991) Biotechnology 9,266-273.

The use of a polvnucleotide of the invention in genetic immunization will preferably emplov a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolffet al.. Hum Mol Genet 1992. 1 :363, Manthorpe et al., Hum. Gene Ther. 1963 : 4, 419), delivery of DNA complexe with specific protein carriers (Wu et al., J Biol Chem 1989 :264, 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS. 1986 : 83, 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science 1989 :243,375), particle bombardment (Tang et al., Nature 1992, 6 : 152. Eisciibraun et at., DNA Cell Biol 1993. 12 : 791) and in vivo infection using cloned retroviral vectors (Seeger et al.. PNAS 1984 : 81. 5849).

AROI-binding molecules and assays This invention also provides a method for identification of molecules, such as binding molecules. that bind AROI polynucleotides and polypeptides. Genes encoding proteins that bind ARO1, can be identified by numerous @rous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory marials such as, for instance, Coligm cl al., Current Protocols in Immunology 1(2): Chapter 5 (1991). Also. a labeled ligand can be photoaffinity linked to a cell extract. Polypeptides of the invention also can be used to assess ARO1 binding capacity of ARO1-binding molecules, in cells or in cell-free preparations.

Polypeptides of me invention may also be used to assess the binding or small molecule substrates and ligands in. for exemple. cclls. cell-free preparations. chemical libraries. and natural product mixtures.

These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics.

Antagonists and Agonists-assays and molecules The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of ARO 1 polypeptides or polynucleotides. such as its interaction with ARO1-binding molecules.

For example, to screen for agonistes or antagonists, a sythetic reaction ii-ux, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof. may be prepared from a cell that expresses a molecule that muids ARO 1. The preparation is incubated wdtli labeled ARO 1 in the absence or the presence of a candidate molecule which may be a ARO 1 agonist or antagonist.

The abilitv of the candidate molecule to bind the binding molecule is reflected in decreased binding of the

labeled ligand. Molecules which bind gratuitously, i. e., without inducing the effects of ARO1 on binding the ARO1 binding molecule, are most likely to be good antagonists. Molecules that bind well and elicit effects that are the same as or closely related to ARO1 are agonsts.

AROl-like effects of potential agonists and antagonists may by measured. for instance, by detennining activity of a reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation. and comparing the effect with that of ARO I or molecules mat elicit the same effects as ARO1. Reporter systems that may be useful in this regard include but are not limited to colonmetric labeled substrate converted into product, a reporter gene that is responsive to changes in ARO I activity, and binding assays known in the art.

Another example of an assay for ARO 1 antagonists is a competitive assay that combines ARO 1 and a potential antagoust witli membrane-bound AROI-binding molecules. recombinant AROI binding molecules. natural substrates or ligands, or substrate or ligand mimetics. under appropriate conditions for a competitive inhibition assay. ARO1 can be labeled, such as by radioactivitv or a colonmetnc compound. such that the number of ARO I molecules bound to a binding molecule or converted to product can bc determined accurately to assess the effectiveness of the potential antagonst.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or cxti Iguish its activait !. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, ithout inducing AROI- induced activities. thereby prevcnting dc action of ARO I by excluding ARO 1 from binding.

Potential antagonists include a small molecule which binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules. such that normal biological activity is prevented. Examples of small moleculcs include but are not limited to small orgmic molecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see Okano.. J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press. Boca Raton. FL (1988), for a description of these molecules).

Preferred potential antagonists include compounds related to and variants of ARO 1.

In a particular aspect the invention provides the use of the polypeptide. polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a fungal pathogen and mammalian host responsible for sequelae of infection. In particular the molecules of the invention may be used: i) in the prevention of adhesion of fungi, in particular Candida fungi, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds ; ii) to block arom multifunctional protein protein-mediated mammalian cell invasion by, for example,

initiating phosphorylation of manunalian tyrosine kinases (Rosenshine et al.. Infect. Immun. 60 : 2211 (1992) ; iii) to block fungal adhesion between mammalian extracellular matrix proteins and fungal arom multifunctional protein proteins which mediate tissue damage ; iv) to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.

Each of the DNA sequences provided herein may be used in the discoverv and development of antifungal compounds. The encoded protein upon expression can be used as a target for the screening of antiftmgal drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to constmct antisense sequences to control the expression of the coding sequence of interest.

The e antagonists and agonistst may be employed for instance to inhibit diseuses such as, for example, candidosis (candidiasis), for example, superficial candidosis; candidosis of the oropharynx. such as, oral thrush (acute pseudomembraneous candodisis), denture stomatitis, angular choilitis (perlèche), Candida leukoplakia (chronic hyperplastic candidosis), midline glossitis (median rbombboi glossitis, glossal central papillary atrophy, "antibiotic sore tongue", acute atrophic candidosis), and miscellaneous forms of oral candidosis : candidosis of me genitalia, such as. vulvovaginal candidosis (vaginai thrush. Candida colpitis), and candidosis of the penis (Candida balanitis, balanoposthitis and urethritis) ; candidosis of the skin. nails and other external sites. such as. Candida intertrigo, napkin (diaper) dermatitis. Candida onychia and paronychia. candidosis of me external ear. and miscellaneous cutaneous forms of candidosis : chronic mucocutaneous candidosis : systemic candidosis ; candidosis of the gastrointestinal tract, such as. esophageai candidosis. gastric candidosis. candidosis of the intestine. Candida cholecystitis, and the "autobrewery swdromc" ("mcitci-sho") ; candidosis of the Unnary Tract. such as. rcnal candidosis (Candida pyeloncphritis), candidosis of the urinary bladder (Candida cystitis), Candida urethritis and prostatitis; cardiovascular Candida infections. such as. candida endocarditis, and myocarditis : Candida endocariditis, Candida myocarditis and pericarditis, Candida phlebitis and thrombophlebitis : Candida infections of the eye, such as. Candida endophthalmitis, Candida infections of the cornea and conjuctiva, and candidosis of the lacrimal sacs (dacryocystitis); candidosis of me central nervous system; Candida meniingitis, cerebral candidosis, and candidosis of the inner ear : Candida infections of bones and joints ; Candida peritonitis, hepatitis and miscellaneous other forms of systemic candidosis, such as. Candida peritonitis. Candida hepatitis and splenitis, Candida pancreatits, Congenital and intrauterine candidosis (Candida chorioamnionitis and funisitis) : disseminated candidosis, such as, in neonates, voung infants, and heroin addicts; Candida allergy, such as. cutaneous Candida allergy and"candidids", Respiratory tract allergy to "Candida", Candida allergy in other bodilv sites,"chronic candidosis"and"the veast connection".

Vaccines Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with ARO 1, or a fragment or variant thereof, adequate to produce antibody to protect said individual from infection, particularly fungal infection and most particularly Candida infections. Yet another aspect of the invention relates to a method of inducing inununological response in an individual which comprises. through gene therapy, delivering gene encoding ARO 1 polypeptide. or a fragment or a variant thereof, for expressing ARO l polvpeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response to produce antibody to protect said individual from disease.

A further aspect of the invention relates to an immunological composition which, when introduced into a host capable or having induced within it an immunological response induces an immunological response in such host to a AROI polynucleotide or protein encoded therefrom. wherein the composition comprises a recombinant ARUI polynucleotide or protein encoded therefrom comprising DNA which codes for and expresses an antigen of said AROI polynucleotide or protein encodedtherefrom.

A ARO 1 polypeptide or a fragment thereof may be fused w ith co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties, Thus fused recombinant protein, preferably further comprises an antigenic co-protein. such as Glutathione-S-transferase (GST) or beta- galactosidase, relatively large co-proteins which solubilise the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.

Provided by this invention are compositions, particularly vaccine compositions. and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences. such as those described in Sato, Y. et al. Science 273 : 352 (1996).

Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of fungal cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection with Candida albicans will be particularly useful for identifying protein epitopes able to provoke a prophvlactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of Candida albicans infection in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of fungi, for example by blocking adherence of fungi to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused. e. g., by mechanical. chemical or thermal damage or by implantation of indwelling devices. or wounds in the mucous membranes, such as the mouth. mammary glands. urethra or vagina.

The present invention also includes a vaccine formulation which comprises the immunogenic recombinant protein together with a suitable carrier. Since the protein may be broken down in the stomach. it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular. intravenous, or intradermal. Formulations suitable for parenteral administration include e aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants. buffers. bacteriostats and solutes which render the formulation instonic with the bodily fluid, preferably the blood. of the individual : and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, scaled ampoules and vials and may be stored in a freeze-dried condition requiring onlv the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for eWancing the immunogenicity of the formulation, such as oil-in water svstems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be rcadily determined by routine experimentation.

Whilc the invention has been described with reference to certain ARO1 polypeptides, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins with additions. deletions or substitutions which do not substantiallv affect the immunogenic properties of the recombinant protein.

Compositions The invention also relates to compositions comprising me polynucleotide or the polypeptides discussed above or the agonists or antagonists. Thus, the polypeptides of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms. such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise. for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaccutically acceptable carrier or excipient. Such carriers may include_ but are not limited to. saline. buffered saline. dextrose. water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.

Kits The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the

invention. Associated with such container (s) can be a notice in the form prescribed bv a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

Administration Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration b topical. oral. anal. vaginal. intravcnous. intraperitoneal. intramuscular. subcutaneouss ultranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of a specific uldication or indications. In general. the compositions are administered in an amount of at least about 10)-ig/kgbodyweight.In most cases they will be administercd i 1 an amount not in excess of about 8 mg/kg bodv weiglt pcr day. Preferably. in most cases. dose is from about 10 ug/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication. its severity. route of administration, complicating conditions and me like.

In therapy or as a prophylactic. the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion. preferably isotonic.

Alternatively the composition may be formulated for topical application for example in the form of ointments creams lotions, eye ointments. eye drops, ear drops, mouthwash. impregnated dressings and sutures and aerosols. and may contain appropriate conventional additives, including, for example, preservatives. solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.

Such carriers may constitute from about 1% to about 98% by weight of the formulation : more usually they will constitute up to about 80% by weight of the formulation.

For administration to manunals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0. 01 mg/kg to 10 mg/kg, typically around 1 mgikg. The physician in any event will determine the actual dosage which w ill be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can. of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

In-dwelling devices include surgical implants, prosthetic devices and catheters, i. e.. devices that are introduced to the body of an individual and remain in position for an extended time. Such

devices include, for example. artificial joints. heart valves, pacemakers. vascular grafts. vascular catheters. cerebrospinal fluid shunts, urinary catheters. continuous ambulator peritoneal dialysis (CAPD) catheters. etc.

The composition of the invention may be administered by injection to achieve a systemic effect against relevant fungi shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device. In addition the composition could also be used to broaden perioperative cover for any surgical technique to prevent Canclida round infections.

Manv orthopaedic surgeons consider that humans with prosthetic joints should be considered for antifungal prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and motality, It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antifungals in this situation.

In addition to the therapy described above. the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of fungi to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to. or in conjunction with, antifungal prophvlaxis.

Altcmativel !. the composition of the invention may be used to bathe an indwelling device inuncdiatclv before insertion. The active agent will preferably be present at a concentration of I Fg/ml to 10mg/ml for bathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventional adjuvants may be emploved to enfance the immune response.

A suitable unit dose for vaccination is 0. 5-5pg/kg of antigen. and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks.

With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.

The antibodies described above may also be used as diagnostic reagents to detect the presence of fungi containing the arom multifunctional protein protein.

In order to facilitate understanding of the following example certain frequently occurring methods and/or terms will be described.

EXAMPLES The present invention is further described by the following examples. These exemplification's. while illustrating certain specific aspects of the invention. do not portray the limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are well known and routine to those of skill in the art. except where othenvise described in detail. Routine molecular biology techniques of the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et al.. MOLECULAR CLONING : A LABORATORYMANUAL. 2nd Ed. : Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N. Y. (1989).

Example I Library Production The polynucleotidc having the DNA sequence given in SEQ ID NO : 1 was obtained from a library of clones of chromosomal DNA of Candida albicans. In some cases the sequencing data from two or more clones containing overlapping Candida albicans DNAs was used to construct the contiguous DNA sequence in SEQ ID NO : 1. Libraries mav be prepared bv routine methods, for example : Methods 1 and 2 below.

Total cellular DNA is isolated from Canclicla alhicanx SCD314 according to standard procedures and size-fractionated by either of two methods.

Method 1 Total cellular DNA is mechanically hsared bv passage through a needle in order to size- fractionate according to standard procedures. DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added. Fragments are ligated into the vecotr Lambda Zapll that has been cut with EcoRI, the library packaged bv standard procedures and E.coli infected with the packaged library. The library is amplified by standard procedures.

Method 2 Total cellular DNA is partially hydrolyzed with a one or combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vcctors (e g Rsal Pall Alul Bshl2351), and such fragments are size-fractionated according to standard procedures. EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI. the library packaged bv standard procedures. and E. coli infected with the packaged library. The library is amplified by standard procedures.

Example 2 AROI Complementation Analysis Candida albicanx ARO I protein is tagged and this tagged version is used in immunofluorescence studies to determine the cellular localization of the protein.

Complementation anal, is used to observe whether the Candida albicans ARO1 clone is able to complement a ARO1-deletion in Candida albicans when expressed from its native promoter (Mol Gen. Genet. 200. 500-502, 1985).

Expression of ARO1 from a Cajclicla albicans promoter is performed to test for complementation (Mol Gen. Genet. 200, 500-502. 1985).

Conditional alleles of AROI are thereby identificd and are used as a basis for a cell based sceens for anti-fungal compounds.