WO2012087004A2

WO2012087004A2 - Use of upr signaling pathway genes ire1 and hxl1 for treatment of fungal infection and meningitis

Info

Publication number: WO2012087004A2
Application number: PCT/KR2011/009862
Authority: WO
Inventors: Yong Sun Bahn; Kwang Woo Jung; Hyun Ah Kang; Seon Ah Cheon
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University; Chung-Ang University Industry-Academy Cooperation Foundation
Priority date: 2010-12-23
Filing date: 2011-12-20
Publication date: 2012-06-28
Also published as: KR101311196B1; KR20120072096A; WO2012087004A3

Abstract

The present invention relates to the use of the UPR signaling pathway genes IRE1 and HXL1 for treatment of fungal infection and meningitis. In the invention, it was newly found that disruption of Ire1 and Hxl1 (HAC1 and XBP1-Like gene 1) proteins, newly identified in Cryptococcus neoformans, genes encoding the proteins, provides an antifungal effect and a meningitis-treating effect. Based on this finding, a candidate which can show a synergistic effect when being co-administered with an existing antifungal agent or meningitis-treating agent can be screened and a novel pharmaceutical composition having an antifungal effect and a meningitis-treating effect can be provided.

Description

USE OF UPR SIGNALING PATHWAY GENES IRE1 AND HXL1 FOR TREATMENT OF FUNGAL INFECTION AND MENINGITIS

The present invention relates to the use of the UPR signaling pathway genes IRE1and HXL1for treatment of fungal infection and meningitis.

The unfolded protein response (UPR) signaling pathway plays an important role in maintaining endoplasmic reticulum (ER) homeostasis under various environmental conditions that cause ER stress in eukaryotic cells. In the yeast UPR signaling pathway, the Ser/Thr kinase/endoribonuclease Ire1 senses ER stress and is auto-phosphorylated and activated, after which it excises an intron from HAC1 mRNA encoding the bZIP transcription factor to activate the transcription factor, so that the cells cope with ER stress. Meanwhile, it was reported that a deletion of HacA (Homologous to Hac1 transcription factor of S. cerevisiae) which is involved in the UPR signaling pathway in the pathogenic fungus Aspergillus fumigatus rendered the fungus sensitive to antifungal agents (see PLoS Pathogens1, Vol 5, Issue 1, January 2009). However, Ire1 or a target transcription factor which is involved in the UPR signaling pathway in the pathogenic fungus Cryptococcus neoformans is not yet known.

In the past, local fungal infections, such as athlete’s foot, Tinea cruris and thrush, frequently occurred, and systemic fungal infections rarely occurred. However, in recent years, systemic fungal infections have frequently occurred such that they account for the fourth highest rate of total infections in hospitals. Antifungal agents developed to date can be broadly classified into azole-based antifungal agents and non-azole-based antifungal agents. The azole-based antifungal agents include ketoconazole, fluconazole, itraconazole, voriconazole and the like, and the non-azole-based antifungal agents include terbinafine, flucytosine, amphotericin B, caspofungin and the like. The azole-based antifungal agents, such as ketoconazole, fluconazole, itraconazole, voriconazole, and allylamine-based antifungal agents, such as naftifine and terbinafine, have similar action mechanisms. These two classes of antifungal agents act to inhibit an enzyme required in the process in which lanosterol is converted into ergosterol, the main component of the fungal cell membrane. To exhibit the above effect, the azole-based antifungal agents inhibit microsomal enzymes, and the allylamine-based antifungal agents inhibit squalene epoxidase. Flucytosine (5-FC) that is a metabolic antagonist inhibiting nucleic acid synthesis shows antifungal activity by non-competitively antagonizing fungal RNA and DNA synthesis, and polyene-based amphotericin B binds to ergosterol in the fungal cell membrane to induce the depolarization of the cell membrane and to form holes which induce loss of intracellular contents, thereby exhibiting antifungal activity. Caspofungin, an echinocandin-based antifungal agent, acts to irreversibly inhibit the formation of the fungal cell wall and differs from the above-mentioned antifungal agents in that it acts on the cell wall. The azole-based drugs, when administered to impaired liver function patients, can cause hepatitis, leading to death, and for this reason, a liver function test should be carried out before administration of the azole-based drugs. It was reported that flucytosine inhibits bone marrow in a dose-dependent manner, shows hepatotoxicity and can cause enterocolitis. Such side effects further increase in the case of impaired renal function patients, and thus monitoring of renal function of the patients is very important. Also, flucytosine must not be prescribed for pregnant women. The typical toxicity of Amphotericin B is glomerular nephrotoxicity resulting from renal artery vasoconstriction, which is dose-dependent. Thus, when the total cumulative dose of Amphotericin B is 4-5 g or more, the possibility of permanent renal function impairment will increase. Further, nephrotoxicity, including the excessive loss of potassium, magnesium and bicarbonate caused by renal tubular toxicity, and a decrease in erythropoietin production, can occur. In addition, symptoms, including thrombophlebitis, rigor, tremor and hyperventilation, can appear due to acute reactions.

As described above, because the existing antifungal drugs cause various side effects depending on the kind of drug, there has been a need to develop a novel treatment method which can reduce such side effects while increasing antifungal activity. Thus, the present inventors have newly identified Ire1 and a related transcription factor in the pathogenic fungus C. neoformansand have found that inhibition of the identified transcription factor provides an antifungal effect and an effect against meningitis, thereby completing the present invention.

It is an object of the present invention to provide novel Ire1 and Hxl1 ( H AC1 and X BP1-Like gene 1) proteins and genes encoding the same andto provide a method for screening a novel antifungal agent or meningitis-treating agent.

Another object of the present invention is to provide a method for screening a novel antifungal agent or meningitis-treating agent which can show a synergistic effect when being co-administered with an existing antifungal agent or meningitis-treating agent.

Yet another object of the present invention is to provide a pharmaceutical composition which shows an antifungal effect or a meningitis-treating effect by inhibiting novel Ire1 and Hxl1 ( H AC1 and X BP1-Like gene 1) proteins and genes encoding the same.

The term "sample"as used herein with reference to the screening method means an unknown candidate that is used in screening to examine whether it influences the expression of a gene or the amount or activity of a protein. Examples of the sample include, but are not limited to, chemical substances, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

The measurement ofa change in the expression of a gene may be carried out according to various methods known in the art, for example, using RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press), cDNA microarray hybridization (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).

According to RT-PCR protocol, total RNA is extracted from the sample-treated cells, and single-stranded cDNA is prepared from the total RNA using oligo dT primer and reverse transcriptase. Then, PCR reaction is performed using the single-stranded cDNA as a template and a gene-specific primer set. The gene-specific primer set is shown in Table 2 below. The PCR amplification product is electrophoresed and the bands are analyzed to determine a change in the expression of the gene.

A change in the amount of protein can be carried out according to various immunoassay methods known in the art. Examples of the immunoassay methods include, but are not limited to, radioimmunoassay, radioimmuno-precipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, and sandwich assay.

The immunoassay or immunostaining method is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. For example, when the method of the present invention is carried out according to the radioimmunoassay method, protein-specific antibodies labeled with radioisotopes (e.g., C¹⁴, I¹²⁵, P³²and S³⁵) may be used.When the method of the present invention is carried out by the ELISA assay, a specific embodiment of the present invention includes the steps of: (i) coating an extract from sample-treated cells on the surface of a solid substrate; (ii) allowing the cellextract to react with Ire1 or a protein-specific antibody as a primary antibody; (iii) allowing the material resulting from step (ii) to react with an enzyme-conjugated secondary antibody; and (iv) measuring the activity of the enzyme. The solid substrate is preferably a hydrocarbon polymer (e.g., polystyrene or polypropylene), glass, a metal or gel, and most preferably a microtiter plate. The enzyme conjugated to the secondary antibody includes, but is not limited to, an enzyme that catalyzes a color-development reaction, a fluorescent reaction, a luminescent reaction or an infrared reaction. Examples of the enzymes include alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme conjugated to the secondary antibody, the substrate used may be a color-development substrate, such as alkaline phosphatase, bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-As-B1-phosphate or ECF (enhanced chemifluorescence). When horseradish peroxidase is used as the enzyme, the substrate used may be chloronaphtol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine), ABTS (2,2'-azine-di[3-ethylbenzthiazoline sulfonate]), or o-phenyldiamine (OPD). In the ELISA assay, the measurement of the final enzymatic activity or signal can be carried out accordingto various methods known in the art. If biotin is used as the label, the signal can be easily detected with streptavidin, and if luciferase is used as the label, the signal can be easily detected with luciferin.

The pharmaceutical composition of the present invention may comprise, as an active ingredient, a chemical substance, a nucleotide, antisense RNA, an siRNA oligonucleotide or a natural extract. The antifungal pharmaceutical composition or antifungal composite formulation of the present invention may comprise, in addition to the active ingredient, pharmaceutically suitable and physiologically acceptable adjuvants. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, flavoring agents, solubilizers, etc. For administration, the antifungal pharmaceutical composition of the present invention may also contain at least one pharmaceutically acceptable carrier, in addition to the active ingredient. Examples of pharmaceutically acceptable carriers, which can be used to formulate the antifungal pharmaceutical composition of the present invention in the form of liquid solutions, include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of two or more thereof. If necessary, the inventive composition may also contain other conventional additives, such as antioxidants, buffers and bacteriostatic agents. Moreover, the inventive composition may additionally contain diluents, dispersants, surfactants, binders and lubricants in order to formulate it into injection formulations, such as aqueous solutions, suspensions and emulsions, pills, capsules, granules and tablets. Furthermore, the inventive composition may preferably be formulated depending on particular diseases and its components, using the method described in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, which is a suitable method in the relevant field of art. The pharmaceutical compositions of the present invention can be formulated as granules, powders, coated tablets, tablets, capsules, suppositories, syrup, juice, suspensions, emulsions, drops, injectable liquids, and sustained-release preparations of active ingredients, etc. The pharmaceutical composition of the present invention can be administered in the conventional manner via the intravenous, intraarterial, intraabdominal, intramusclar, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal route. As used herein, the term "effective amount" of the active ingredient of the pharmaceutical composition means the amount required for prevention or treatment of disease. Thus, the effective amount can vary depending on various factors, including the kind of disease, the severity of disease, the kind and content of active ingredient and other components contained in the composition, the kind of formulation, the patient's age, weight, physical condition, sex and diet, and administration time, administration route, the secretion rate of the composition, administration period, and the kind of drug used in combination with the composition. For example, when the inhibitor of the present invention is administered to adult patients once or several times per day, it may be administered at doses of 0.1 ng/kg to 10 g/kg for a compound, 0.1 ng/kg to 10 g/kg for a polypeptide, a protein or an antibody, and 0.1 ng/kg to 10 g/kg for an antisense oligonucleotide, siRNA, shRNAi, or miRNA.

As used herein, the term "subject"is intended to include a human, an orangutan, a chimpanzee, a mouse, a rat, a dog, a cow, a chicken, a pig, a goat, a sheep, etc., but is not limited thereto.

The term "antisense oligonucleotide" used herein refers to DNA, RNA or its derivatives, that contain nucleic acid sequences complementary to the sequences of a target mRNA, characterized in that they bind to the target mRNA and interfere with its translation to protein. The antisense nucleic acid is 6-100, preferably 8-60, more preferably 10-40 nucleotides in length. The antisense oligonucleotide may at least one modification in its base, sugar or backbone for its higher inhibition efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55(1995)). The modified nucleic acid backbone may comprise phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic or heterocyclic intersugar linkages. The antisense oligonucleotide may also contain one or more substituted sugar moieties. The antisense nucleic acid may also include modified bases. Examples of the modified bases include hypoxanthine, 6-methyladenine, 5-Me pyrimidines (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N₆(6-aminohexyl)adenine, and 2,6-diaminopurine. In addition, the antisense nucleic acids of the present invention may be chemically bonded to one or more moieties or conjugates enhancing the activity and cellular uptake of the antisense nucleic acids. For example, lipophilic moieties include, but are not limited to, a cholesterol moiety, a cholesteryl moiety, cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine chain, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, an octadecylamine moiety and a hexylamino- carbonyl-oxycholesterol moiety. Methods of preparing oligonucleotides comprising lipophilic moieties are well known in the art (see U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acids may have enhanced stability in the presence of nucleases and enhanced binding affinity to target mRNA. The modifications described above enhance stability against nuclease degradation and increase affinity of the antisense oligonucleotide toward its target mRNA. Antisense RNA may be synthesized in vitro by a conventional method and administered to the body, or may be synthesized in vivo. A method for synthesizing antisense RNA in vitro employs RNA polymerase I. A method for synthesizing antisense RNA in vivo involves performing transcription of antisense RNA using a vector containing a multicloning site (MCS) in the opposite direction. Such antisense RNA preferably contains a translation stop codon in its sequence to block translation into a peptide sequence.

The term "siRNA" as used herein refers to a nucleic acid molecule mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914). The siRNA to inhibit expression of a target gene provides effective gene knock-down method or gene therapy method. It was first used in plants, insects, Drosophila melanogaster and parasites and recently has been used for mammalian cell research (8-10). The siRNA molecule of the present invention may consist of a sense RNA strand (having sequence corresponding to mRNA) and an antisense RNA strand (having sequence complementary to mRNA) and form a duplex structure. Alternatively, the siRNA molecule of the present invention may have a single strand structure comprising self-complementary sense and antisense strands. The siRNA of the present invention is not restricted to an RNA duplex of which two strands are completely paired and may comprise a non-paired portion such as a mismatched portion with non-complementary bases and bulge with no opposite bases. The overall length of the siRNA is 10-100 nucleotides, preferably, 15-80 nucleotides, and more preferably, 20-70 nucleotides. The siRNA may comprise either a blunt or cohesive end so long as it enables to silence the BLT2expression due to RNAi effect. The cohesive end may be prepared in 3'-end overhanging structure or 5'-end overhanging structure. The siRNA may be constructed by inserting a short nucleotide sequence (e.g., about 5-15 nt) between self-complementary sense and antisense strands. The siRNA expressed forms a hairpin structure by intramolecular hybridization, resulting in the formation of stem-and-loop structure. The stem-and-loop structure is processed in vitro or in vivo to generate active siRNA molecule mediating RNAi.

Meanwhile, the term "shRNA" (small hairpin RNA) refers to shRNA which is prepared in the following manner. An oligo DNA having sense sequence and its complimentary nonsense sequence of target gene siRNA linked via a linker having 3 to 10 bases is synthesized. Then, the resulting oligo DNA is cloned into a plasmid vector or lentivirus, which is a retrovirus, or adenovirus to express, thereby forming a loop sequence of the shRNA. The resulting sequence is cleaved by a Dicer in the viral cell to generate siRNA, which will exhibit RNAi effects. shRNA exhibits RNAi effects for a relatively long time, as compared to siRNA.

In one embodiment, the present invention provides a method for screening an antifungal agent, the method comprising the steps of: (a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed; (b) measuring the amount or activity of the protein; and (c) determining the sample as an antifungal agent wherein the amount or activity of the Hxl1 protein is measured to be down-regulated. In another embodiment, the present invention provides a method for screening an antifungal agent, the method comprising the steps of: (a) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed; (b) measuring the expression of the gene; and (c) determining the sample as an antifungal agent wherein the expression of the gene is measured to be down-regulated. In yet another aspect, the present invention provides a method for screening an antifungal agent for co-administration, the method comprising the steps of: (a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with an antifungal agent, and measuring the amount or activity of the protein; (b) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed and the said antifungal agent, and measuring the amount or activity of the protein; (c) comparing the value measured in step (a) with the value measured in step (b); and determining the sample as an antifungal agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a). In yet another embodiment, the present invention provides a method for screening an antifungal agent for co-administration, the method comprising the steps of: SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with an antifungal agent, and measuring the expression of the gene; (b) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed and the said antifungal agent, and measuring the expression of the gene; (c) comparing the value measured in step (a) with the value measured in step (b); and determining the sample as an antifungal agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a). In these embodiments, SEQ ID NO: 1 is the amino acid sequence of the mRNA spliced by UPR activation, and SEQ ID NO: 2 is the sequence of the cDNA of the mRNA spliced by UPR activation. Also, SEQ ID NO: 3 is the amino acid sequence of the Ire1 protein, and SEQ ID NO: 4 is the nucleotide sequence of the gene encoding the Ire1 protein.

In one embodiment of the inventive method for screening the antifungal agent for co-administration, the antifungal agent is an azole-based antifungal agent or a non-azole-based antifungal agent. In this embodiment, the azole-based antifungal agent for co-administration is any one or more of fluconazole, itraconazole, voriconazole and ketoconazole. In this embodiment, the non-azole-based antifungal agent for co-administration is amphotericin B or fludioxonil.

In one embodiment of the inventive method for screening the antifungal agent, step (a) is carried out at a temperature ranging from 35℃ to 40℃, and the cells in step (a) are C. neoformans.

In one embodiment, the present invention provides an antifungal pharmaceutical composition comprising, as an active ingredient, an antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to a nucleotide sequence set forth in SEQ ID NO: 2 or 4, wherein the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 88 to 617 of the nucleotide sequence of SEQ ID NO: 2, and the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 1935-2421 of the nucleotide sequence of SEQ ID NO: 4.

In one embodiment, the present invention provides a method for screening a meningitis-treating agent, the method comprising the steps of: (a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed; (b) measuring the amount or activity of the protein; and (c) determining the sample as a meningitis-treating agent wherein the amount or activity of the Hxl1 protein is measured to be down-regulated. In another embodiment, the present invention provides a method for screening a meningitis-treating agent, the method comprising the steps of: (a) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed; (b) measuring the expression of the gene; and (c) determining the sample as a meningitis-treating agent wherein the expression of the gene is measured to be down-regulated. In yet another aspect, the present invention provides a method for screening a meningitis-treating agent for co-administration, the method comprising the steps of: (a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a meningitis-treating agent, and measuring the amount or activity of the protein; (b) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed and the said meningitis-treating agent, and measuring the amount or activity of the protein; (c) comparing the value measured in step (a) with the value measured in step (b); and determining the sample as a meningitis-treating agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a). In yet another embodiment, the present invention provides a method for screening a meningitis-treating agent for co-administration, the method comprising the steps of: (a) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1gene set forth in SEQ ID NO: 4 with a meningitis-treating agent, and measuring the expression of the gene; (b) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1gene set forth in SEQ ID NO: 4 with a sample to be analyzed and the said meningitis-treating agent, and measuring the expression of the gene; (c) comparing the value measured in step (a) with the value measured in step (b); and determining the sample as a meningitis-treating agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a).

In one embodiment of the inventive method for screening the meningitis-treating agent for co-administration, the meningitis-treating agent is an azole-based or non-azole-based meningitis-treating agent. In this embodiment, the azole-based meningitis-treating agentfor co-administration is any one or more of fluconazole, itraconazole, voriconazole and ketoconazole. In this embodiment, the non-azole-based meningitis-treating agent for co-administration is amphotericin B or fludioxonil.

In one embodiment of the inventive method for screening the meningitis-treating agent, step (a) is carried out at a temperature ranging from 35℃ to 40℃, and the cells in step (a) are Cryptococcus neoformans.

In one embodiment, the present invention provides a pharmaceutical composition for treating meningitis, the composition comprising, as an active ingredient, an antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to a nucleotide sequence set forth in SEQ ID NO: 2 or 4, wherein the antisense orsiRNA oligonucleotide has a sequence complementary to nucleotides 88 to 617 of the nucleotide sequence of SEQ ID NO: 2, and the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 1935-2421 of the nucleotide sequence of SEQ ID NO: 4.

In one embodiment, the present invention provides: an Hxl1 ( H AC1 and X BP1- Like gene 1) protein set forth in SEQ ID NO: 1; an HXL1 gene encoding the protein of SEQ ID NO: 1; an HXL1 gene set forth in SEQ ID NO: 2; and a host containing a deletion of the gene of SEQ ID NO: 2.

The term "antifungal agent"as used herein is meant to include inorganic antifungal agents, organic natural extract-based antifungal agents, organic aliphatic compound-based antifungal agents, and organic aromatic compound-based antifungal agents, which serve to inhibit the propagation of bacteria and/or fungi. Examples of the inorganic antifungal agents include, but are not limited to, chlorine compounds (especially sodium hypochlorite), peroxides (especially hydrogen peroxide),boric acid compounds (especially boric acid and sodium borate), copper compounds (especially copper sulfate), zinc compounds (especially zinc sulfate and zinc chloride), sulfur-based compounds (especially sulfur, calcium sulfate, and hydrated sulfur), calcium compounds (especially calcium oxide), silver compounds (especially thiosulfite silver complexes, and silver nitrate), iodine, sodium silicon fluoride, and the like. Examples of the organic natural extract-based antifungal agents include hinokithiol, Phyllostachys pubescens extracts, creosote oil, and the like.

The term "meningitis" as used herein is meant to include various inflammatory diseases occurring in the subarachnoid space between the arachnoid and the pia mater, for example, those caused by invasion of viruses or bacteria into the subarachnoid space, inflammation caused by a certain chemical substance, and those caused by the spread of cancer cells into the cerebrospinal fluid space.

The present invention makes it possible to screen an effective candidate having an antifungal effect or a meningitis-treating effect, which inhibits novel Ire1 and Hxl1 ( H AC1 and X BP1- Like gene 1) proteins and genes encoding the same. Furthermore, the present invention provides a method for screening a candidate which can show a synergistic effect when being co-administered with an existing antifungal agent or meningitis-treating agent. In addition, the present invention can provide a pharmaceutical composition which shows an antifungal effect or a meningitis-treating effect by inhibiting the novel Ire1 and Hxl1 ( H AC1 and X BP1-Like gene 1) proteins and the genes encoding the same.

FIG. 1 is a schematic diagram showing a method of preparing primers for performing PCR.

FIG. 2 shows the results of identifying the target gene of Ire1 sensor kinase/ribonuclease by RT-PCR. As shown in FIG. 2, a specific splicing pattern was confirmed by RT-PCR1 and RT-PCR2, and KAR2 and ACT1 were used as controls.

FIG. 3 shows a change in the specific splicing pattern of the HXL1 gene in an ire1Δ mutant strain. After treatment with DTT and tunicamycin which cause UPR stress, it was confirmed by RT-PCR2 that a change in the specific splicing pattern of the HXL1 in the ire1Δ mutant strain, suggesting that HXL1 is the target of Ire1 sensor kinase/ribonclease.

FIG. 4 shows a phylogenetic tree of target proteins of Ire1 sensor kinase. In FIG. 4, An: Aspergillus nidulans, Af: Aspergillus fumigatus, Tr: Trichoderma reesei, Sc: Saccharomyces cerevisiae, Ca: Candida albicans, Yl: Yarrowia lipolytica, Hs: Homo sapiens, Mm: Mus musculus, Ce: Caenorhabditis elegans, and Cn: Cryptococcus neoformans.

FIG. 5shows the effect of a deletion of the UPR signaling pathway gene on temperature sensitivity. It can be seen that a deletion of the UPR signaling pathway gene IRE1 or HXL1 leads to an increase in temperature sensitivity. Particularly, it can be seen that the hxl1Δ mutant strain shows temperature sensitivity at a lower temperature than the ire1Δmutant strain. The following strains were used in the experiment: wild type (WT), ire1Δ (YSB552), ire1Δ+IRE1 (YSB1000), hxl1Δ (YSB723), hxl1Δ+HXL1 (YSB762), ire1Δ+HXL1 ^u(YSB1125), ire1Δ+HXL1 ^s(YSB1127), mpk1Δ (KK3), cna1Δ (KK1), and ras1Δ (YSB53).

FIG. 6 shows the effect of a deletion of the UPR signaling pathway gene on antifungal agent sensitivity. It was found that a deletion of the UPR signaling pathway gene IRE1 or HXL1 led to an increase in sensitivity to the antifungal agents. The following drug concentrations were used in the experiment: ketoconazole, 0.1 μg/ml fluconazole, 14 μg/ml itraconazole, 0.5 μg/ml amphotericin B, 0.8 μg/ml and fludioxonil, 0.5 μg/ml. Also, the following strains were used in the experiment: wild type (WT), ire1Δ (YSB552), ire1Δ+IRE1 (YSB1000), hxl1Δ (YSB723), hxl1Δ+HXL1 (YSB762), ire1Δ+HXL1 ^u(YSB1125), ire1Δ+HXL1 ^s(YSB1127).

FIG. 7 shows an siRNA design for inhibiting IRE1 gene expression. An ACT1 promoter and a GAL7promoter were introduced at the ends of the sense and antisense strands of the IRE1 gene, and a linker was inserted into the gene such that the sense and antisense strands could form a stem-loop. The linker may be the GFP protein as mentioned as the references.

FIG. 8 shows an siRNA design for inhibiting HXL1 gene expression. An ACT1 promoter and a GAL7 promoter were introduced at the ends of the sense and antisense strands of the HXL1, and a linker was inserted into the gene such that the sense and antisense strands could form a stem-loop. The linker may be the GFP protein as mentioned as the references.

FIG. 9 shows the effect of a deletion of the UPR signaling pathway gene IRE1 on pathogenicity in vivo. It shows that a deletion of the UPR signaling pathway gene IRE1 led to a decrease in pathogenicity in vivo. The following strains were used in the experiment: wild type (WT), ire1Δ (YSB552), and ire1Δ+IRE1 (YSB1000).

FIG. 10 shows the effect of a deletion of the UPR signaling pathway gene HXL1 on pathogenicity in vivo. It shows that a deletion of the UPR signaling pathway gene HXL1 led to a decrease in pathogenicity in vivo. The following strains were used in the experiment: wild type (WT), hxl1Δ (YSB723), and hxl1Δ+HXL1 (YSB762).

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

<Strains and incubation conditions>

In this Example, C. neoformans strains as shown in Table 1 below were used. As liquid medium, yeast-peptone-dextrose (YPD) was used, and the strains were incubated at 30℃ at 210 rpm.

Table 1

Strain	Genotype	Parent strain
C. neoformans
H99	Serotype A MATα
YSB552	MATα ire1Δ::NAT-STM#224*	H99
YSB554	MATα ire1Δ::NAT-STM#224	H99
YSB723	MATα hxl1Δ::NAT-STM#229	H99
YSB730	MATα hxl1Δ::NAT-STM#229	H99
YSB1000	MATα ire1Δ::NAT IRE1-NEO	YSB552
YSB1005	MATα ire1Δ::NAT IRE1-NEO	YSB554
YSB1125	MATα ire1Δ::NAT HXL1-HXL1 ^u -NEO	YSB552
YSB743	MATα ire1Δ::NAT HXL1-HXL1 ^u -NEO	YSB554
YSB1127	MATα ire1Δ::NAT HXL1-HXL1 ^s -NEO	YSB552
YSB747	MATα ire1Δ::NAT HXL1-HXL1 ^s -NEO	YSB554
YSB762	MATα hxl1Δ::NAT HXL1 ^u -NEO	YSB723
KK1	MATα cna1Δ::NAT-STM#117	H99
KK3	MATα mpk1Δ::NAT-STM#150	H99
YSB53	MATα ras1Δ::NAT-STM#150	H99

(* Each NAT-STM# means Nat^r marker with a unique signature tag)

<Stress sensitivity test method>

According to the method described in "Bahn YS, Kojima K, Cox GM, Heitman J (2005) Mol Biol Cell 16: 2285-2300", and "Bahn YS, Kojima K, CoxGM, Heitman J (2006) Mol Biol Cell 17: 3122-3135", each of the strains was incubated in 5 ml of YPD medium at 30℃ for 16 hours, washed, and then serially diluted in dH₂O (degree of dilution: 1 to 10⁴). Then, each of the incubated strains was spotted on a solid YPD medium containing an indicated concentration of an antifungal agent.

Specifically, in order to examine sensitivity to antifungal agents, each of the strains was spotted on an YPD medium containing each of polyene-based drug(amphotericin B (Sigma)), azole-based drug (fluconazole (Sigma), itraconazole (Sigma), ketoconazole (Sigma)), and the phenylpyrrole-based drug Fludioxonil. After spotting, the strains were incubated at 30℃and photographed for 2-4 days.

In order to examine temperature sensitivity, in the same manner as described above, each of the strains was incubated in 5 ml of YPD at 30℃ for 16 hours, washed, diluted serially in dH₂O (degree of dilution: 1 to 10⁴), and then spotted on solid YPD medium. After spotting, each of the strains was incubated at each of 30℃, 35℃ and 37℃ and photographed for 2-4 days.

<RNA extraction and RT-PCR process>

To extract RNA, each of the strains was incubated in YPD medium at 30℃ for 16 hours. Then, each strain was inoculated into 100 ml of fresh YPD medium and adjusted to an optical density of 0.15 at 600 nm (OD₆₀₀), after which each strain was incubated at 30℃ until it reached an OD₆₀₀of 0.5. To obtain a zero-time sample, 50 ml was sampled from 100 ml of the incubation medium and washed twice with DEPC-treated dH₂O, followed by rapid freezing in liquid nitrogen. The remaining 50 ml of the incubation medium was treated with 8 μg/mlof tunicamycin or 20 mM of DTT, and after 1 or 2 hours, the strains were harvested. Each of the harvested cells was washed twice with DEPC-treated dH₂O, followed by rapid freezing in liquid nitrogen. Total RNA was extracted from the strain cells using a RNeasy Mini Kit (Qiagen), and cDNA was synthesized from the total RNA using MMLV reverse transcriptase (Invitrogen). The cDNA was subjected to RT-PCR (reverse transcriptase polymerase chain reaction) using Maxime™ PCR PreMix (i-Taq) (iNtRON Biotechnology).

<Method for Disruption of UPR signaling pathway genes>

For gene disruption, information on the genomic DNA structure of each gene was obtained from the genomic database(http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) of C. neoformansserotype A. According to the method described in "Davidson RC, et al. (2002) A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology148:2607-2615" and "Kim MS, et al. (2009) An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers. Biochem Biophys Res Commun 390:983-988", IRE1 (CNAG_03670.2) and HXL1 (CNAG_06134.2) in a C. neoformansserotype A H99 strain were disrupted by overlap PCR and double-joint PCR with split markers and biolistic transformation. Primers required for disruption of the genes are shown in Table 2 below. Specifically, gel-extracted deletion cassettes produced by overlap PCR or double-joint PCR were coated on gold microcarrier beads (0.6 μm Bio-Rad) and transformed into the serotype A H99 strain. Strains growing well in Nourseothricin-containing YPD medium were primarily selected through diagnostic PCR, after which transformed strains containing a deletion of the gene were confirmed through Southern blot analysis using gene-specific probes.

Table 2

Primer	Sequence (5' to 3)^a	Intended use
B1644	GCCCCATCATCATAATCAC	Primer for disruptionof IRE1
B1645	GCTCACTGGCCGTCGTTTTACACTATGTGTCCATCTGAGGC	The same as above
B1646	CATGGTCATAGCTGTTTCCTGAGTGAGTTGAGGGAGGAAAG	The same as above
B1647	GAAGAAGAGCGTCAAGAAGG	The same as above
B1648	AGGAATACGAGGTTTATCGG	Primer for diagnosis ofIRE1
B1683	AGCATTAGGGGTGTAGGTG	Probe primer for IRE1
B1881	GTTTGAGGCTGGTAAAAAGG	Primer for disruptionof HXL1
B1882	GCTCACTGGCCGTCGTTTTACATGGGGAATGAAAGCGTG	The same as above
B1883	CATGGTCATAGCTGTTTCCTGAAGGGGCGAGAGTAGTTCAG	The same as above
B1884	GACTGTAAAGGAGGGCATAAG	The same as above
B1880	AACTCTTCTCAGCCTTCGG	Primer for diagnosis ofHXL1
B1885	CGTTCTCCGTCTTGATAGC	Probe primer for HXL1
M13Fe	GTAAAACGACGGCCAGTGAGC	Primer for disruptive marker
M13Re	CAGGAAACAGCTATGACCATG	Primer for disruptivemarker
B1969	CGCGCGGCCGCGCACAGGATTACTTTTGGGTGATG	Complement of IRE1
B1970	CGCGCGGCCGCAGTTGGAAAAGGAGCGTCC	The same as above
B1454	AAGGTGTTCCCCGACGACGAATCG	Primer for double-jointPCR
B1455	AACTCCGTCGCGAGCCCCATCAAC	Prime for double-jointPCR
C01	ATACAGCCAGTGTCCTTCCC	Primer for RT-PCR ofCNAG_03976.2 of H99(RT-PCR1)
C02	ATGGATATCAGTTGCGGGAT	The same as above
C03	AGAGATTGAGCTCCTCCG	Primer for RT-PCR ofCNAG_03976.2 of H99(RT-PCR2)
C04	TTACCAGACACTGACACC	The same as above
C05	ATGTCCGCGATCGATTAC	Primer for RT-PCR of CNAG_07560.2 of H99 (RT-PCR1)
C06	CCTTCTTCTCAGCAGCAGTC	The same as above
C07	TTAACGGCTGCGTCAATC	Primer for RT-PCR of CNAG_07560.2 of H99 (RT-PCR2)
C08	TCTTCTTCTATCGACCCG	The same as above
C09	TTTGGCAATGACGACTCAAG	Primer for RT-PCR of CNAG_07940.2 of H99 (RT-PCR1)
C10	CCTGTAACGCTCTGTTCTCC	The same as above
C11	TCATGGGCTGTTGATTCC	Primer for RT-PCR of CNAG_07940.2 of H99 (RT-PCR2)
C12	TAAAGGAAGGTTCCGGTG	The same as above
C13	AGTGCACTGATGGCGTCA	Primer for RT-PCR of CNAG_00871.2 of H99 (RT-PCR1)
C14	AAAGCATAGACAACGGCG	The same as above
C15	CACTCGGCACCGTTATGT	Primer for RT-PCR of CNAG_00871.2 of H99 (RT-PCR2)
C16	TGGCAAATGCGTAGCTTC	The same as above
C17	ATGGCTACCGCTGTCGCT	Primer for RT-PCR of CNAG_06134.2 of H99 (RT-PCR1)
C18	TGATTCGCGGTTACGGAT	The same as above
C19	CACTCCATTCCTTTCTGC	Primer for RT-PCR ofCNAG_06134.2 of H99(RT-PCR2)
C20	CGTAACTCCACTGTGTCC	The same as above
C25	TGCAGAAGATGGCGTTGC	Primer for RT-PCR of IRE1 of all serotypes
C26	ACACTCCCGCCTTTATAC	The same as above
C27	TCGATGCCAATGGTATCC	Primer for RT-PCR ofCNAG_06443.2
C28	TCATGGCTGAAAGGCATC	The same as above
C33	AGCCTTCTCTCCTTGGTC	Primer for RT-PCR of ACT1
C34	ACGATTGAGGGACCAGAC	The same as above
C35	GTCGTTAACCTTCGGAGGCTTTTACAC	Complement of HXL1
C36	CTCGAGCATATGGCTAGCTGCGAGGATGGGAATAGG	The same as above
C37	GCTAGCCATATGCTCGAGGGAAGAAACAAAATAACCAAC	The same as above
C38	CACGGTACCAATATATCATGCCCTCCCG	The same as above

^a The underlined primer sequences are restriction enzyme portions for subsequence subcloning, and the sequences indicated by bold letters are complementary sequences for fusion PCR.

<Preparation of complemented strain for deletion of UPR signaling pathway gene>

In order to examine sensitivity to antifungal agents caused by deletions of the UPR signaling pathway genes, a complemented strain for each of the transformed strains containing a deletion of the UPR signaling pathway gene was produced. In order to prepare an IRE1 complemented strain, an IRE1 gene fragment comprising a 0.78-kb promoter, a 3.52-kb ORF (Open Reading Frame) and a 0.53-kb terminator was amplified by PCR using primers containing restriction enzyme sites. The PCR product was subcloned into a pTOP-V2 plasmid (purchased from Enzynomics), and the DNA base sequence thereof was analyzed. The 4.8-kb IRE1 gene insert was subcloned into a pJAF12 vector (provided from James A. Fraser, Centre for Infectious Disease Research, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia) having a NEO^r marker (Neomycin/G418-resistant marker) using a plasmid which base sequences are matched. The subcloned plasmid was digested with Mlu1 restriction enzyme, and the IRE1-deleted mutant strain was transformed with the digested plasmid.

Likewise, in order to prepare a HXL1 complemented strain, a HXL1 gene fragment comprising a 1-kb promoter, a 1.8-kb ORF (Open ReadingFrame) and a 0.6-kb terminator was amplified by PCR using primers (C35/C38) and subcloned into a pJAFS1 vector (from which the Sac1 restriction enzyme site of pJAF12 had been removed) having a NEO^r marker. The subcloned plasmid was digested with Sac1 restriction enzyme, the HXL1-deleted mutant strain was transformed with the resulting plasmid.

<Transformation of HXL1 splicing gene and unsplicing gene into IRE1 -deleted mutant strain>

In order to examine whether sensitivity to antifungal agents resulting from IRE1 deletion is HXL1-dependent, the spliced mRNA and unspliced mRNA of the HXL1gene were obtained from cDNA derived from total RNA isolated from cells treated or untreated with the UPR (unfolded protein response) derivative tunicamycin. DNAs were obtained from the mRNAs and subcloned into pJAFS1-HXL1PT plasmids (from which the SacI restriction enzyme site of pJAF12 had been removed) having a HXL1 promoter and a terminator, thereby obtaining a pJAFS1-HXL1u plasmid and a pJAFS1-HXL1s plasmid. Each of the plasmids was digested with Sac1 restriction enzyme and transformed into the IRE1-deleted mutant strain.

<Examination of pathogenicity of HXL1 gene - deleted mutant and IRE1 gene-deleted mutant in vivo through Cryptococcosis mouse model>

In order to examine the effects of Ire1 sensor kinase/ribonuclease and its target gene HXL1(which play an important role in the UPR signaling pathway) on pathogenicity in vivo, 4-6-week-old A/Jcr mice (Jackson Laboratory, 18-22 g) were used. Each of wild type, ire1Δ, hxl1Δ mutant strains and the restored strains was incubated in YPD medium at 24℃ for 16 hours, after which each strain was washed with PBS (phosphate buffered saline) and then adjusted to a cell density of 10⁶ cells/ml. 10 mice were used for each of the wild type strain, the mutant strain and the restored strain and infected by intranasal infection of 10⁵cells. The survival of the mice was observed twice a day for 6 weeks.

<Example 1> Identification of target gene HXL1 of IRE1

In order to find the target gene of Ire1 kinase/ribonuclease in the UPR signaling pathway, candidate genes were searched through the serotype A C. neoformans genome database (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) using the bZIP domain sequence of Hac1 transcription factor known as the target in S. cerevisiae. As a result, the following five candidate genes were selected: CNAG_00871.2, CNAG_06134.2, CNAG_07560.2, CNAG_07940.2 and CNAG_03976.2.

Ire1 kinase/ribonuclease is characterized in that it splices the target mRNA in an unconventional manner. Thus, in order to examine the splicing pattern, RT-PCR primers as shown in Table 2 above were prepared. The experiment was performed by two stages, that is, RT-PCR1 and RT-PCR2 (see FIG. 1). The target gene was identified from changes in splicing patterns which appeared in RT-PCR1 and RT-PCR2 when treated with DTT and tunicamycin which cause UPR stress (see FIG. 2). The experimental results showed that CNAG_06134.2 was specifically spliced. In order to examine whether CNAG_06134.2 is spliced in the ire1Δ mutant strain, CNAG_06134.2 was treated with the UPR stress-causing DTT and tunicamycin in the same manner as above and subjected to RT-PCR2. As a result, CNAG_06134.2 was specifically spliced in the wild type strain, but was not spliced in the ire1Δ mutant strain (see FIG. 3). Such results demonstrated that CNAG_06134.2 mRNA is the target gene of Ire1 sensor kinase/ribonuclease. In order to examine the protein sequence similarity between CNAG_06134.2 identified through these experimental results and the target protein of Ire1 sensor kinase/ribonuclease known in the prior art, phylogenetic tree analysis was performed. As a result, CNAG_06134.2 had a significantly low sequence similarity to the known target protein of Ire1 sensor kinase/ribonuclease (see FIG. 4). Although the CNAG_06134.2 gene had a significantly low sequence similarity to the known target protein, the gene was named "HXL1 ( H AC1 and X BP1-Like gene 1)"based on splicing patterns similar to those of the Hac1 transcription factor in fungi and the Xbp1 transcription factor in humans.

<Example 2> Examination of temperature sensitivity resulting from inhibition of UPR signaling pathway of ire1 Δ mutant strain and hxl1 Δ mutant strain

Pathogenic determinants having effects on the pathogenicity of C. neoformansinclude capsules interfering with phagocytosis, antioxidant melanin, and the capability to grow at the body temperature of humans which are hosts. Among them, the capability to grow at the host temperature of 37℃ can be said to be a very important factor in causing pathogenicity.

In order to examine the effect of this temperature sensitivity on the UPR signaling pathway, a temperature sensitivity test was carried out. As a result, the growth of both the ire1Δ mutant strain and the hxl1Δ mutant strain at 37℃ was significantly inhibited compared to the growth of the wild type strain.

Even when the ire1Δ mutant strain was transformed with the HXL1 splicing gene, the growth thereof at 37℃ was not restored, like the case of the wild type strain. Also, the growth of the hxl1Δ mutant strain was significantly reduced even at 35℃, indicating that the hxl1Δ mutant strain showed a higher temperature sensitivity (see FIG. 5).

<Example 3> Examination of effects of existing antifungal agents resulting from inhibition of UPR signaling pathway of ire1 Δ mutant strain and hxl1 Δ mutant strain

Whether C. neoformans mutant strains having a mutation in the UPR signaling pathway are sensitive to antifungal agents was examined. As a result, it was shown that both the ire1Δ mutant strain and the hxl1Δmutant strain were more sensitive to polyene-based amphotericin B than the wild type strain. Also, the amphotericin B sensitivity of the ire1Δmutant strain transformed with the HXL1splicing gene was restored to that of the wild type strain. The amphotericin B sensitivity of the ire1Δmutant strain was shown to be dependent on Hxl1 (FIG. 6).

The drug sensitivities of the mutant strains to azole-based drugs (including fluconazole, itraconazole and ketoconazole) were tested. As a result, the mutant strains were significantly more sensitive to the drugs than the wild type strain. Particularly, the hxl1Δ mutant strain showed more than 10 times higher sensitivity to fluconazole than the ire1Δ mutant strain. This fact suggests that Hxl1 is regulated not only by Ire1, but also by other up-regulating proteins. Also, it could be seen that the drug sensitivity of the ire1Δ mutant strain transformed with the HXL1 splicing gene was restored to the level of the wild type strain. Likewise, this suggests that the sensitivity of the ire1Δ mutant strains to the azole-based drugs also depends on Hxl1 in the UPR signaling pathway (FIG. 6).

Meanwhile, the results of a drug sensitivity test for phenylpyrrole-based fludioxonil indicated that both the ire1Δ mutant strain and the hxl1Δmutant strain were sensitive to fludioxonil. Also, like in the case of sensitivity to fluconazole, the hxl1Δmutant strain showed about 100 times higher sensitivity to fludioxonil than the ire1Δmutant strain. As mentioned above, this suggests that Hxl1 is regulated not only by Ire1, but also by other up-regulating proteins (FIG. 6).

<Example 4> Examination of antifungal effects resulting from inhibition of UPR signaling pathway by siRNA against IRE1 and HXL1 genes

To analyze the silencing of the expression of IRE1 and HXL1gene, siRNA can be synthesized using Silencer^TM siRNA cocktail kit (RNase III; Ambion). Oligonucleotides used to synthesize dsRNA against IRE1 are as follows: 5'-GATCTCAGATACTATCATTGGTTTTGGATC-3', and 5'- CAAGTTGTTCGCCGTCGGCGCAAAGGAT-3. Also, oligonucleotides used to synthesize dsRNA against HXL1 are as follows: 5'- CCTATCAAGCGTCCTCGTCAATCTAGT -3', and 5'- CCTATCAAGCGTCCTCGTCAATCTAGT -3'.

The base sequences of siRNA had a length of 431 bp (nucleotides 1935-2421 of SEQ ID NO: 4) for IRE1 and a length of 530 bp (nucleotides 88 -617 of SEQ ID NO: 3) for HXL1 (see Ahn JH, et al: Identification of the genes differentially expressed in human dendritic cell subsets bycDNA subtraction and microarray analysis. Blood 2002, 100(5):1742-1754, and Yang YH et al: Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variations. Nucleic Acids Res 2002, 30(4): e15. 20, 21). Designs of IRE1 and HXL1siRNAs are shown in FIGS. 7 and 8 (see Hong Liu, et al: RNA interference in the pathogenic fungus C. neoformans. Genetics, 2002, 160:463-470). Each of the above-designed siRNAs was linearized and transformed into the wild type strain by electroporation. Silencing of the expression of the IRE1 or HXL1 gene by such siRNA showed effects, such as antifungal agent sensitivity, pathogenicity and temperature sensitivity, like the deletion of the genes.

<Example 5> Examination of effect of UPR signaling pathway inhibition resulting from deletion of IRE1 gene and HXL1 gene on pathogenicity in vivo

The pathogenicities of the C. neoformans mutant strains having a mutation in the UPR signaling pathway were examined using a mouse model. As a result, it could be seen that the pathogenicities of the ire1Δ and hxl1Δ mutant strains having a mutation in the UPR signaling pathway were significantly inhibited compared to that of the wild type strain. This suggests that the UPR signaling pathway plays an important role in the pathogenicity of C. neoformans (FIGS. 9 and 10).

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Sequence ID No. 1 represents amino acid sequence of Hxl1 protein, sequence ID No. 2 represents nucleotide sequence of Hxl1 gene. Sequence ID No. 3 represents amino acid sequence of Ire protein, sequence ID No. 4 represents nucleotide sequence of IRE1 gene.

Claims

A method for screening an antifungal agent, the method comprising the steps of:

(a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed;

(b) measuring the amount or activity of the protein; and

(c) determining the sample as an antifungal agent wherein the amount or activity of the Hxl1 protein is measured to be down-regulated.
A method for screening an antifungal agent, the method comprising the steps of:

(a) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed;

(b) measuring the expression of the gene; and

(c) determining the sample as an antifungal agent whrerein the expression of the gene is measured to be down-regulated.
A method for screening an antifungal agent for co-administration, the method comprising the steps of:

(a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with an antifungal agent, and measuring the amount or activity of the protein;

(b) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed and the said antifungal agent, and measuring the amount or activity of the protein;

(c) comparing the value measured in step (a) with the value measured in step (b);

and determining the sample as an antifungal agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a).
A method for screening an antifungal agent for co-administration, the method comprising the steps of:

(a) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with an antifungal agent, and measuring the expression of the gene;

(b) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed and the said antifungal agent, and measuring the expression of the gene;

(c) comparing the value measured in step (a) with the value measured in step (b);

and determining the sample as an antifungal agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a).
The method of claim 3 or 4, wherein the antifungal agent is an azole-based antifungal agent or a non-azole-based antifungal agent.
The method of claim 3 or 4, wherein the azole-based antifungal agent is any one or more of fluconazole, itraconazole, voriconazole and ketoconazole.
The method of claim 3 or 4, wherein the non-azole-based antifungal agent is amphotericin B or fludioxonil.
The method of claim 1 or 2, wherein step (a) is carried out at a temperature ranging from 35℃ to 40℃.
The method of claim 1 or 2, wherein the cells in step (a) are C. neoformans.
The method of claim 3 or 4, wherein steps (a) and (b) are carried out at a temperature ranging from 35℃ to 40℃.
The method of claim 3 or 4, wherein the cells in step (a) and (b) are C. neoformans.
An antifungal pharmaceutical composition comprising, as an active ingredient, an antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to a nucleotide sequence set forth in SEQ ID NO: 2 or 4.
The antifungal pharmaceutical composition of claim 12, wherein the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 88 to 617 of the nucleotide sequence of SEQ ID NO: 2.
The antifungal pharmaceutical composition of claim 12, wherein the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 1935-2421 of the nucleotide sequence of SEQ ID NO: 4.
A method for screening a meningitis-treating agent, the method comprising the steps of:

(a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed;

(b) measuring the amount or activity of the protein; and

(c) determining the sample as a meningitis-treating agent wherein the amount or activity of the Hxl1 protein is measured to be down-regulated.
A method for screening a meningitis-treating agent, the method comprising the steps of:

(a) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed;

(b) measuring the expression of the gene; and

(c) determining the sample as a meningitis-treating agent wherein the expression of the gene is measured to be down-regulated.
A method for screening a meningitis-treating agent for co-administration, the method comprising the steps of:

(a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a meningitis-treating agent, and measuring the amount or activity of the protein;

(b) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed and the said meningitis-treating agent, and measuring the amount or activity of the protein;

(c) comparing the value measured in step (a) with the value measured in step (b);

and determining the sample as a meningitis-treating agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a).
A method for screening a meningitis-treating agent for co-administration, the method comprising the steps of:

(a) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a meningitis-treating agent, and measuring the expression of the gene;

(b) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed and the said meningitis-treating agent, and measuring the expression of the gene;

(c) comparing the value measured in step (a) with the value measured in step (b);

and determining the sample as a meningitis-treating agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a).
The method of claim 17 or 18, wherein the meningitis-treating agent is an azole-based or non-azole-based meningitis-treating agent.
The method of claim 17 or 18, wherein the azole-based meningitis-treating agent is any one or more of fluconazole, itraconazole, voriconazole and ketoconazole.
The method of claim 17 or 18, wherein the non-azole-based meningitis-treating agent is amphotericin B or fludioxonil.
The method of claim 15 or 16, wherein step (a) is carried out at a temperature ranging from 35℃ to 40℃.
The method of claim 15 or 16, wherein the cells in step (a) are C. neoformans.
The method of claim 17 or 18, wherein step (a) is carried out at a temperature ranging from 35℃ to 40℃.
The method of claim 17 or 18, wherein the cells in step (a) and step (b) are C neoformans.
A pharmaceutical composition for treating meningitis, comprising as an active ingredient, an antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to a nucleotide sequence set forth in SEQ ID NO: 2 or 4.
The pharmaceutical composition of claim 26, wherein the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 88 to 617 of the nucleotide sequence of SEQ ID NO: 2.
The pharmaceutical composition of claim 26, wherein the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 1935-2421 of the nucleotide sequence of SEQ ID NO: 4.
A Hxl1 (HAC1 and XBP1-Like gene 1) protein set forth in SEQ ID NO: 1.
A HXL1 gene encoding the protein of claim 29.
The HXL1 gene of claim 30, wherein the HXL1 gene is set forth in SEQ ID NO: 2.
A host containing a deletion of the gene of claim 31.