KR102041684B1 - Use of Hsf1 gene for treating mycoses or meningoencephalitis - Google Patents

Abstract

The present invention relates to the use of the Hsf1 gene for the treatment of fungal infections or meningitis.

Description

Use of Hsf1 gene for treating mycoses or meningoencephalitis

The present invention relates to the use of the Hsf1 gene for the treatment of fungal infections or meningitis.

The response and adaptation to the host's physiological temperature, 37 ° C., is a key toxic property of most human fungal pathogens that infect the host through the respiratory tract in its natural environment at ambient temperature, Cryptococcus neoformans One of the pathogens. The basidiomycetes fungus (basidiomycetous fungus exist in a variety of environmental places and produce infectious yeast cells or spores through positive and single differentiation, which are then inhaled through the host's respiratory system and initially in large quantities in the lungs. Cryptococcus neoformans is one of the deadly meninges and encephalitis that causes more than 600,000 deaths each year, causing infection to continue to spread into the bloodstream, invade the central nervous system, and eventually death. The ability to detect and adapt to elevated temperatures (about 25 ° C to 37 ° C) during this transmission process is an important factor for pathogens to establish the early reproduction of the lungs. Therefore, since most mutations with severe defects in heat resistance tend to be strongly weakened, signaling cascades that control heat resistance in molds are used as a means of identifying potential antifungal agents, and much research has been conducted. have. The temperature-sensing signaling lines in Cryptococcus neoformans include calmodulin / calcineurin, Ras, HOG, Msi1 like proteins, protein kinase C1 (Pkc1) / Mpk1 MAPK and unfolded protein response (UPR) pathways.

Unlike other temperature sensing signaling elements described above, protein kinase Sch9 inhibits Cryptococcus neoformans heat resistance. Sch9 deletion increases the heat resistance of Cryptococcus neoformans. Thus, the sch9 mutant tolerates well up to 41 ° C, unlike the wild type (WT). Sch9 also negatively regulates the production of polysaccharide capsules, which is also a major virulence factor of Cryptococcus neoformans. Nevertheless, the toxicity of sch9 mutants is weakened and can be more susceptible to oxidative stress. sch9 expression is induced by oxidative stress. Although the direct or indirect correlation to Sch9's cAMP signal transduction pathway is not clear, Sch9's role in heat resistance and capsule synthesis appears to depend on protein kinase A (PKA). Therefore, it is not known how Sch9 regulates the heat resistance of Liptococcus neoformans.

Sch9 functions in yeast ( Saccharomyces cerevisiae ), much research is being done. Sch9 is a Ser / Thr protein kinase that plays an important role in nutrient sensing, stress response and chronological life-span regulation. Targets of rapamycin complex 1 (TORC1), an important nutrient sensor in eukaryotes, directly phosphorylate Sch9. Sch9 will probably maintain the optimal activity of the RNA polymerase by promoting the recruitment of the catalytic subunit Rpa190. Sch9 also properly processes 35S rRNA into 25S, 18S and 5.8S rRNAs and is essential for processome components such as Rps6. Sch9 has a synthetic relationship with cAMP / protein kinase A (PKA), another nutrient-sensing pathway. Sch9 plays an important role in the regulation of cellular stress through interaction with Rim15 kinase or Sko1-Hog1 complex. Sch9 activated by TORC1 promotes phosphorylation of Rim15 and promotes cellular maintenance. Upon inhibition of TORC1 and Sch9, Rim15 is translocated into the nucleus to activate the Msn2 / 4 transcription factor, which governs the general stress response. Sch9 also appears to act as a chromatin-related transcriptional activator complex consisting of Sch9, Sko1 and Hog1 in response to osmotic stress. It was demonstrated that Sch9 interacts with Sko1 and Hog1 and Sch9 phosphorylates Sko1. Sch9 is also heterologous to mammalian protein kinase B ( PKB ) / Akt with a pleckstrin homology (PH) domain that binds to the lipid secondary messenger phosphatidyl inositol-3,4,5-trisphosphate (PIP3) Because it is homosexual, it is involved in lipid signaling. Upon binding to PIP3 , PKB is phosphorylated and activated by phosphoinositide-dependent kinase 1 ( PDK1 ) and TORC2 . Yeast has two PDK1 heterologies, Pkh1 / 2, which are necessary for the function of Sch9. Sch9 is also required for the regulation of genes associated with induction of mitochondrial function, tricarboxylic acid circulation and autophagy. Loss of protein kinase Sch9 increases the heat resistance of Cryptococcus neoformans, but the regulatory mechanism is unknown. The present invention studied Sch9 dependent and Sch9 independent signaling processes involved in Cryptococcus neoformans heat resistance and potential molecular mechanisms for Sch9 mediated heat resistance in order to identify downstream target genes. The present invention was completed by confirming that overexpression of Hsf1 promotes heat resistance of Cryptococcus neoformans in association with resistance.

An object of the present invention is to provide a method for screening an antimicrobial agent or a meningitis therapeutic agent using a strain comprising a Hsf1 protein or a gene encoding the same.

It is an object of the present invention to provide a method for screening an antimicrobial agent comprising the following steps.

 (a) transforming the bacterium with a recombinant vector comprising a promoter, lacZ and pNEO activated by Hsf1 (Heat shock fator 1)

 (b) treating the transformed bacteria with antimicrobial candidates

 (c) determining that the candidate is an antimicrobial agent when it is determined that the transformed bacteria treated with the candidate material have fewer blue colonies than the transformed bacteria not treated with the candidate material.

Candidates used while referring to the screening methods of the present invention mean unknown candidates used in screening to examine whether they affect the expression level of a gene or affect the amount or activity of a protein. Such candidates include, but are not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

In the present invention, the recombinant expression vector includes a plasmid, cosmid or phage capable of synthesizing a protein encoded by each recombinant gene carried by the vector. Preferred vectors are vectors capable of self-replicating and expressing the nucleic acid to which they are bound.

The promoter activated by Hsf1 is SSA1 It may be a promoter. The promoter activated by Hsf1 may be an SSA1 promoter and may be an HSE gene in the SSA1 promoter.

The transformed bacteria may be Cryptococcus neoformans.

Still another object of the present invention is to provide an antimicrobial agent selected by the above screening method.

Another object of the present invention to provide a method for screening a pharmaceutical composition for the prevention or treatment of brain diseases comprising the following steps.

(a) SSA1 promoter (promoter), transfection step the recombinant vector containing the LacZ and pNEO the fungus

 (b) treating the transformed bacteria with antimicrobial candidates

 (c) when it is determined that the transformed bacteria treated with the candidate has fewer blue colonies than the transformed bacteria not treated with the candidate, the candidate is a pharmaceutical for preventing or treating brain diseases. Determining the composition.

The transformed bacteria may be Cryptococcus neoformans.

The brain disease may be characterized as being a cognitive impairment including dementia and Parkinson's disease.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of brain diseases selected by the screening method.

The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisenses, siRNA oligonucleotides, and natural extracts as active ingredients. The antifungal pharmaceutical composition or the antifungal complex preparation of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include an excipient, a disintegrant, a sweetener, a binder, a coating agent. Solubilizers such as swelling agents, lubricants, lubricants or flavoring agents can be used. The antifungal pharmaceutical composition of the present invention can be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate in injectable forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art. Pharmaceutical formulation forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. Can be. The pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered. An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to prevent or treat a disease. Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. For example, in adults, when administered once or several times a day, the inhibitor of the present invention is administered once or several times a day, when the compound is 0.1ng / kg to 10g / kg, a polypeptide, In the case of protein or antibody, 0.1ng / kg ~ 10g / kg, antisense oligonucleotide, siRNA, shRNAi, miRNA can be administered at a dose of 0.01ng / kg ~ 10g / kg.

In the present invention, the subject includes, but is not limited to, humans, orangutans, chimpanzees, mice, rats, dogs, cattle, chickens, pigs, goats, sheep, and the like.

As used herein, the term "antisense oligonucleotide" refers to DNA, RNA or derivatives thereof that contain nucleic acid sequences complementary to the sequence of a particular mRNA, and binds to complementary sequences in the mRNA to inhibit translation of the mRNA into a protein. It works. The antisense nucleic acid has a length of 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases. The antisense nucleic acid can be modified at the position of one or more bases, sugars or backbones to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5 (3): 343-55 (1995)). . The nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like. In addition, antisense nucleic acids may comprise one or more substituted sugars. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-Me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylamino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides comprising a fat soluble moiety and methods of preparation are already well known in the art (see US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA. Antisense oligonucleotides can be synthesized in vitro by conventional methods to be administered in vivo or to allow antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the multiple restriction enzyme cloning site (MCS). Such antisense RNA is desirable to ensure that there is a translation stop codon in the sequence so that it is not translated into the peptide sequence.

As used herein, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene suppression (see WO00 / 44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409). And WO 00/44914). siRNA is provided as an efficient gene knockdown method or gene therapy method because it can inhibit the expression of the target gene. siRNA was first discovered in plants, worms, fruit flies and parasites, but has recently been applied to mammalian cell research by developing / using siRNAs (8-10). The siRNA molecules of the present invention may have a structure in which a sense strand (a sequence corresponding to an mRNA sequence) and an antisense strand (a sequence complementary to an mRNA sequence) are positioned opposite to each other to form a double strand. In addition, according to another embodiment, siRNA molecules of the present invention may have a single chain structure having self-complementary sense and antisense strands. siRNAs are not limited to the complete pairing of double-stranded RNA portions paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), and the like. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases. The siRNA terminal structure can be either blunt or cohesive, as long as the expression of the gene can be inhibited by the RNAi effect. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures. The siRNA molecules of the present invention may have a form in which a short nucleotide sequence (eg, about 5 to 15 bases) is inserted between a self-complementary sense and an antisense strand, in which case by expression of the nucleotide sequence The formed siRNA molecules form a hairpin structure by intramolecular hybridization, and form a stem-and-loop structure as a whole. This stem-and-loop structure is in in vitro or in It is processed in vivo to produce an active siRNA molecule capable of mediating RNAi.

Meanwhile, in the present invention, the term "shRNA (small hairpin RNA)" refers to a oligo DNA linking 3-10 base linkers between the sense of the target gene siRNA and complementary nonsense, and then cloned into a plasmid vector. Or expressing by inserting shRNA into the retroviruses lentivirus and adenovirus, a looped hairpin structure of short hairpin RNA is produced and converted into siRNA by Dicer in the cell Say what it represents. The shRNA has a relatively long RNAi effect compared to siRNA.

The present invention has the effect of searching for candidates having an antimicrobial or meningitis therapeutic effect that inhibits the Hsf1 protein and the gene encoding the same. In detail, the pNEO-SSA1pro-LacZ recombinant vector is transformed into Cryptococcus neoformans, which is treated with Cryptococcus neoformans candidates having antimicrobial or meningitis therapeutic effects. Hsf 1 gene, which is involved in the heat resistance of pathogenic fungi, is inhibited, Hsf1 decreases binding to HSE of the SSA1 promoter, LacZ gene expression decreases, and β-galactosidase activity decreases in X-gal. In the presence of white colonies. This system uses a colorimetric method (a candidate that has an antibacterial or meningitis effect is white / an antibacterial or a candidate that has no therapeutic effect on meningitis is blue) and thus a new compound that modulates Hsf1 (a compound that inhibits or activates Hsf1). It is effective and can be searched at high speed.

1 during the temperature riseSCH9 Dependencies andSCH9 It is the overall fragment profile of the independent gene. 1A is a result of gene clustering and functional classification of temperature-regulated genes using KOG (Eukaryotic Orthologous Groups of protein; http://www.ncbi.nlm.nih.gov/COG/) database. A total of 1831 genes (ANOVA test, P <0.05), whose expression was differentially regulated with increasing temperature, are classified as a description of KOG function. The inserted table of FIG. 1B shows wild type (WT) (H99) andsch9Here is a summary of patterns of gene expression changes in Δ (YSB619) strains. 1CSch9It is the result of functional categories of genes that are differentially regulated by. Compared to wild type (WT) (H99) strainsch9A total of 313 genes with significantly different basal and temperature mediated increase / decrease expression levels in Δ mutants were classified as KOG function descriptions.
Figure 2 is the result of identifying a new temperature control signal transduction pathway in Cryptococcus neoformans. The temperature control pathway of Cryptococcus neoformans by CryptoNet was predicted (http://www.inetbio.org/cryptonet/). Nine transcription factors and 17 kinases were used to elucidate the temperature control signaling pathway. Links between genes for which functional correlations are predicted.
Figure 3 is a result of the heat shock protein (heat shock protein) and ergosterol (ergosterol) biosynthesis gene is regulated by the temperature rise. Figure 3A shows a wild type (WT) strain after raising the temperature from 25 ℃ to 37 ℃sch9In Δ strainHSP104 Gene expression The result shows that it is derived. Gene expression analysis using qRT-PCR was performed with wild-type (WT) (H99) grown at 120 ° C. to 37 ° C. for 120 min.sch9From whole RNA isolated from Δ strainHSP104 It was performed using specific primers.HSP104 Expression levelACT1 Corrected by expression. 3B shows that Hsp104 is required for growth even in extreme high temperature conditions (55 ° C.). Wild Type (WT),hsp104(YSB2261) and hsp104 deficiency repair strains (hsp104Δ +HSP104YSB4689) at 30 ° C. in YPD liquid medium. Incubate for 16 hours, serial dilution (1 to 10⁴ Dilution) and plated on YPD solid medium. YPD solid medium was incubated at 55 ° C. for the indicated time and photographed after further incubation at 30 ° C. for 2 days. Figure 3C is a result showing that the gene expression of ERG11 and ERG2 is reduced by the temperature rise. qRT-PCR showed wild type (WT) and temperature rise from 25 ° C to 37 ° C.sch9This was done using total RNA extracted from Δ strain. For qRT-PCRERG11 AndERG2 Expression isACT1 Corrected by expression. 3DERG11 ManifestationSch9 Results are shown to be induced independently of sterol depletion. qRT-PCR analysis showed fluconazole treatment (10 μg / mL, 90 minutes) or untreated wild flavor (WT) andsch9Total RNA extracted from Δ strain was used. using qRT-PCRERG11Expression analysis was wild type (WT) andsch9Of the total RNA extracted from the Δ strainERG11 It was performed using specific primers.ERG11 Expression levelACT1 Corrected by expression.
4 isHSF1The results show a partial decrease in Sch9 dependent expression as the temperature rises. 4AHSF1Results show that expression decreases gradually in a partially Sch9-dependent manner with increasing temperature. Logarithmic multiplier at OD (OD₆₀₀ Wild type (WT) grown to about 1.0) andsch9The Δ strain was further incubated at 37 ° C. or 40 ° C. and then total RNA was extracted at the indicated time points. Gene expression analysis using qRT-PCRACT1 By manifestationHSF1 Expression was corrected. 4B shows that Hsf1 protein levels also decrease in a Sch9-dependent manner in part with increasing temperature. For Western blot analysis, whole cell lysates were cultured in the same manner as in FIG. 4A.HSF1: 4xFLAG (YSB3160) and sch9ΔHSF1: 4xFLAG (YSB3338) was isolated from the strain. Western blot membranes were first developed in hybridization with FLAG antibodies. After deprobing, the same membranes were rehybridized with β-actin antibodies as loading controls. Protein expression of Hsf1 was applied to the control (0 hours) by correcting the value divided by β-actin expression (Hsf1 / β-actin) to 1.0. 4C shows the result of the electrophoretic mobile phase of Hsf1 with increasing temperature. For western blotsHSF1: 4xFLAG And sch9ΔHSF1: 4xFLAG Whole cell extract of the strain was used. Black arrows represent phosphorylated Hsf1-4xFLAG protein (P-Hsf1) and white arrows represent dephosphorylated Hsf1: 4xFLAG protein (Hsf1). 4D shows the results of the electrophoretic mobile phase of Hsf1: 4xFLAG due to temperature rise. For the preparation of whole cell extracts, the logarithmic multiplier (OD at 25 ° C.₆₀₀ I grew up to approximately 1.0)HSF1: 4xFLAG The strain was further incubated for 30 minutes at 25 ℃ or 37 ℃ and then sampled. Whole cell extracts were incubated at 30 ° C. for 1 hour with or without gamma phosphatase and phosphatase inhibitors. Western blot membranes were hybridized with FLAG antibodies. 4FHSP90This is a result showing that the Sch9 is induced independently by the temperature rise. The same RNA used in FIG. 4A was used for qRT-PCR using HSP90 specific primers. 4FHSF1Overexpression in Cryptococcus neoformansHSP90It is a result showing that it affects the expression of. For quantitative RT-PCRHSP90 Expression levelACT1 Corrected by expression.
5 isHSF1The results show that Cryptococcus is necessary for Neoformans' viability. 5A is P _CTR4 : HSF1 Schematic representation of a promoter replacement strain.HSF1 The structure in which the CTR4 gene promoter and the NAT cassette were inserted before the ATG start codon of the gene was illustrated. The letter H shown in the diagram was used for Southern blot analysis.HindIII restriction enzyme site. 5B shows the result that Hsf1 is required for the growth of Cryptococcus neoformans. Wild type strain (WT) (H99) and P _CTR4 : HSF1  (YSB2340, YSB2341, YSB2342 and YSB2343) strains were incubated for 16 hours at 30 ° C. in YPD liquid medium, and 200 μM vasocuproindaisulfonic acid (BCS) or 25 μM CuSO₄Inoculated in YNB solid medium containingHSF1 Growth was confirmed by expression induction or reduction. Incubated for 2 more days at 30 ° C. in YND solid medium and photographed. 5C shows a diploid Cryptococcus neoformans strain.HSF1 Schematic representation before and after gene target substitution. The function of the HSF1 type DNA binding domain was replaced with a NAT cassette. FIG. 5D is a specific primer for detecting only NAT cassettes (ALID2271-ai37 and ALID2272-ai270), heterozygotesHSFOfhsf1Two amplification products (2592 and 2452 bp, respectively) that are only amplified in Δ are the result of PCR analysis. 5E is a heterozygoteHSFOfhsf1Seven colonies from 46 spores isolated from Δ were grown in four media types for the gene isolation model. All progeny sensitive to nourseothricin hsf1 :: NAT Deleting strain indicates that it cannot grow, whichHSF1 It suggests that the gene is essential for the viability of Cryptococcus neoformans.
6HSF1 Overexpression of Cryptococcus neoformans This results in promoting heat resistance. 6A P _H3 : HSF1Is a schematic representation for the construction of overexpressed strains. Constructs containing a promoter of the histone H3 gene and a NEO drug resistance cassetteHSF1 The gene was inserted before the ATG start codon. The letter K shown in the plot is used for Southern blot analysis.KpnI restriction enzyme site. 6B is P _H3 : HSF1 Southern blot analysis for identification of strains. Wild type (WT) (H99) (lane 1), P _H3 : HSF1Genome DNA was isolated from YSB2200 (lane 2), YSB2201 (lane 3), and YSB2202 (lane 4) strains, and digested with Kpn1 and used for Southern blot analysis. 6C is P _H3 : HSF1 In strainHSF1This result is confirmed by Northern blot analysis. Wild type (WT) and P _H3 : HSF1 Total RNA of strains (YSB2200, 2201, and 2202 of lanes 1 to 3) were used for Northern blot analysis. The membrane is radiolabelledHSF1 Hybridization with specific probes (B and C). 6D isHSF1 Overexpression increases the heat resistance of Cryptococcus neoformans. Wild Type (WT),hog1Δ (YSB64),sch9Δ (YSB619 and YSB620), and P _H3 : HSF1 (YSB2200 and YSB2201) strains were grown in YPD solid medium. Cells were incubated for 3 days at the indicated temperature and photographed. 6EHSF1 Overexpressionsch9The results show that the heat resistance of the Δ strain is slightly increased. Wild Type (WT),hog1Δ (YSB64),sch9Δ (YSB619), P _H3 : HSF1 (YSB2200, YSB2201 and YSB2202), andsch9Δ P _H3 : HSF1 (YSB2269 and YSB2270) strains were incubated for 16 hours at 30 ° C. in YPD liquid medium, and serially diluted (1 to 10⁴Dilution), inoculated in YPD solid medium, and photographed after further incubation for 4 days at the indicated temperature. 6F shows HSF1 overexpressionHSP104 Results show that it promotes expression. Wild type (WT), P _H3 : HSF1 (YSB2200) andhsp104Total RNA of Δ (YSB2260) strain was used for Northern blot analysis. The membrane is radiolabelledHSP104 Hybridization with specific probes. 6GHSF1 OverexpressionSSA1 AndSSA2 Results show increasing the expression level. For quantitative RT-PCR (qRT-PCR),SSA1 AndSSA2Expression isACT1 Corrected by expression. 6H shows that Ssa1 is important for Cryptococcus neoformans heat resistance. Nocturnal (WT),ssa1Δ (YSB2235) andssa1Δ +SSA1 (YSB4702) strains were incubated in YPD liquid medium at 30 ° C. for 16 hours and serial dilution (1 to 10⁴ Dilution), and inoculated in YPD solid medium, photographed incubation for 2 days at the indicated temperature. Two phenotypic images separated by horizontal white lines were analyzed on the same medium.
7 shows that Hsf1 plays two roles in oxidative stress response and adaptation. 7AHSF1 OverexpressionSRX1 Results show that it reduces the level of basal expression. 7B is H₂O₂ViaSRX1Is a result showing that Hsf1 does not regulate the expression of. Log Season (OD₆₀₀ Wild type (WT) and P grown to about 1.0) _H3 : HSF1 (YSB2200, YSB2201) strain was 2.5 mM H₂O₂Further incubation for 30 minutes in YPD medium containing, and the total RNA was extracted for Northern blot analysis. 7CHSF1 The results show that overexpression increases susceptibility to oxidative stress. HSF1 (YSB2200 and YSB2201) andsch9Δ P _H3 : HSF1 (YSB2269 and YSB2270) strains were incubated in YPD liquid medium at 30 ° C. for 16 hours, serial dilution (1 to 104 dilution), and the indicated concentrations of H₂O₂was inoculated in YPD solid medium containing tBOOH or diamide. Solid medium was incubated at 30 ° C. for 2 days and photographed. Phenotypic images separated by horizontal white lines were analyzed in the same medium. 7DHSF1 Results show that expression is induced by peroxidative stress. Logistics at 30 ℃ (OD₆₀₀ Wild type (WT) grown to about 1.0) andsch9Δ (YSB619) strain 2.5 mM H₂O₂Further incubation for 30 minutes in YPD liquid medium containing 2.5 mM diamide or 0.6 mM tBOOH. The total RNA was extracted from cells treated with the reagents and untreated cells for Northern blot analysis. 7EHSF1 The results show that overexpression does not affect the virulence of Cryptococcus neoformans. Nocturnal (WT) and P _H3 : HSF1 Strains (YSB2200 and YSB2201) were used for the wax moth virulence assay. Phosphate buffered saline (PBS) was used as an uninfected control for injection.
8 is a result of the chromatin immunoprecipitation (ChIP) PCR analysis of the direct target gene of Hsf1. 8A is a heat shock protein (HSP78) And chaperone gene (STI1,SSA1,SSA2 AndKAR2Is a schematic representation of HSEs in the promoter region. Red letters on the sequence represent conserved Hsf1 binding motifs (nGAAn or nTTCn) and yellow letters represent low conservative Hsf1 binding motifs. HSE predicted by using the Clustal X2 software to align the promoter region sequence of each gene with a complete type, gap type or step type motif. 8BSTI1,SSA1,SSA2,HSP78 AndKAR2Is the direct target of Hsf1. Quantitative PCR (qRT-PCR) was performed in three replicates. 8C is the result showing that Hsf1 does not regulate its own transcription.
FIG. 9 is a schematic diagram illustrating a regulation mechanism of Sch9 and Hsf1 in the heat resistance of Cryptococcus neoformans. Cryptococcus neoformans Sch9 modulates heat resistance in an Hsf1 dependent and Hsf1 independent manner. In response to a rise in temperatureHSF1 Expression of the transcript is partially reduced by Sch9-dependent. However, the fact that Hsf1 protein expression is positively regulated by Sch9 under normal growth conditions suggests that Sch9 may be involved in Hsf1 stability. Hsf1 is required for growth and regulation related to oxidative stress response and heat resistance. Hsf1 can regulate both heat resistance, positive and negative. Hsf1 binds directly to the step-type thermal shock element (HSE) in the promoter region of the heat shock protein (HSP) even under unstressed conditions and this binding increases significantly at elevated temperatures. Hsf1 is known as a subgene of the UPR pathwayKAR2To increase the expression. The UPR pathway is also involved in the heat resistance of Cryptococcus neoformans. In conclusion, Sch9 is involved in heat resistance by modulating Hsf1, the UPR pathway, and other unknown HSP factors in Cryptococcus neoformans.
10 confirmed through Hsf1 chromatin immunoprecipitation (ChIP) -PCR analysis to build an Hsf1 target drug screening systemSSA1 Promoter (SSA1pro) andLacZ Reporter gene system was used.LacZ The plasmid was amplified from pUC19,SSA1 The promoter region was amplified from Cryptococcus neoformans (genomic DNA) by PCR using SSA1-specific primer combinations (B7240 / B7241 and B7197 / B7198). Cloning each PCR amplification product to plasmid pTOP-V2 (Enzynomics) PLacZ and pSSA1pro were prepared.EcoRV andNotFrom pLacZ by I cleavageLacZ Fragments were isolated and purified and subcloned into plasmid pNEO to produce pNEO-LacZ. 10A isolates the SSA1pro fragment and removes pSSA1proApaI andNotThis is a schematic diagram of cleavage with I restriction enzyme, purification and subcloning into plasmid pNEO-LacZ to generate pNEO-SSA1pro-LacZ. The product was introduced into the wild type (WT) (H99) strain by bioistic transformation. NEO resistant Cryptococcus neoformans strain was isolated. 10B shows that the Hsf1 binding activity for HSE of the SSA1 promoter increases with temperature change from 25 ° C. to 37 ° C. and shows that the added indicator (X-gal) shows a darker blue.
FIG. 11 shows the results of a summary of temperature control transcription factors and kinases of Cryptococcus neoformans. Growth data and in vivo toxicity data are from previous reports. RMS (relative median survival) is the mutant median survival day divided by the wild-type median survival day. RMS scores for two independent strains for each transcriptional regulator or kinase mutant are described as mean values.
Signature-tagged mutagensis (STM) analysis was carried out by infecting mice with a respiratory infection by mixing mutant strains with specific sequence tag binding. Lungs extracted after one day are lysed with PBS and incubated for 3 days at 30 ° C. in YPD solid medium containing chloramphenitol. After recovering the cells in the medium, the output genome DNA was extracted. In order to extract the input genome DNA, the mutant strains bound to the specific nucleotide sequence tag were mixed and incubated in YPD solid medium at 30 ° C. for 3 days, and cells were recovered from the medium and genome DNA was extracted. STM score refers to the ratio of values comparing delta Ct (ΔCt) of input and output genome DNA. STM score for each mutant strain is Log₂2^{- ΔΔC}It was calculated using the mutation of.
Figure 12 shows that the heat shock protein and ergosterol biosynthesis genes are regulated by temperature rise. 12AHSP104With these wild type (WT) cellssch9The results show that the cells are induced by increasing the temperature from 25 ℃ to 37 ℃ in Δ cells. Northern blot analysis showed wild type (WT) (H99) and temperature rise from 25 ° C to 37 ° C.sch9Performed using total RNA extracted from Δ (YSB619) strain. Each membrane is radiolabelledHSP104 Hybridization with specific probes. Ethidium bromide staining of rRNA was used as loading control. 12B shows the temperature riseERG11andERG2It is a result showing that the expression of is reduced. Northern blot analysis showed wild type (WT) and temperature rises from 25 ° C to 37 ° C.sch9Performed using total RNA extracted from Δ strain. Each membrane is radiolabelledERG11 orERG2 Hybridization with specific probes.
13 ishsp104The result of the preparation of the Δ variant. Figure 13Ahsp104Scheme of production of Δ mutations. K for Southern Brat AnalysisKpnI restriction enzyme cleavage site. Figure 13BKpnGenomic DNA digested with I andHSP104 By Southern blot analysis using specific probeshsp104This is the result of confirming the correct genotype of the Δ mutants (YSB2260, YSB2261, YSB2262 and YSB2263, respectively, labeled 1-4).
14 except for extreme thermal shockHsp104The results show that they are not involved in various environmental stress responses. Wild type (WT) (H99) strain, IΔ (YSB64) andhsp104Δ (YSB2260, YSB2261) strains were incubated for 16 hours at 30 ° C. in YPD liquid medium and serial dilution (1 to 10⁴And the phenotype was observed in YPD solid medium containing reagents. The medium was further incubated at 30 ° C. for 2 days and photographed. The heat resistance test was inoculated with the strain to YPD solid medium incubated for 2 days at 37 ℃ or 39 ℃ and photographed.
15 isHSF1The results show that this temperature is down-regulated depending on the Sch9. Figure 15AHSF1 Results show that expression levels decrease at high temperatures. Total RNA of the microarray used in Figure 1 was used for Northern blot analysis. Radioactively labeled membraneHSF1 Hybridization with specific probes. 15B shows the logarithmic (OD at 30 ° C.₆₀₀ Wild type (WT) (H99) grown to about 1.0) andsch9Δ (YSB619) strains were further incubated at 37 ° C. or 40 ° C. and then sampled for total RNA extraction at the indicated time points. Northern blot membranes are radiolabelledHSF1 Hybridization with specific probes. Ethidium bromide staining of rRNA was used as a loading control.
16 isHSP90This results in an upregulation upon temperature rise in a manner independent of Sch9. Logistics at 30 ℃ (OD₆₀₀ Wild type (WT) (H99) incubated with about 1.0) andsch9Δ (YSB619) strains were further incubated at 40 ° C. and then sampled for total RNA extraction at the indicated time points. Northern blot membranes are radiolabelledHSP90 Hybridization with specific probes. Ethidium bromide staining of rRNA was used as a loading control.
17 is P _CTR4 : HSF1Southern blot analysis of the strain. AndHindGenomic DNA cleaved with IIIHSF1P by Southern blot analysis using specific probes _CTR4 : HSF1The exact genotype of the strains (YSB2340, YSB2341, YSB2342 and YSB2343, respectively, represented by 1 to 4) was confirmed.
FIG. 18 shows that Ssa1 is important for the heat resistance of Cryptococcus neoformans of FIG. 11. Wild type (WT) (H99), ssa1Δ (YSB2235) and ssa2Δ (YSB2236 and YSB2237) strains were incubated for 16 hours at 30 ° C. in YPD medium, and serially diluted (1 to 10)⁴ Dilution), and drip into YPD agar medium, Further incubation for 2 days at the indicated temperature and photographed.
19 is a list of Cryptococcus neoformans used in the study.
20 is a list of primers used in the study.
21 is Cryptococcus is a list of chaperone proteins regulated by an elevated temperature of neoformans.
FIG. 22 is a list of heat shock proteins and chaperone-like genes controlled by elevated temperature of Cryptococcus neoformans.
FIG. 23 is a list of ergosterol biosynthesis genes regulated by elevated temperature of Cryptococcus neoformans.
FIG. 24 is a view of Cryptococcus neoformansHSF1A list of HSEs in the 1kb upper region of.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Strains and Culture Conditions

The strains and primers used in this example are shown in FIGS. 19 and 20. Unless otherwise indicated, the cells were cultured in yeast extract-peptone-dextrose (YPD) medium. Yeast nitrogen base (YNB) medium containing 25 μM CuSO ₄ or 200 μM bathocuproinedisulfonic acid (BCS) was used to reduce or induce HSF1 expression in P _CTR4 : HSF1 strain. Diploid Cryptococcus neoformans strain AI187 (ade2 / ADE2, ura5 / URA5 and MAT a / MATα) was used as parent strain to prepare heterozygous HSF1 / hsf1 mutations. This strain was produced through the fusion of two haploid nutritive elements of opposite mating type and maintained in YPD solid medium.

Total RNA Isolation and DNA Microarray Analysis

For DNA microarray analysis, total RNA was extracted as follows: wild type (WT) ( H99 ), sch9 Δ, ire1 Δ and hxl1 Δ mutant strains were incubated for 16 hours at 30 ° C. in 50 mL YPD liquid medium. Thereafter, 5 mL of overnight culture was inoculated with 100 mL of fresh YPD liquid medium and incubated for 5-6 hours at 25 ° C. until absorbance (OD) reached approximately 1.0 at 600 nm (OD ₆₀₀ = 1.0). did. For 0 hour sampling, 50 mL of 100 mL culture was taken. The remaining 50 mL of culture medium was pelleted by centrifugation and the supernatant was discarded. Preheated (37 ° C. or 40 ° C.) fresh YPD liquid medium was added to the remaining pellets and further incubated at 37 ° C. or 40 ° C. for 30 minutes. The culture was then pelleted by centrifugation, frozen in liquid nitrogen and lyophilized for 16 hours. As biological replicates for DNA microarrays, three independent samples for each strain were prepared for total RNA extraction. Total RNA was extracted with RiboEx reagent (Geneall Biotechnology) as described above. As a control, total RNA prepared from wild type and mutant cells was pooled (pooled reference RNA).

For DNA microarray analysis using constituent HSF1 overexpressing strains, total RNA was extracted as follows: wild type (WT) (H99) and constituent HSF1 overexpressing strains (P _H3 : HSF1 , YSB2200) in YPD liquid medium30 Incubated for 16 hours at ℃. 5 mL of this culture was reinoculated into 95 mL of fresh YPD liquid medium. At 600 nm, absorbance (OD) reaches approximately 1.0 (OD ₆₀₀ = 1.0) The culture was further incubated at 30 ° C. The culture was immediately centrifuged, frozen in liquid nitrogen and lyophilized for 16 hours. As biological replicates for DNA microarrays, three independent samples for each strain were prepared for total RNA extraction. Total RNA was prepared using easy-BLUE reagents (Invitrogen, Carlsbad, CA, USA) as described previously. As a control, total RNA prepared from wild type and HSF1 overexpressing strains was collected (gathered reference RNA).

For cDNA synthesis, total RNA concentration was adjusted to 1 μg / μl with DEPC treated water and 15 μl total RNA was used. The cDNA was synthesized using reverse transcriptase (Fermentas) and labeled with Cy5 / Cy3 labeling agent (Amersham). Cryptococcus neoformans antigen type D 70-mer microarray slides containing 7,946 probes (Duke University, NC, USA) were used for DNA microarray analysis. Of the 6,980 genes in the wild-type strain, 6,302 genes were matched to 6,756 spots (including multiple spots for a single gene) on the JEC21 gene chip, with e-values ranging from 1e ^{- 6} (90% coverage) through blast search. Each S. cerevisiae gene name or ID was identified by blast irradiation (e value range: e ^{- 6} ) using the antigenic A gene sequence. Three independent DNA microphone arrays with three independent biological replicates were performed. After hybridization and washing, the microarray slides were scanned with a GenePix 4000B scanner (Axon Instrument) using GenePix Pro software (version 4.0), and the Acuity software (version 4.0) and GenePix array list (Gal) files ( http : // genome. wustl.edu/activity/ma/cneoformans ). Data sent from GenePix software for hierarchical and statistical analysis uses LOWESS standardization, reliable gene filtering (95% filtering), hierarchical clustering, zero transformation, ANOVA analysis (P <0.05), and Microsoft Excel software (Microsoft). Were analyzed using Acuity software.

Construction of hsp104, ssa1, ssa2, hsp104 + HSP104 and ssa1 + SSA1 strains

The HSP104 gene (CNAG_07347) was destroyed by homologous replacement of the open reading frame with a DNA fragment containing a unique drug resistance gene marker. Primer pairs used for amplification of the 5-terminal and 3-terminal contiguous regions of the HSP104 gene were B5125 / B5126 and B5127 / B5128, respectively. NAT STM # 125 dominant marker was amplified with M13Fe / M13Re, a primer pair for HSP104 deletion. For the second PCR producing a continuous fragment, the hsp104 Δ :: NAT structure was amplified using the B5125 / B5128 primer pair, and three gels extracted the DNA fragment of the first round PCR into the template. Cryptococcus neoformans antigen type A MATα strain (H99) was biologically transformed with a deletion allele. To identify the desired hsp104 Δ variant, PCR was performed using primer pair B79 / B5124 and Southern blot analysis was performed using gene specific probes with primer pair B5125 / B5129.

SSA1 The gene (CNAG — 01727) was cleaved using the same approach as HSP104 . For first round PCR, SSA1 Primer pairs used for amplification of the 5-terminal and 3-terminal contiguous regions of the gene were B5135 / B5136 and B5137 / B5138, respectively. NAT STM # 169 dominant markers were amplified with primer pair M13Fe / M13Re. For nested PCR, the ssa1 Δ allele was amplified using B5135 / B5138 primer pairs on three gel extracted DNA fragments from the first round PCR. Cryptococcus neoformans wild type strains were biologically transformed with a deletion allele. To identify the desired ssa1 Δ variant, Southern blot analysis was performed using PCR using primer pair B79 / B5139 and gene specific probes amplified with primer pair B5135 / B5140.

The SSA2 gene (CNAG — 01750) was deleted as follows. For the first round PCR, the primer pairs used for amplification of 5- and 3-terminal contiguous regions of the SSA2 gene were B5219 / B5220 and B5221 / B5222, respectively. NAT STM # 177 dominant markers were amplified with primer pair M13Fe / M13Re. During the second round of nested PCR, the ssa2Δ construct was amplified using B5219 / B5222 primer pairs and three gel extracted DNA fragments from the first round PCR. Cryptococcus neoformans wild type strains were biologically transformed with a deletion allele. Southern blot analysis was performed using PCR using primer pair B79 / B5223 and gene specific probes amplified with primer pair B5219 / B5224 to identify the desired ssa2 Δ variant.

To complement the HSP104 Δ mutant with the wild type HSP104 gene, HSP104 (CNAG_07437) was PCR amplified by two fragments F1 and F2 with B7785 / B8001 and B8002 / B8003 primer pairs, respectively. Each fragment was cloned into a TOPCLoner TA vector to make pHSP104-F1 and pHSP104-F2 and confirmed by sequencing. HSP104-F2 delivered to Stu I and Xba I was cloned into pHSP104-F1 to prepare pHSP104. PJAF12-HSP104 was constructed by cloning Xho I and Xba I cleaved HSP104 into pJAF12. Plasmid pJAF12-HSP104 was linearized with Nsi I and introduced into hsp104 Δ (YSB2261) strain by biological modification. Ectopic insertion of HSP104 has been confirmed by PCR using the primer pair of B7789 / B8001.

To complement the ssa1 Δ variant with the wild type SSA1 gene, SSA1 (CNAG — 01727) was PCR amplified with fragments F1 and F2 using B7778 / B8004 and B8005 / B8006 primer pairs, respectively. Each fragment was cloned into a TOPCLoner TA vector to construct pSSA1-F1 and pSSA1-F2 and confirmed by sequencing. PSSA1 was constructed by cloning SSA1-F2 digested with Xho I and Xba I to pSSA1-F1. By cloning the SSA1 cut with Kpn I and Not I to prepare a pJAF12 pJAF12-SSA1. Plasmid pJAF12-SSA1 was cleaved with Nsi I and linearized and introduced into ssa1 Δ (YSB2235) strain by biolistic transformation. Ectopic insertion of SSA1 was confirmed by PCR using primer pair B7783 / B8004.

P _CTR4 : HSF1  Construction of Promoter Alternative Strains

Promoter replacement strain for HSF1 (CNAG_07460) was constructed as follows using a promoter of CTR4. As the first round of PCR, the 5-terminal contiguous regions (HSF1 promoter region spanning the region -756 to -1 compared to the ATG start codons (+1 to +3)) and 3-terminal contiguous regions (HSF1 gene, +1 to +971) was amplified with primer pairs B1892 / B5190 and B5193 / B5145, respectively. The bound NAT-CTR4 promoter DNA was amplified by PCR using two primers B354 and B355. During the second round of nested PCR, the P _CTR4 : HSF1 construct was amplified with B1892 and B5145 primers on three gel extracted DNA fragments in the first round. Cryptococcus neoformans wild type strains were biologically transformed with a deletion allele. Stable transformants were selected in YPD solid medium containing nourseothricin (100 ug / mL). P _CTR4 : HSF1 Strains were identified by screening PCR and Southern blot analysis using HSF1-specific probes amplified with primers B5144 and B5145.

Construction of Component HSF1 Overexpressing Strains

Component HSF1 overexpressing strains were prepared using histone H3 promoter of Cryptococcus neoformans antigen type D JEC21 strain. During the first PCR, 5-terminal contiguous regions [HSF1 promoter region] and 3-terminal contiguous regions (HSF1 gene, +, spanning from -756 to -1 with respect to ATG start codons (+1 to +3) 1 to +971) were amplified with primer pairs B1892 / B5190 and B5191 / B5145, respectively. NEO-H3 promoter fragments were amplified with primer pair B4017 / B4018. During the second round of overlap PCR, the P _H3 : HSF1 construct was amplified using primer pair B1892 / B5145. Cryptococcus neoformans wild type strains were biologically transformed with gel extracted DNA. Transformants were selected in YPD medium containing G418 (50 μg / mL). P _H3 : HSF1 strains were identified by Southern blot analysis using PCR and HSF1- specific probes amplified with primers B5144 and B5145. HSF1 expression levels in P _H3 : HSF1 strains were determined by Northern blot analysis using HSF1-specific probes amplified by PCR using primers B5067 and B5068.

HSF1 : 4xFLAG  Construction of Epitope Tagged Strains

HSF1: 4 × FLAG tagged strains were constructed using plasmids containing 4 × FLAG and NEO dominant selection markers. During the first round, primer pairs B5460 / B6141 and B6142 / B5740 were used to amplify the 5-terminal contiguous region (C-terminal region of HSF1) and 3 'contiguous region (HSF1 terminator region). 4xFLAG - NEO construct was amplified with primer pair B5431 / B1966. In the second overlap PCR, HSF1 : 4xFLAG : NEO Constructs were amplified using B5460 / B5740 primer pairs from three gel extracted DNA fragments extracted in the first round PCR. Wild type and sch9 Δ strains (YSB619, YSB620) were biologically transformed with the construct. Stable transformants were selected in YPD medium containing G418 (50 μg / mL). HSF1 : 4xFLAG and sch9Δ HSF1: 4xFLAG strains were confirmed by diagnostic PCR and Southern blot analysis . Production of the Hsf1 4xFLAG tag protein was performed with monoclonal anti-FLAG M2 antibody and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) produced in mice (Sigma-Aldrich, St Louis, MO, USA). Confirmed.

Hsf1 phosphorylation and λ phosphatase assay

HSF1: 4xFLAG strain was carried out in 50 mL of YPD liquid medium at 30 ° C. for 16 hours. Cultures were diluted 1/10 in 50 mL of fresh YPD liquid medium and incubated at 25 ° C. until OD ₆₀₀ reached about 1.0. 25 mL of the culture was incubated at 25 ° C. and the remaining 25 mL of the culture was incubated at 37 ° C. for 30 minutes. Cells were sampled by centrifugation and whole cell extracts were prepared with lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100 supplemented with protease inhibitor cocktails]. Samples were placed in PMP buffer [50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, 1 mM MnCl2 and λ protein phosphatase (New England BioLabs) 400 units (phosphatase inhibitor cocktail) 30 ° C. Incubated for 1 hour at. Proteins were analyzed on 8% SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad). Membranes were hybridized with mouse monoclonal FLAG M2 antibody (Sigma), then reacted with mouse IgG antibody bound with horseradish peroxidase and assayed with ECL Western blotting detection reagent.

Structure of HSF1 / hsf1 Mutants in Heterozygous

For target substitution of the HSF1 gene, the hsf1 :: NAT structure consisted of a 1.5kb flaking region fused with a NAT cassette via in vivo recombination of S. cerevisiae in the FY834 strain, and the Neurospora crassa And three DNA fragments, plasmid pRS426, a high throughput strategy used to generate gene replacement constructs in Cryptococcus neoformans. HSF1 5-terminal and 3-terminal contiguous regions were amplified using primers ALID2267-ALID-2268 and ALID2269-ALID2270 of Cryptococcus neoformans KN99α genomic DNA, and the NAT cassette was plasmid pAI3 with primer ai006-ai290. Amplified. Primers were designed to replace the 2017 bp region of the HSF1 gene, including the entire HSF type DNA binding domain. The construct was amplified from Saccharomyces cerevisiae transformants using primers ALID1229 and ALID1230 homologous to plasmid pRS426. Alternative alleles were precipitated in the gold beads and Cryptococcus neoformans strain AI187 was biologically transformed. The transformants are selected from YPD + nourseothricin (100 ug / mL), bind to screening primers (ALID2271 and ALID2272) located outside the homologous recombination region, and by PCR using primers specific for the NAT gene (ai37 and ai270). Screening for homologous recombination events. Primers used are shown in FIG. 20. The heterozygous strain HSF1 / hsf1 Δ was cultured in MS medium (Murashige-Skoog medium) to induce meiosis and spores. After incubation for 3 weeks in cancer conditions, 46 monocyte spore spores of the mixed population were inoculated into YPD solid medium. Four genetic markers [nourseothinin resistance (NAT ^R ) or susceptibility (NAT ^S ), which grow the colonies for 3-4 days at 30 ° C., transfer to 96-well plates containing 100 μL of YPD and separate during meiosis, ura5 / URA5, ade2 / ADE2 and MAT a / MATα] while adding 3 μl of the cell suspension to YPD + nourseothricin (100 μg / mL), YNB + adenine (20 mg / L) or YNB + uracil (40 mg / L) Tested. The hybrid marker was scored by crossing the haploid progeny on MS medium supplemented with adenine and uracil with the KN99 a and KN99α strains and assessing the carrier formation under a microscope.

Chromatin immunoprecipitation (ChIP) analysis

HSF1: 4xFLAG (YSB3160) strains were incubated for 16 hours and then diluted in two 50 mL cultures with an OD ₆₀₀ of 0.2. Cells were incubated at 30 ° C. for 3 hours. One culture was kept at 37 ° C., the other at 30 ° C. for 30 minutes. Formaldehyde was added at a final rate of 1% and incubated for 20 minutes at room temperature for crosslinking. Crosslinking was terminated by adding 125 mM glycine to the final concentration and incubating for 5 minutes at room temperature. The cells were pelleted and washed once with phosphate buffered saline (PBS) containing 10 mL of 125 mM glycine. The washed pellet was resuspended in 1 mL ChIP lysis buffer (50 mM HEPES-KOH, pH 7.5; 140 mM NaCl; 1% Triton-X 100; 1 mM EDTA) and contained glass beads washed with about 200 μl of acid. Transferred to bead beating tube. Cells were lysed by stirring three times in Mini Bead Beater 15 (BioSpec) for 1 min and cooling on ice for 1 min. The lysate was then transferred to a 15 mL conical tube and 1 mL of chip lysis buffer added. The lysates were then sonicated five times (30 sec ON / 1 min OFF) to cleave the DNA. The supernatant was then centrifuged at 20,000 × g at 4 ° C. for 10 minutes to transfer the supernatant to an Eppendorf tube and once again centrifuged to make the lysate clearer. The resulting supernatant was used for the ChIP experiment. 30 μl of the supernatant was aliquoted into new tubes, named Whole Cell Extract (WCE), and stored at 4 ° C. for the duration of the experiment. To prepare immunoprecipitated samples, 250 μl of lysate was added to 250 μl of cold chip lysis buffer and 120 μl of 3.6 M NaCl. 50 uL of Protein A Agarose (Pierce) beads were added to the sample and the mixture was spun at 4 ° C. for 1 hour while spinning to remove proteins that bind non-specifically with the beads. During this time, FLAG beads (EZview Red Anti-FLAG M2 Affinity Gel, Sigma-Aldrich) were blocked by incubating for 1 hour in PBS with 5% skim milk. The lysate containing agarose beads was centrifuged at 10,000 xg for 4 min at 4 ° C and the supernatant was transferred to a new tube. Nonspecific binding blocked FLAG beads were washed three times with chip lysis buffer and then resuspended in 100 μl. Blocked FLAG Bead Slurry 50 Μl was added to the lysate and incubated for 16 h at 4 ° C. with rotation. The next day, the beads were placed twice in 1 mL ChIP lysis buffer, once in 1 mL ChIP lysis buffer containing 0.5 M NaCl, 1 mL ChIP wash buffer (10 mM Tris-HCl, pH 8.0, 0.75 M LiCl; 0.5% NP -40, 1 mM EDTA) and then treated twice at 4 ℃ in 1 mL TE. Immunoprecipitated Hsf1-4xFLAG was eluted with 150 uL of ChIP elution buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS) and incubated at 65 ° C. for 10 minutes. The tubes were then centrifuged at 10,000 × g for 1 minute and 125 uL of the supernatant was transferred to a new tube. 95 μl of ChIP elution buffer was added to the WCE. The IP and WCE tubes were then incubated at 65 ° C. for 16 hours to break formaldehyde crosslinks. The next day, 5 μl Proteinase K (BioLine) was added to the sample and incubated at 37 ° C. for 90 minutes. Samples were purified using the Qiagen PCR purification kit and eluted in 50 uL of water according to the manufacturer's instructions (Qiagen, Hilden, Germany).

Honeycomb Moth Toxicity Analysis

Cryptococcus neoformans wild type and P _H3 : HSF1 to prepare for inoculation Strains (YSB2200 and YSB2201) were incubated for 16 hours at 30 ° C. in YPD liquid medium, washed three times with PBS, and then resuspended in PBS for cell counting with a hemocytometer. Honeycomb moth at the final larval stage ( Galleria mellonella ) Larvae were used within 7 days of shipment (Vanderhorst Wholesale Inc., St. Marys, OH, USA). Fifteen honeycomb moth larvae weighing 200-300 mg in each group were randomly selected. 4 of 10 ⁶ cells / mL (4,000 Cryptococcus neoformans cells / larvae) via terminal protopes of the larvae using a 100 μl Hamilton syringe equipped with a 10 μl needle and repeat dispenser (PB600-1, Hamilton). Μl was inoculated. PBS was injected into the uninfected control. After injection, the larvae were incubated in a culture dish in a humidified plastic container and observed daily. The larvae were considered dead when there was no movement when touched. Individuals who became pupa during the experiment were excluded from statistical analysis. Survival curves were calculated using GraphPad Prism 6 (GraphPad, San Diego, CA, USA) and statistically analyzed by Log-rank (Mantel-Cox) test.

Hsf1-targeted drug screening system construction

To construct the Hsf1-target drug screening system, the SSA1 promoter ( SSA1 pro) and LacZ reporter gene systems identified by Hsf1 chromosome immunoprecipitation (ChIP) -PCR analysis were used. The LacZ gene was amplified from plasmid pUC19 and the SSA1 promoter region was genome of Cryptococcus neoformans by PCR using primer pairs B7240 / B7241 (SEQ ID NOs: 2 and 3) and B7197 and B7198 (SEQ ID NOs: 4 and 5), respectively. Amplified from DNA. Each PCR product was cloned into plasmid pTOP-V2 (Enzynomics) to prepare pLacZ and pSSA1pro. LacZ fragments were isolated and purified from pLacZ by cleavage with Eco RV and Not I and subcloned into plasmid pNEO to generate pNEO-LacZ. SSA1 pro fragments were isolated and pSSA1pro was digested with Apa I and Not I to purify and subcloned into plasmid pNEO-LacZ to generate pNEO-SSA1pro-LacZ (SEQ ID NO: 1). The product was then introduced into wild type strains by bioistic transformation. NEO resistant Cryptococcus neoformans strain was isolated.

Example  One. Cryptococcus In Neoformans Sch9 -Dependent heat resistance Regulen  ( regulon To reveal Transcript  analysis

Comparative transcriptome analysis of antigenic type A wild type and sch9 mutants was performed while raising the temperature from 25 ° C. to 37 ° C. or 40 ° C. using a DNA microarray to reveal Sch9 mediated heat-resistant signaling mechanism in Cryptococcus neoformans. It was. Microarray data of wild type strains were analyzed to identify temperature control genes of Cryptococcus neoformans. The 1831 genes in the genes monitored by the DNA microarray exhibited significant different expression patterns during temperature changes from 25 ° C. to 37 ° C. or 40 ° C. (FIG. 1A) (ANOVA test, P <0.05). Of these, 1671 genes showed more than two-fold induction or reduction at 37 ° C (1691 genes at 40 ° C). The results strongly suggest that a significant portion of the Cryptococcus neoformans genome has been adjusted for different growth temperatures.

Next, we compared the transcription profile of the sch9 mutant with the wild type transformant profile during temperature upshifts to find Sch9 dependent genes and Sch9 independent genes. As a result of analyzing the transcription profile, the Sch9 expression level was very low in the sch9 mutant compared to the wild type strain (−5.713 at 25 ° C., −2.98 at 37 ° C., −6.614 at 40 ° C., log ₂ scale). There are three types of Sch9 dependent genes. The first class is genes affected by the basal expression level of Sch9. The second class is genes whose temperature induced or temperature inhibited expression levels were affected by Sch9. The third class is genes that affect both basal and temperature-induced / inhibited expression levels. For the comparison, the ANOVA test was first carried out by using the data of sch9 at 25 ℃ in wild-type, 37 ℃ in sch9 and 37 ℃ in wild-type, 25 ℃ and confirm the gene using P <0.05 cut off. A total of 2909 genes significantly affected expression in this state. We performed further analysis of these genes.

For the first time, the basic transcription profile (25 ° C) between wild type strain and sch9 mutant was compared. A total of 1032 genes (except SCH9 itself) showed more than two-fold induction (583 genes) or reduction (449 genes) in sch9 mutants compared to wild-type strains. The results indicate that Sch9 has a significant effect on the basal expression of various genes in Cryptococcus neoformans.

Next, the transcription profiles were shown to show an increase or decrease in expression at 25 ° C. to 37 ° C. between the wild type strain and the sch9 mutant. The genes were divided into four groups according to the expression levels, which were significantly increased by more than 2 times or less than 2 times in wild-type and sch9 Δ upon temperature rise; Two or more or more than two times significantly reduced groups. Approximately 76% of the genes (2218 out of 2909) in the wild-type and sch9 Δ strains showed similar expression patterns (Figure 1B). Expression of a total of 246 genes was reduced or induced in sch9 Δ mutants but not in wild type strains. In contrast, expression of 427 genes was reduced or induced in wild-type strains, but not in sch9 Δ mutant strains. Interestingly, 12 genes were derived from wild-type strains, but their expression was reduced in the s ch9 Δ mutant, while 6 genes were regulated in the opposite direction (Figure 1B). It was found that 18 genes are regulated by the Sch9 pathway. We searched for genes whose base and induction / decrease of transcription levels with increasing temperature are regulated by Sch9. To identify these gene categories, we compared the results of basal transcript profiles at 25 ° C. and transcript profiles induced or reduced upon temperature rise from 25 ° C. to 37 ° C. A total of 313 genes were identified in this group, including genes involved in post-translational and chaperone functions, signal transduction, carbohydrate metabolism and transport, or intracellular exchange and secretion (Figure 1C). The types of genes involved are as follows. Steen et al. Demonstrated that tags representing cyclophilin A ( CPA1 and CPA2 ) were expressed at higher levels at 37 ° C than 25 ° C. Microarray data also confirmed the above findings. Kraus et al . Found that expression of a number of genes, including SLG1 , MGA2 , CLC1 , RDS1 , SMG1 , TPS1 and RIM15 , was induced at 37 ° C and that expression of genes such as ILV5 , ILV2 and URA2 were inhibited at 37 ° C. In the present invention, it was found that SLG1 , MGA2 , RDS1 , SMG1 , TPS1 and RIM15 expression were induced during temperature rise, while ILV2 , ILV5 and URA2 expression were inhibited. Chow etc Many thermal shock genes ( KAR2 , HSC8202 , HSP104 , HSP78 , SSA4 , STI101 , SSE2 And LHS1 ) is demonstrated. Array analysis of the present invention confirmed that the expression of some heat shock genes including KAR2 , HSP104 , HSP78 was strongly induced by temperature changes, and HSP12 , SSB2 , SSC1 , SSA1 like genes (CNAG_01727), SSE1 , AHA1 , CDC37 and HSP10 also. The effect was shown. Chow et al. Reported that the expression of genes involved in ergosterol biosynthesis ( ERG3 , ERG4 , ERG502 , ERG25 and OSH6 ) was inhibited by heat. In the present invention, the expression of other ergosterol biosynthesis genes such as ERG2 , ERG5 , ERG6 , ERG8 , ERG10 , ERG11 , ERG13 , ERG20, and other ergosterol biosynthesis genes such as ERG2 , ERG4 , ERG25, etc. ( ERG3 , ERG4 and ERG25 ), ERG24 , NCP1 And MVD1 was confirmed to be suppressed at high temperatures. In conclusion, Sch9 was found to act as a master regulator of genes under basal and elevated temperature conditions.

Example 2 Identification of Heat-Resistant Controlled Signaling System in Cryptococcus Neoformans

Microarray data revealed that 1671 genes (25 ° C. to 37 ° C.) and 1691 genes (25 ° C. to 40 ° C.) were induced or reduced more than twofold at elevated temperatures in wild-type strains. To identify the heat resistant regulatory signaling system in Cryptococcus neoformans, the temperature control genes were compared with previous large transcription factors and kinase expression based studies. We found that 9 transcription factors and 17 kinases are not only regulated at the level of transcription by temperature rise but also involved in the heat resistance of Cryptococcus neoformans (FIG. 11). Most of these transcription factors (8 of 9) and kinases (14 of 17) genes were also involved in Cryptococcus neoformans virulence or infectivity. This suggests that genes involved in adaptation to temperature rise are critical for Cryptococcus neoformans virulence. CryptoNet (http://www.inetbio.org/cryptonet/), a stochastic functional gene network of Cryptococcus neoformans, has recently demonstrated the interconnection of transcription factors and kinases that regulate heat resistance. 2) It was found that several signal transduction pathways cooperate to enhance the heat resistance of pathogens. This includes reported signal paths such as Hog1 and Mpk1 MAPK, calcineurin, Rim101 / Nrg1, UPR and RAM paths (Figure 2). Interestingly, a new heat-resistant regulatory pathway, the NAD kinase pathway, was discovered in Cryptococcus neoformans. Saccharomyces cerevisiae has three NAD kinases: Utr1, Yef1, and Pos5. Utr1 and Yef1 are known as cytoplasmic NAD kinases and Pos5 is known as mitochondrial NAD kinase. The inventors found that UTR1 and POS5 are more than twice induced at elevated temperatures and require UTR1 and POS5 when grown at high temperature in Cryptococcus neoformans (FIG. 11). YEF1 was also induced by temperature rise but was not involved in the heat resistance of Cryptococcus neoformans (FIG. 19).

Example  3. Cryptococcus Neoformans  Diverse Molecules for High Temperature Adaptation Chaperon  And Thermal shock  Protein Induction Confirmation

DNA microarray data showed that the genes whose expression was most dramatically regulated during temperature upshift belonged to the molecular chaperone group (FIG. 21). Molecular chaperones are proteins that not only play a role in mediating the proper folding of newly synthesized polypeptides under physiological conditions, but also help prevent protein denaturation or refold the denatured proteins under stress conditions. Microarray data showed that the cytoplasmic, endoplasmic reticulum-, and mitochondria-resident molecular chaperone genes were all upregulated at elevated temperatures (FIG. 21). Genes encoding heat shock proteins (Hsp) Hsp104 (CNAG_07347), Ydj1p / Hsp40 (CNAG_03944) and Ssa1 / Hsp70 (CNAG_01750) as cytoplasmic molecular chaperone complexes were upregulated during temperature upshifts. Genes encoding the ER resident molecule chaperones LHS1 (CNAG_03899) and KAR2 (CNAG_06443) were also upregulated to support previous findings that the UPR pathway of ER is activated in response to temperature rise. Interestingly, protein refolding in the endoplasmic reticulum is known to require cytoplasmic chaperone Hsp104. In addition, mitochondrial resident molecular chaperone gene SSC1 (CNAG_05199) was also induced (FIG. 21). Sch9 appears to be involved in basal expression levels of some of these molecular chaperone genes but does not significantly regulate induction upon temperature rise (FIG. 21). Of these genes, HSP104 was the most expressed gene and was selected for verification of microarray data. It was found that HSP104 expression was strongly induced within 10 minutes upon temperature rise and that induction was maintained for at least 2 hours (FIG. 3A). HSP104 was shown to show transcripts that were selectively conjugated at elevated temperatures (FIG. 12A). HSP104 expression was also induced in sch9 mutants with increasing temperature, but rapid induction was slightly delayed, indicating that Sch9 plays a small role in HSP104 induction. To determine whether Hsp104 plays a role in heat resistance, we removed HSP104 from Cryptococcus neoformans (FIG. 13) and analyzed the hsp104 mutant phenotype. Interestingly, the hsp104 mutant did not show growth defects at 25 ° C. and 39 ° C. and was as resistant to most environmental stress and antifungal agents as tested in the present invention (FIG. 14). However, the hsp104 mutant was much more susceptible at extreme temperatures (55 ° C) than the wild type strain (Figure 3B). Complementing the hsp104 mutant with the HSP104 gene of the wild-type strain restores heat shock resistance, suggesting that Hsp104 is required for the response and adaptation of Cryptococcus neoformans to severe thermal shocks rather than mild thermal stresses. In addition to this cell molecule chaperone, many genes encoding other HSP and chaperone-like proteins have been identified as temperature control genes of Cryptococcus neoformans (FIG. 22). This includes HSP10 , HSP12 , HSP60 , HSP78 And HSP90 is included. Similar to the regulation of molecular chaperones, Sch9 was primarily involved in regulating basal expression levels of many HSP genes (FIG. 22). In conclusion, the various cellular molecules chaperone and HSP are both upregulated during the high temperature adaptation process of Cryptococcus neoformans.

Example  4. against temperature rise Cryptococcus Neoformans Ribosomes Translation and Confirmation of Ergosterol Biosynthesis Gene Suppression

The microarray data of the present invention showed that the expression of genes involved in ribosomal biosynthesis and translation decreased significantly during temperature rise (Figure 1A). In addition, most of the ergosterol biosynthesis genes were significantly down regulated during temperature up-shifts in both wild type and sch9 mutant strains (FIG. 23). The transcript levels of ERG11 and ERG2 were assessed by qRT-PCR and Northern blot analysis to confirm this result by showing a significant decrease in expression levels in wild-type and sch9 mutant strains during temperature rise (Figures 3C and 12B). Previously, sch9 mutants have been reported to be more susceptible to azole drugs such as fluconazole and ketoconazole compared to wild type strains. We next assessed whether ERG11 induction for azole treatment was due to Sch9. Similar levels of ERG11 expression induced by fluconazole in wild type strains and sch9 mutants were observed (FIG. 3D). In conclusion, ergosterol biosynthesis is strictly regulated in temperature upshifts and sterol-depleted conditions in a Sch9 independent manner.

Example  5. Cryptococcus In Neoformans  As a temperature control gene Thermal shock  Factor 1 ( Hsf1 )and Of Hsp90  Sympathy

The most notable but unexpected result in the array analysis is that the gene encoding HSF1 (CNAG_07460) was down regulated upon temperature rise of Cryptococcus neoformans (FIG. 22). Hsf1 is a transcription factor that coordinates transcriptional activation and synthesis of HSPs in response to environmental stress. Hsf1 is known to be expressed as a component in eukaryotes of most tissues and cell types, and is primarily regulated by post-translational mechanisms. Thus, downregulation of HSF1 with temperature rise in Cryptococcus neoformans is a counter-intuitive and surprising result as many HSP and chaperone genes appear to be upregulated during temperature rise as described above. In contrast, HSP90 (CNAG_06150.2), a putative molecular chaperone that can be controlled by Hsf1, was induced by temperature rise.

To confirm gene expression results in microarray experiments, we performed a Northern blot analysis. Consistent with the data, HSF1 was down regulated during temperature rise (37 ° C. to 25 ° C. or 25 ° C. to 40 ° C.) of wild type strains (FIG. In sch9 mutants, HSF1 was not regulated more down, especially at elevated temperature, especially at 40 ° C. (FIG. 15A). To further analyze the expression pattern of HSF1 , we monitored HSF1 expression at different time points during the temperature rise (Figure 4A). HSF1 in the wild type strain was confirmed to be incrementally adjusted downward after moving up the temperature from 25 ℃ to 37 ℃ (Fig. 4A). In the sch9 mutant, HSF1 had lower baseline expression levels of the mutant than the wild type but was down regulated with increasing temperature (Figure 4A).

To test whether Hsf1 protein levels decrease similarly to HSF1 transcript levels during temperature rise, strains expressing the epitope marker Hsf1 ( HSF1: 4xFLAG ) in wild-type and sch9 backgrounds were generated and western blot analysis was performed. Hsf1 protein levels also decreased slowly with temperature rise (up to 50%) and were maintained in the HSF1: 4xFLAG strain (Figure 4B). Interestingly, sch9 Δ Hsf1 protein levels in the HSF1: 4xFLAG strain were slightly lower at baseline conditions (about 70%), but sch9 Δ There was no significant change in the temperature rise of HSF1: 4xFLAG . The data show that Sch9 plays a role in HSF1 down regulation during temperature rise but may be required for HSF1 mRNA stability under basal conditions.

To determine whether Hsf1 is phosphorylated during temperature rise, we monitored the electrophoretic mobile phase change of Hsf1: 4xFLAG by Western blot analysis. We have Hsf1: 4xFLAG protein HSF1: 4xFLAG and sch9Δ HSF1: 4xFLAG It was found that in both strains the mobile phase decreased after the temperature rise (FIG. 4C). This suggests that Hsf1 is phosphorylated with increasing temperature. In the sch9 Δ mutant, Hsf1 phosphorylation appeared to be slightly delayed. To determine if the mobile phase reduction of Hsf1: 4xFLAG was due to phosphorylation, Hsf1: 4xFLAG, which was immunoisolated in the presence or absence of a phosphatase inhibitor, was incubated with λ phosphatase and analyzed by Western blot analysis. The reduced mobile phase of Hsf1: 4xFLAG due to temperature rise was reversed in λ phosphatase but not in λ phosphatase with phosphatase inhibitors (Figure 4D). In conclusion, Hsf1 is phosphorylated with increasing temperature of Cryptococcus neoformans.

One well known Hsf1 target gene in other organisms is HSP90 . HSP90 transcription is activated by Hsf1, which is negatively regulated by interaction with Hsp90 as a self-regulating loop. Interestingly, HSP90 expression was found to be induced rapidly in both wild-type and sch9 mutant strains by temperature rise, which is in stark contrast to the HSF1 expression pattern (FIG. 4E and FIG. 16). Overexpression of HSF1 did not affect the basal expression level of HSP90 , but increased the induced expression level of HSP90 with increasing temperature (FIG. 4F). These results suggest that increased amounts of phosphorylated Hsf1 protein in response to high temperatures can enhance HSP90 induction. Taken together, the data of the present invention show that the expression of HSF1 and HSP90 is differentially regulated upon temperature rise of Cryptococcus neoformans.

Example 6. Hsf1 Investigation of the growth of Cryptococcus neoformans

It is not yet clear whether Hsf1 promotes or inhibits the heat resistance of Cryptococcus neoformans. Given the fact that HSF1 is downregulated during temperature upshifts, Hsf1 can inhibit genes associated with Cryptococcus neoformans heat resistance. In this case, although Hsf1 is known to be essential for the viability of most eukaryotes reported so far, HSF1 destruction can increase Cryptococcus neoformans heat resistance. Thus we attempted to destroy the HSF1 gene in Cryptococcus neoformans. However, repeated attempts to delete HSF1 were unsuccessful, suggesting that HSF1 is indispensable for the growth of Cryptococcus neoformans. Two complementary strategies were carried out to confirm the necessity of Hsf1. First, conditional null HSF1 mutations were generated using a copper regulated promoter of the CTR4 gene (P _CTR4 : HSF1) as previously described (FIGS. 5A and 17). P _CTR4 : HSF1 under the induction of specific copper chelator bathocuproinedisulfonic acid (BCS) Strains as well as wild type strains were grown (FIG. 5B). However, under the inhibition conditions by CuSO ₄ , the growth of P _CTR4 : HSF1 strain was severely impaired, indicating that Hsf1 is critical for the viability of Cryptococcus neoformans (Figure 5B).

The second approach is to generate a heterozygous hsf1 Δ / HSF1 strain in the diploid background, and then analyze haploid progeny derived after meiosis for the presence or absence of NAT- dominant markers. To generate heterozygous HSF1 / hsf1 Δ, the hsf1 Δ :: NAT deletion allele was generated according to a recently reported strategy, and the diploid strain AI187, well characterized in Cryptococcus neoformans, was transformed by biological mutation. It became. Expression of the mutated copy of the target gene and HSF1 is shown in FIG. 5C. Nourseothricin resistant (NAT ^R ) transformants were screened by PCR for correct homologous recombination events, and one transformant that produced two amplification products of expected size was selected as heterozygous hsf1 Δ / HSF1 strain ( 5D). Mutations of the heterozygotes were grown in spore derived media for classical genetic analysis of the resulting offspring. 43 spores were dissected and seven germinated were tested for four markers (NAT ^R or nourseothricin susceptibility (NAT ^S ), ura5 or URA5, ade2 or ADE2, MAT a or MATα) that were separated upon meiosis. All seven offspring could not grow on selection medium containing nourseothricin (Figure 5E), confirming that the HSF1 gene is required for Cryptococcus neoformans viability.

Example 7. Confirmation of Cryptococcus neoformans heat resistance promotion of Hsf1

When Hsf1 acts as a Cryptococcus neoformans heat resistant activator, overexpression can improve pathogen viability at high temperatures. In order to solve the above hypothesis, the inventors found that the histone H3 promoter (P _H3 : HSF1) is active as a constituent. Strains; 6A and 6B) were used to construct HSF1 overexpressing strains. HSF1 overexpression in the P _H3 : HSF1 strain was confirmed by Northern blot analysis (FIG. 6C). Surprisingly, HSF1 overexpression significantly increased the growth rate of Cryptococcus neoformans at high temperatures (Figure 6D) and strongly suggests that Hsf1 acts as a heat-resistant activator of Cryptococcus neoformans. In addition, mutants sch9: HSF1 overexpressed in (P _H3 HSF1 sch9Δ) is which further increases the heat resistance of the sch9 mutant (FIG. 6E).

To further study the positive role of Hsf1 in Cryptococcus neoformans heat resistance, we determined whether HSF1 overexpression can induce the expression of temperature response genes. To this end, we selected HSP104 and two SSA1 like genes. Expression of these genes is most induced by elevated temperatures (Figure 21). Northern blot analysis showed that HSP104 expression was induced by HSF1 overexpression (FIG. 6F), suggesting that Hsf1 promotes HSP104 expression and is required for heat shock resistance. Cryptococcus neoformans contains two SSA1 like genes. One is CNAG_01727 and the other is CNAG_01750. Adler et al. Initially characterized CNAG_01727 (Ssa1) heterologous to Hsa70 member protein Ssa1 in Saccharomyces cerevisiae. Interestingly, Ssa1 is a laccase DNA binding transcriptional co-activator of Cryptococcus neoformans. CNAG_01750 also encrypt Ssa1 like proteins such as Cryptosporidium in SSA2 caucus's only neo-Fort. To distinguish between SSA1 and SSA2 transcriptional regulatory patterns, qRT-PCR was performed with primer sets specific to each gene (FIG. 6G). Basal SSA1 expression levels were much higher than that of SSA2 (FIG. 6G). To address the role of Ssa1 and Ssa2 in Cryptococcus neoformans heat resistance, we generated ssa1 Δ and ssa2 Δ variants. Interestingly, SSA2 expression has been shown to be increased in the absence of SSA1 (in ssa1 mutations), suggesting that Ssa2 may complement Hsp70 function in Cryptococcus neoformans. Both SSA1 and SSA2 expression was strongly induced in the P _H3 : HSF1 strain, suggesting strongly that Hsf1 promotes the expression of the Hsp70 gene (Figure 6G). The ssa1 mutant showed very strong temperature sensitivity (FIG. 6H and FIG. 18), and the ssa1 + SSA1 recovery strain restored wild-type levels of heat resistance, suggesting that Ssa1 is the major Hsp70 of Cryptococcus neoformans. Taken together, Hsf1 can promote Cryptococcus neoformans heat resistance by inducing two major HSPs , Hsp104 and Ssa1.

Example 8 Gene Identification in Response to Temperature and Oxidative Stress of Hsf1 Activators and Inhibitors

To further elucidate the downstream network hosted by Hsf1, we performed additional DNA microarray analysis using HSF1 overexpressed strains. We found that a total of 722 genes were differentially regulated to statistically significant levels (P <0.05, ANOVA). 56 of these genes are P _H3 : HSF1 More than two-fold induction or reduction was seen in the strain. Sixteen of the 56 Hsf1 reactive genes are heterologous in Saccharomyces cerevisiae. Microarray data also support that Hsf1 has the dual function of inhibitors and activators of genes required for Cryptococcus neoformans heat resistance. Many genes expressed by temperature rise (25 ° C. to 37 ° C.) were down regulated by HSF1 overexpression. This includes HSP12 , a low molecular weight heat shock protein. In particular, expression of SRX1 encoding sulfiredoxin was downregulated by HSF1 overexpression. We have recently shown that SRX1 is rapidly induced by peroxidative stress and is required for the reuse of redoxin peroxide (Tsox1) and oxidative stress response and adaptation. qRT-PCR analysis confirmed that basal SRX1 expression levels were reduced by HSF1 overexpression (FIG. 7A). However, peroxide-dependent SRX1 induction was normally observed in P _H3 : HSF1 strains such as wild type strains (FIG. 7B), indicating that Hsf1 only inhibits basal expression of SRX1 . The P _H3 : HSF1 strain was confirmed to be resistant to H ₂ O ₂ and tBOOH similarly to the wild type strain (FIG. 7C). Nevertheless, HSF1 overexpression further exacerbated the resistance of sch9 mutants to H ₂ O ₂ (FIG. 7C). In addition, HSF1 overexpression increased sensitivity to thiol specific oxidants, diamides (FIG. 7C), indicating that Hsf1 plays a negative role in the oxidative stress response. However, we also found that HSF1 expression was actually induced by oxidative stress (FIG. 7D). The fact that HSF1 overexpression decreases the resistance to oxidative stress is due to P _H3 : HSF1 It means that the investigation should be carried out as to whether the strain has weakened virulence. In the insect host model system using honeycomb moth ( Galleria mellonella ) (set at 37 ° C), the PP _H3 : HSF1 strain showed wild-type toxicity (Figure 7E), and HSF1 overexpression affected Cryptococcus neoformans toxicity. Indicates that it did not.

Example  9. Hsf1 is Cryptococcus In Neoformans  Identification of conserved or poorly conserved HSE binding in the promoter region of genes controlled by temperature.

In eukaryotic cells, Hsf1 binds directly to heat shock elements (HSEs) that are conserved in the promoter region of the target HSP gene. To date, three types of HSE have been reported: Perfect (P)-, Gap (G)-and Step (S) types. P-type HSE consists of an nGAAn unit consisting of at least three contiguous inverse repeats (eg, nTTCnnGAAnnTTCn). G-type HSE has two inverted nGAAn units, followed by another unit after a 5 bp gap (eg, nTTCnnGAAn (5 bp) nGAAn). S-type HSE consists of three direct repeats of nTTCn (or nGAAn) with 5 bp inserted. To determine whether Cryptococcus neoformans Hsf1 can bind with known HSE, we examined the promoter region of the Hsf1 target gene identified by DNA microarray analysis and HSE studies in Saccharomyces cerevisiae. Four of these genes ( SSA1 , SSA2 , HSP78 , KAR2 ) had an S-type like HSE and one gene ( STI1 ) had Perfect (P) or Gap (G) like HSE (FIG. 8A). Only SSA2 had highly conserved S-type HSE (gTTCtcgagagTTCtggtggaTTCg). To test whether Hsf1 interacts with these binding sites, chromatin-immunoprecipitation (ChIP) was performed on a Cryptococcus neoformans strain expressing Hsf1-4xFLAG , followed by a primer adjacent to the estimated HSE. Quantitative PCR was performed. In particular, Hsf1 was shown to bind to the promoter region of four genes with S-type HSE ( SSA1 , SSA2 , HSP78 and KAR2 ) even at 30 ° C. (FIG. 8B). However, increasing the temperature to 37 ° C dramatically increased its binding capacity, suggesting that Hsf1 binds directly to the target gene (Figure 8B). Even Hsf1 binding ability to STI1 in which Perfect / Gap-type HSE is present was increased despite the low binding capacity due to temperature rise. In contrast, Hsf1 did not bind nonspecific genes (ACT1 and β-tubulin genes) at 30 ° C. or 37 ° C., indicating that Hsf1 binding is specific for its binding element. In conclusion, Hsf1 was found to bind strongly to S-type HSE in Cryptococcus neoformans than Perfect (P) or Gap (G) HSE.

Next, we investigated whether Hsf1 regulates its transcription to explain why HSF1 expression is downregulated during the temperature rise of Cryptococcus neoformans. Candida albicans (Candida In albicans ), Hsp90 interacts with Hsf1 and negatively regulates Hsf1 activity. Thus, increased expression of Hsp90 may inhibit Hsf1, which reduces the induction of HSF1 . To solve this hypothesis, we first identified whether the promoter region of HSF1 contains HSE. By aligning using Clustal X2, several conserved HSEs were found in the 1 kb upstream region of HSF1 (Figure 24). Perfect (P) -type 1 (-394 to 382), reverse sequence Perfect (P) -type 1 (-795 to -783), Gap (G) -type 3 (-134 to 117), Step ( S) type (-197 to 175) and reverse sequence Step (S) -type (-410 to -388) were local well-conserved HSEs. We have identified the location of adjacent genes in the genome and excluded these presumed HSEs from being located on the coding sequence of adjacent genes. By comparing the position of the HSF1 promoter region with the position of the HSE, we found that two HSEs, Gap (G) -type 3 and Step (S) -type HSE, are located within the HSF1 promoter region. Chromatin-immunoprecipitation using primers for the promoter region (-229 to -130) of HSF1 to cover HSE and gap type HSE to determine whether Hsf1 can bind to HSEs; ChIP) was performed. Hsf1 does not appear to bind in the promoter region with the HSE at both 30 ° C. and 37 ° C. conditions (Figure 8C). In conclusion, HSF1 expression did not appear to be automatically regulated during temperature rise in Cryptococcus neoformans.

Example 10. Hsf-1 Target Drug Screening System.

To identify compounds that disrupt the DNA binding and / or transcriptional activity of Hsf1, we constructed an Hsf1 target drug screening system with a LacZ reporter system (strains containing pNEO-SSA1pro-LacZ) (FIG. 10A). Hsf1, a transcription factor essential for the viability of Cryptococcus neoformans, binds to the thermal shock element (HSE) in the SSA1 promoter found by ChIP-PCR analysis using the Hsf1-FLAG tag protein. When HSF1 binds to the HSE of the SSA1 promoter, LacZ gene was expressed in the system and blue colonies appeared in the presence of X-gal due to β-galactosidase activity (FIG. 10B). We have previously shown that the Hsf1 binding activity of the SSA1 promoter to HSE increases with temperature changes from 25 ° C to 37 ° C (Figure 10B). Thus, the expression of the LacZ gene is increased and the colonies produce darker blue at 37 ° C (Figure 10B). Compounds that inhibit Hsf1 binding or Hsf1 trans activating activity make the colony or liquid culture white or pale blue (Figure 10B). The screening system thus makes it possible to identify new compounds that modulate Hsf1 (compounds that inhibit or activate Hsf1), especially since the pNEO-SSA1pro-LacZ system is colorimetric and facilitates efficient high-speed mass screening.

<110> Industry-Academic Cooperation Foundation. Yonsei university <120> Screening system for Hsf1 tageting drug using Hsf1 promoter-LacZ          reporter system of Cryptococcus neoformans <130> 1061687 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 3359 <212> DNA <213> Artificial Sequence <220> <223> the NEO-SSA1pro-LacZ fragment of pNEO-SSA1pro-LacZ <400> 1 tctagaggat ccgcatgcag gattcgagtg gcatggtgtg cactgagtgt atggttgtcg 60 gaggagagga tgatggtaac aacaatagca gcaacgtcac tcgacgcgcg tccggtgtgc 120 cacacggggt aacgccgagt cgccgtcagg gtcgccgaga ccactctcac agcgtcaccg 180 ttggcaccag ctcagcttac agcttctatc ctccgccagc atccacatac atcccctata 240 ccgcatcccc cacccactgc ccaaggtgag tcatcttccc gcccccttcc cttgcccgcc 300 actcagtcct ccatcctcca ctaatccacc ttatcgcacc caccgcctat cgcacatccg 360 agcacaatgc tgggcctgcc aggggctgct agatggtgct ctccccacgc tgatctgcat 420 gccggccatt ggatcatggg tgctaggtgc tgggtgctgg atgttggatg ctggatgctg 480 ggtgcacgct tggtcatttc cttccaggat tgacggtcgc cgagaggacg acgtggcgtt 540 cgacaacgag ggccgatagc accgcatcgc ctcgacctgc atccatctcg ccttgtcctt 600 ttggtgcaac aatccatccg tgctggtgcc acacgcatag ctggaagaga tggatgtgcg 660 ttgaacagag ctgccgtcag gactttttgg tgcacggacc ctattgtcct ccccaatctt 720 caccgcgtct cctaatatgc agcctctttg ctaattgtct ttttccatta gtaaactcgc 780 ccaacatgtc tatgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg 840 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt 900 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc 960 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt 1020 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag 1080 tgccggggca ggatctcctg tcatcccacc ttgctcctgc cgagaaagta tccatcatgg 1140 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag 1200 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg 1260 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc 1320 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca 1380 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc 1440 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg 1500 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct 1560 atcgccttct tgacgagttc ttctgagtga aggcggtaag gggttaattt tccttagagg 1620 gtgtatatat acatattaga gaagtgatac aattttagca cactgcgaat tcgagacaga 1680 catcgtgtca atcatctttt ttgacattat atgcccatta atctatctac agacaacaat 1740 accatccttc ccaccctcag caacgccgtt gaatcctcag gatcttcatg gctccggatc 1800 ctctagaaag ggcgaattct gcagatacac tatgcggcat cagagcagat tgtactgaga 1860 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg 1920 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 1980 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca 2040 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattcgagct cggtacccgg 2100 ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catgcggccg 2160 ctttgtctat ttatctatta aagctttgag tgtaagggaa tgtaatgaga ttttttgtca 2220 acaaatgaaa aggtgaagag aagaaagaga gaaaaagaaa gggaatataa atacggaaat 2280 cgacggaagg cgggggaagg gaggggaagc ttgtcgtcac tggtggtagg gagagccacg 2340 aattccgggg acttcttccc ccttctcgca ccaaatcgaa tgtggaggca aagcatcgaa 2400 aatgccagaa caatcgcgtc ttatgtgggg gatcgcataa cgacggccat gtccaccgcg 2460 gcgggcaccg ccgctttgct tcttcgttca ttcatttaat tgatgactgc atcatcagaa 2520 gagaccgcaa accgacacac cggcaagact gctacttctt taccacatca agcaacaccc 2580 agcaacttca gaagggccat tcaatgcaga aatcaaatat agtaaaggta caacatatta 2640 tcgcctggag cccttcttct tcttcttccc gtcctcatca gagtcacctc tgtctgtcct 2700 gtgcttctcg tcggctggtt ggtaaatccc cgtcagcttt atgctcacac atcaatgacg 2760 gtatcgaatc ttcactcacc cctcttagct ttacgttcaa gctcgtccca atcttcaccc 2820 tcatcagaga ggctctcagc agtacctgag tcggaatcgc cctcgactat gataagtcag 2880 caaaagtaat cagacccaag caggatacac tgacagtcag attcgctatc ctcgtcactg 2940 ccgctggatt catcaaagac atcactatcg ccttcaaatt cagagccttc ctcagattcc 3000 tctgacccat cgtcctagat cgatcgtcag agttgctcaa caaaaaagga taaagtggta 3060 atcttacaga gcctgaacca gtgaggaaat tccatccgcc ctccgcgtag aacgcatggg 3120 ggtcttcgtt gacagtcttc atgatcgctg gccaagaaag attgacaggg ccttcggata 3180 tcggcacatc acaagagcta gatggagtta gttacggaag ataggcatgt gggtattaca 3240 ggcactactc actcaagcca ctccttcacg ttgtcaaggt gcgcaactgg gatggaattg 3300 atatgaatag gcggcttctt gaggtcttgg agtacaaaaa ccatatcaaa attgggccc 3359 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification for SSA1 promoter region named          B7240 <400> 2 gcggccgcat gaccatgatt acgccaag 28 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification for SSA1 promoter region named          B7241 <400> 3 gatatcctat gcggcatcag agcaga 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification for SSA1 promoter region named          B7197 <400> 4 gggcccaatt ttgatatggt ttttgt 26 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplification for SSA1 promoter region named          B7198 <400> 5 gcggccgctt tgtctattta tctattaa 28

Claims (8)

(a) transforming the bacterium into a recombinant vector comprising a SSA1 promoter, LacZ and pNEO;
(b) treating the transformed bacteria with antimicrobial candidates;
(c) determining that the candidate is an antimicrobial agent when it is determined that the transformed bacteria treated with the candidate material have fewer blue colonies than the transformed bacteria not treated with the candidate material;
Screening method for antimicrobial agents showing antimicrobial activity against Cryptococcus neoformans (Cryptococcus neoformans) comprising a. delete The method of claim 1, wherein the transformed bacteria is Cryptococcus neoformans. delete (a) transforming the bacterium with a recombinant vector comprising an SSA1 promoter, LacZ and pNEO;
(b) treating the transformed bacteria with antimicrobial candidates;
(c) when it is determined that the transformed bacteria treated with the candidate has fewer blue colonies than the transformed bacteria not treated with the candidate, the candidate is a pharmaceutical for preventing or treating brain diseases. Determining that it is a composition;
Screening method of the pharmaceutical composition for the prevention or treatment of meningitis comprising a. The method of claim 5, wherein the transformed bacteria is Cryptococcus neoformans (Cryptococcus neoformans), the screening method of the pharmaceutical composition for the prevention or treatment of meningitis. delete delete

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