Analysis of part of the Trichophyton rubrum ESTs

Science in China Ser. C Life Sciences 2004 Vol.47 No.5 389- -395389Analysis of part of the Trichophyton rubrum ESTsWANG Lingling', MA Li", LENG Wenchuan', YANG Jian2, ZHU Junping', DONG Jie',XUE Ying', WAN Zhe', LI Ruoyu3 & JIN Qi11. State Key Laboratory For Molecular Virology and Genetics Enginering, Beijing 100176, China;2. Chinese National Human Genome Center, Beijing, Beijing 100176, China;3. Research Centre for Medical Mycology, Beijing 100034, ChinaCorrespondence should be addressed to Jin Qi (email: zdsys @ sina.com)Received May 30, 2003; revised August 30, 2003Abstract Trichophyton rubrum (T. rubrum) is the most common of the superficial fungi. In aneffort to better understand the genetic and biochemical makeup of T. rubrum, we generatedcDNA libraries from 3 growth stages and used these to isolate 4002 unique expressed sequencetags (ESTs). Sequence comparisons with the Genbank database allowed 1226 of the ESTs to beassigned putative functions or matched with homologs from other organisms. Of the remainingESTs, 989 were only weakly similar to known sequences and 1787 had no identifiable functions,suggesting that they represent novel genes. We further analyzed the presence of several im-portant genes involved in the growth, metabolism, signal transduction, pathogenesis and drugresistance in T. rubrum. This information was used to newly elucidate important metabolic path-ways in T. rubrum. Taken together, our results should form the molecular basis for continued re-search on the physiological processes and pathogenic mechanisms of T. rubrum, and may leadto a better understanding of fungal drug resistance and identification of new drug targets.Keywords: Trichophyton rubrum, functional genomics, cDNA library, expressed sequence Tags, drug targets.DOI: 10.1360/03yc0107Superficial fungi are the etiologic pathogens ofclinical drugs are not particularly effective; therefore itvarious dermatophytoses, such as tinea capitis, tineais important that we study the pathogeneses of super-corporis, tinea inguinalis, tinea manus, tinea unguiumficial fungi and use this data to develop new therapeu-and tinea pedis. These widespread infections affect uptic agents and identify novel drug targets.to 25% of the world's population, a percentage thathas continued to increase in recent years. T. rubrum is .To date, functional fungal genomics have notthe most common superficial fungus, accounting for atbeen extensively studied; fungal genomes containleast 60% of all infections of this type. T. rubrum canvarious non-coding regions, including regulatoryinduce dermatophytoses in different parts of the hu-components, introns and repeat sequences, makingman skin, and can also cause deeper infections such asthese genomes much larger and more complicated thankerion, abscesses and granulomas. Superficialthose of bacteria. Thus, the sequencing of entire fungalfungi-induced dermatophytoses are obstinate andgenomes has not proven sufficient for elucidation ofreadily relapse and re-infect. The commonly usedthe o\中国煤化工and expression pa-YHCNMHG* These authors contributed equally to this work.tPresent address: Institute of Molecular Immunology, the First Military Medical University, Guangzhou 510515. China.Copyright by Science in China Press 2004390Science in China Ser. C Life Sciencestterns of these organisms.The mixtures were centrifuged and the supermatantAdams et al. used EST analysis as a new apwas discarded. The pelleted mycelia were washedproach for studying the functions of genes in organ-twice with PBS.isms, including those with completely sequenced ge-1.3 Isolation and purification of total RNA andnomes. With the continued progress of the human gmRNAnome project, the use of EST technology has spread toTotal RNA and mRNA were isolated and purifiedmany fields, including homolog identification, genefollowing the protocols of the RNeasy Plant Mini Kitexpression profiling and novel gene detection. ESTand the Oligotex mRNA Mini Kit， respectivelyanalysis techniques have accelerated the identification(QIAGEN). .and analysis of novel genes, hence facilitating researchon various eukaryotic organisms.1.4 Construction of the cDNA libraryHere, we report the construction and analysis of 3The cDNA libraries were constructed followingT. rubrum cDNA libraries derived from various de-the protocols of the SUPERSCRIPT M Plasmid Sys-velopmental stages. We obtained 4002 first-passtem with GATEWAYTM Technology for cDNA Syn-unique ESTS and used sequence analysis and databasethesis and Cloning (Invitrogen).searching to identify known genes, define putative1.5 Isolation and purification of plasmids, and se-novel genes, and propose some possible metabolicquencing of clonesnetworks present in this superficial fungus. These re-The cDNA plasmids were isolated by the Milli-sults may provide the molecular basis for further re-search on the metabolism, pathogenesis and drug re-pore method using MADV filter plates. Sequencingwas performed with a generic T7 primer located 5'sistance of T. rubrum.upstream of the inserted segments, following the pro-1 Methods and materialstocol of the PRISM Big Dye Terminator Kit on an.1 Preparation of strains and materialsABI3700 automated sequencer.T. rubrum (strain BMU01672) was isolated from1.6 Bioinformatic analysesnail scraps of a patient suffering from tinea unguium.(i) Sequence data processing. The trace filesThe patient was seen at a clinic in the West City Sec-from the sequencer were basecalled by Phred with thetion of Beijing, China, in October of 2000. Strain ref-quality value set at >Q15. Then, the cross_ matcherence samples were stored at the Research Centre forsoftware from the Phrap package (ttp://www.phrap.Medical Mycology, Beijing. The strain was confirmedorg) was used to remove vector sequences. The polyAas T rubrum by morphologic identification, as well astails were clipped from some of the ESTs with thby PCR amplification and sequencing of the 18S ri-Trimmest program from the EMBOSS package.bosomal DNA and ItS regions. The potato glucoseagar, yeast extract, peptone and D-glucose used for the(i) EST clustering and re- assembly. Processedstrain cultures were bought from Difco.ESTs longer than 200 bp were clustered by comparingall base pairs using BLASTN, and collcting those1.2 Preparation of cell cultureswith >95% identity over regions longer than 40 bpPotato glucose agar (39 g/L) was inoculated withwith unmatched overhangs. <20 bp. The sequencesa few hyphae of T. rubrum and incubated at 27.5°Ccontained in each cluster were assembled using CAP3for 2~3 weeks. The hyphae were then placed in 100to identify the consensus ESTs.mL aliquots of YPG medium (10 g/L yeast extract, 20中国煤化工lstruction of the meta-g/L peptone, 10 g/L D-glucose) and incubated in a boclustered EST con-27.5C bath shaker for 7, 10 and 14 days, respectively.serTYHC N M H Gd with potential func-Analysis of part of the Trichophyton rubrum ESTs391tions through homologous comparisons by BLASTXassociated with 1226 ESTs could be assigned putativesearches of the GenBank non-redundant (NR) proteinfunctions, or had identifiable homologs in other or-database. ESTs were further classified according to theganisms (~42% had homologies exceeding 60%). Ad-NCBI Clusters of Orthologous Groups of Proteins da-ditional 989 ESTs were identified as weakly corre-tabase. The metabolic pathway networks of T. rubrumsponding to a known function or homolog, and 1787were partially reconstructed by searching for knownESTs had no identifiable function and were thought topathway homologs found in the Kyoto Encyclopediarepresent novel genes.of Genes and Genomes database.2.3 The analysis of possible signal transduction sys-2 Resultstems2.1 cDNA library constructionExamination of the genes and gene families iden-"hree cDNA libraries were constructed astified in this work revealed that a number of the iso-representing different growth phases (7 d, 10d and 14lated ESTs represent genes involved in signal trans-d). A total of 13038 plasmids were sequenced in oneduction (table 2). We identified members of severaldirection, yielding 4002 ESTs after cluster analysis.signal transduction pathways found in mammalianThe ESTs ranged in length from 202 to 3237 bp, withanimals, such as factors involved in the mito-an average length of 810 bp. The identified ESTsgen-activated protein kinase (MAPK) pathway. Fur-summed to 3241620 bp in total, with a G+C% ofTable 2 Some possible genes involved in Trichophylon rubrum signaltransduction53. .40%.Gene numberProductsE value2.2 EST comparison with the NR databaseF4. _012. _54b Serin/threonine protein kinases1.00E-61F3_ _003B. _70 Serine/threonine protein kinases5.00E-14The ESTs were analyzed by comparison with theF3_ 028B_ 84 Signal transduction histidine kinase5.00E-18NR database, and the encoded putative proteins wereF3_ _041B_ 16 Signal transduction histidine kinase2.00E-72classified in COGs (table 1). The predicted proteinsS008R_ .15Serine/threonine protein kinases2.00E-50F3_ _033B. 91Diadenosine traphospatase and related 9,00E-27Table 1serine/threonine protein phosphatasesInformation storage and processing___ Number PercentS0I0R_ 025.00E-20Translation, ribosomal structure and biogenesis148 3.71%F3_ 012B. _73 PAS/PAC domain6.00E-20Transcription2.71%F3_ 076B_ 88regulatory subunit of cAMP-dependent1.00E-10DNA replication, recombination and repair14 1.10%protein kinasesCellular processesF3. _080B. 52 Serine/threonine protein kinases5.00E-17Cell division and chromosome partitioning10 0.25%Diadenosine tetraphosphatase and relatedPostranslational modification, protein turnover,F3_ _049B_ _039.00E-16)8 2.46%chaperF3_ 060B. _74 Signal transduction histidine kinase2.00E-24Cell envelope biogenesis, outer membrane32 0.80%F3_ _044B. 37Phosphate starvation-inducible protein2.00E-47Cell motility and secretion40 1.00%PhoH, predicted ATPaseInorganic ion transport and metabolism1243.11%F3_ _081B_ 18 Signal transduction histidine kinase1.00E-23Signal transduction mechanisms79 1.98%F3_ 072B_ 92 Signal transduction histidine kinase2.00E-14MetabolismF3_ _093B. _84Predicted integral membrane sensor1.00E-41Energy production and conversion124 3.11%domainCarbohydrate transport and metabolism112 2.81%F3_ 053B_66 CheY like reever domain8.00E-20Amino acid transport and metabolism209 5.24%F3 _071B. _67RIO-like serin/threonine protein kinase 3.00E-29Nucleotide transport and metabolism370.93%F4_ _001 _67b PAS/PAC domain5.00E-26Coenzyme metabolism57 1.43%Diadenosine tctraphosphatase and relatedLipid metabolism611.53%F4_ _003_ 48b .2.00E-68Secondary metabolites biosynthesis, transport32 2.06%F4_ 009. _62b Serine/threonine protein kinases1.00E-21and catabolismPoorly characterizedF5. 0051- steins5.00E-40General function prediction only989 24.81%F5_ 0061中国煤化i8.00E- 42Function unknown1787 45.19%C0368_YHCNMHGnase1.00E-79392Science in China Ser, C Life Sciencesthermore, the T rubrum cDNA libraries contained aare closely associated with fungal drug resistance (ta-large number of signal transduction histidine kinases,ble 4). These genes included the cation/multidrug ef-suggesting that this fungus may contain theflux pump, the ABC multi- drug transport system andtwo-component signal transduction system (TCST),members of the major facilitator superfamily.which is not known to be present in mammalian ani-Table 4 Some possible drug resistance genes in Trichophyton rubrummals.Gene numberProductsE value2.4 The analysis of metabolic pathways in T. rubrumC0210. ContiglABC-type mulidrug/protein/lipid1.00E-30transport systemThe identifiable ESTs were used to assemble pu-F4_ _015_ _04bABC-type multidrug/protein/ipid2.00E-23tative metabolic pathways by bioinformatic methods.transpport systemThe T. rubrum cDNA libraries contained genes en-S004R. _735.00E-14coding putative proteins involved in 113 metabolicF3_ _022B. _70ABC-type mulidrug/protein/ipid3.00E-45pathways (table 3), including the near-universal path-F3_ 038B. 56 .7.00E-31ways for metabolizing saccharides, amino acids, andlipids, as well as other fungi-specific pathways such asF3_ _068B. 811.00E-19sterol metabolism and the Glycosylphosphatidylinosi-F3_ 088B_ _653.00E-20tol (GPI) anchor protein synthesis pathway, which iscrucial to cell wall assembly.F3_ 071B 931.00E-17ABC- type multidrug/protein/lipidTable 3 Some possible metablism pathways in Trichophyton rubrumF4_ 009_90b7.00E-21MapMetabolism pathwayNumber Total PercentF4. _042b. _02ABCtype mutidru/prtein/ipid1.00E-12map00190 Oxidative phosphorylation1286%transpon systemC0421 ContiglMultidrug resistance eflux pump5.00E-16map00563 soyosypalidylinositoio267%(GPI)-anchor biosynthesisC0536_ .Contigl Multidrug resistance eflux pump9.00E-54map0062 Fatty acid biosynthesis563%F4_ 007 _36bMultidrug resistance efflux pump8.00E-27(path 2)F3_ 005B. _70 .Cation/mulidrug efflux pump4.00E-12AminoacyI-tRNAmap00970biosynthesis1362%F3_ _011B. 58Cation/multidrug eflux pump1.00E-44map00020 Citrate cycle61%F3_ 065B. 05Cation/mutidrug efflux pump5.00E-31map00540 Lplysacaride60%F3_ 045B. 48Cation/multidrug fflux pump1.00E-52F3. _085B. 691.00E-66map001 30 Ubiquinone biosynthesis57%C0679_ Contigl1.00E-64map00720Reductive carboxylate cycle354%Permeases of the major faciliator(CO2 fixation)F3_ _037B _35Valine, leucine andsuperfamilymap00290isoleucine biosynthesis853%map00632Benzoate degradation63 Discussionvia CoA ligationmap00710 Cartbon fixation2352%Fungi can be divided into monocellular and fila-map00010 Glycolysis/Gluconeogenesis2050%mentous fungi. T. rubrum (a member of Trichophyton,map00440 Aminophosphonate47%metabolismHyphomycetales，Hyphomycetes， Fungi Imperfecti)Selenoamino acid metabo-45%belongs to the latter group, which differs from thelismmap00071 Fatty acid metabolism43%monocellular fungi in cell components and modes ofmap00251 Glutamate metabolism42%propagation. Current fungal research has primarilymap00100 Sterol biosynthesis25%focused on monocellular yeast and two model fila-mentous fungi, Emericella nidulans and NeurosporaI filamentous fungi, T.2.5 Analysis of the multi-drug resistance genes中国煤化工supeficial fungi group,In the T. rubrum ESTs, we identified the presenceYHC N M H Gque physiological proc-of various drug efflux genes (E<1.0X 10~-"), whichesses. With the recent advances in functional genomics,Analysis of part of the Trichophyton rubrum ESTsit has become possible to study various organisms atcluding components of the 80S ribosome, and genesthe whole-genome level. Because superficial fungi areinvolved in DNA replication, RNA transcriptionalboth numerous and clinically relevant, we suggest thatregulation, and protein translation initiation, elonga-it is of great importance that T. rubrum be examined attion and termination. We additionally identified en-this level in the hopes that we will be able to identifyzymes related to cell wall synthesis, such as chitinasepathogenic mechanisms and/or new drug targets, thusand β-glucan synthetase. These enzymes, which canaccelerate the research on fungi.degrade old cell wall components and synthesize newHere, we report for the first time the construtioncell walls, are closely related to the growth of fila-of 3 cDNA libraries representing different growthmentous fungi. The presence of these enzymes in Tphases of T. rubrum, (7d, 10d and 14d, respectively).rubrum confirms the hypothesis that the main ele-From these libraries, 13038 cDNA clones were se-ments of the filamentous fungal cell wall are hexosequenced and clustered to yield 4002 unique ESTs. Theand/or aminohexose-based polysaccharides; this dif-ESTs were then functionally classified by COG, whichfers from the cell walls of gram-positive andrevealed possible functions for these genes in signalgram-negative bacteria, wherein the cell walls consiststorage and processing, and allowed us to elucidateof peptidoglycan and lipopolysaccharideslipoids. Thissome of the physiological and metabolic processes thatdifference may be of important therapeutic relevance.occur in T. rubrum.As T. rubrum does not share cell wall compo-In addition to a large number of structural genesnents with bacteria, and mammalian animal cells dorelated to chromosome replication, transcription andnot possess cell walls, the components of the cell wallmay thus become the main target of antifungal agents.protein translation, the isolated ESTs included fragIn addition to the enzymes mentioned above, we ana-ments of important functional genes involved inlyzed other proteins related to cell wall synthesis andgrowth and metabolism, signal transduction, patho-found ESTs encoding members of physiological proc-genesis and drug resistance. Indeed, we were able toesses that are critical to fungal cell wall assembly,identify members of 113 metabolic pathways. Thesesuch as the GPI anchor protein synthesis pathway. GPIincluded the near-universal pathways for the metabo-anchor proteins exist extensively in eukaryotic cells,lism of saccharides, amino acids and lipids, as well asand function to attach proteins to the surfaces of allfungispecific pathways such as ergosterol synthesiseukaryotic cell, plasma membranes and cell walls.metabolism. Through these novel identifications, ourMost of the gene families involved in this pathway arework will not only help researchers elucidate themechanism of pathogenesis and identify proper drughighly conserved, with the exception of the Gpi7 fam-targets, but also greatly promote the understanding ofily, which seems to be fungus-specific. In the ESTsidentified herein, we identified homologs to severalT. rubrum propagation and metabolism.proteins involved in the GPI anchor protein synthesisBut, our understanding of other filamentous fungipathway, suggesting that this pathway exists in T. ru-is limited. Even the total fungal gene database is muchbrum. The identification of the crucial enzymes in thissmaller than other organisms, such as bacteria, plantspathway, especially members of the GPI 7 family, mayand mammalian animals etc. Therefore, it is difficultbe useful in identifying new drug targets related to theto elucidate the entire biology of T. rubrum, sincecell wall')!many of these ESTs had no identifiable homologsT. rubrum is a humanphilic fungi. Its regulatoryand/or functions.systems are significantly less complicated than thoseCompared to prokaryotes such as bacteria, fungiof mar: that the growth andhave some clearly eukaryotic characteristics that aremainte中国煤化工e rglated by hostreflected in our data. Our isolated ESTs included manyspecififYHC N M H Gdition requirements.genes shared between T. rubrum and eukaryotes, in-Thus, this fungus may only respond to external stimu-Science in China Ser C Life Sciences394lation in order to maintain survival. This hypothesis isthat for ergosterol metabolism and synthesis. The keysupported by our EST analysis. Parts of the T. rubrumenzyme in the pathway, 14a sterol demethylase, is thesignal transduction systems identified in our ESTtaget of azoles, which have bee widely used forpools are less elaborate than those found in Homotreatment of fungal infections.sapiens, suggesting that I rubrum contains fewer di-T rubrum is one of the major human pathogenicferent transduction pathways and regulatory modes.superficial fungi. Therefore it is of great importance toInterestingly, some of the identified pathways arestudy the virulence facors and pathogenic mecha-similar to the corresponding pathways in bacteria andnisms of this fungus. It has been reported that infec-some plants. For example, we identified a number oftion is tiggered by the adherence of T. rubrum arthro-signal transduction histidine kinases, suggesting that aspores upon contact with the horny layer of the skin.TCST system may be the major response regulatoryThe spores then grow and the mycelia form. The lon-systems in T rubrum. TCST systems serve as the prin-gitudinal growth of hypha can penetrate into the deepcipal mechanisms for coordinating responses to envi-part of the homy layer, while breadthwise growth canronmental changes in bacteria and some plants. Theseaggravate skin damage. During the ifection process T.systems typically consist of manydifferent sensoryrubrum secretes proteinases that degrade skin proteinshistidine kinases and response regulators, which reactsuch as keratin, collagen and elastin. This degradationto ertraellular stimulation by assembling new cellnot only provides the nutrtion required for fungalwall. These systems have also been recently reportedgrowth and metabolismn, but also helps the infectionin some pathogenic fungi, such as Arerius fumiga-invade into deeper tsses. These secreted proteinasesus4s5 and Candida albicansh?l, suggesting thatTCSTare considered the major criteria for determining themay be one of the major signal transduction systems invirulence factor of a fungust* -". However, expressionfungi. Further resarch on these systems will help usof proteinase encoding genes in T rubrum has notto understand the interaction between T. rubrum andbeen previously investigated. In the 4002 ESTS iso-its human host, and may provide important clues to thelated in this study, we identified a great number ofstudy of other fungi.secreted proteins including cllagenase; we believeOne of the possible reasons why fungi require athat further study of our cDNA libraries will revealstrict growth environment is the specificity of theiradditional important virulence-associated genecs andmetabolic substrates. Our EST analysis revealed theperhaps lead to the development of new therapeuticpresence of a number of membrane transporters, in-strategies.cluding: 1) sugar-transportingproteins such aCurrent antifungal agents may be limited by twosugar-binding proteins, fucose pemease, and proteinscommon problems. Firs, fungi are eukaryotes, andthat transport mono- and oligo saccharides; 2) ABCthus share many cell structures and metabolic proc-type transporters, which participate in amino acidesses with the host clls. Thus, drugs may damage hosttransport; and 3) proteins involved in ransportingcells due to the unsatisfactory selectivity of an anti-DNA synthesis precursors. These resuts may indeedfungal agent. Fortunately, this problem can be over-provide new information on some of the importantcome by seking new drug targets. Second, with ex-environmental, metabolic and growth requirements oftensive use of the various antifungal agents, drug re-T. rubrum. Indeed, we used a variety of bioinformaticsistant pathogenic strains may develop.techniques to identify 113 metabolic pathways repre-sented in our ESTS. Most of these are physiologicalThe mechanisms by which fungi obtain drug re-metabolic pathways responsible for growth mainte-sistance are very comolicated. In addition to conven-中国煤化工gical adaptation to anti-nance, such as glycolyis, amino acid synthesis, lipidacid synthes and coenzyme syothesis pathways. Inc N M H G and R plasmid transduc-adio we ietd fnigsecie pahways such as tion, Curent rsarch also sggests that fingal drngAnalysis of part of the Trichophyton rubrum ESTS395resistastance; may be closely related to drug efflux pumps2. Kitagaki, H, Wu, H., Shimoi, H., Ito, K., Two homologous genes,12. 131. These pumps can efctively remove drugs fromDCWI (YKL046c) and DFG5, are essential for cell growth andthe fungal cells, leading to non-lethal intercellularencode glycosylphosphatidylinositol (GPl)-anchored membraneproteins required for cell wall biogenesis in Saccharomyces cerre-drug concentrations and subsequent drug resistance.visiae, Mol. Microbiol, 2002, 46(4): 1011-1022.This mechanism is also common in bacteria and tumor3. Richard, M, De Groot, P.. Courtin, 0. et al, GPI7 afectscells. There are two kinds of drug efflux pumps: ATPcell-wall protein anchorage in Saccharomyces cerevisiae andbinding cassette transporters (ABCT), and members ofCandida albicans, Microbiology, 2002, 148(P1 7): 2125--2133.the major facilitator superfamily (MFS). Members of4. Clemons, K. V., Miller, T. K, Selitennikoff, C. P. et al, fos-l, aboth of these families were identified in the isolated T.putative histidine kinase as a virulence factor for systemic asper-rubrum ESTs, suggesting that this fungus may acquiregllosis,s Med. Mycol, 2002, 40(3): 259- -262.5. Pott, G B.. Miller, T. K.. Bartlet, J. A. et al. The isolaion ofdrug resistance through drug efflux pump mechanisms.FOS-I, a gene encoding a putative two component histidineThis indicates that it may be promising to developkinase from Asperilus fumigatus, Fungal Genet. Biol, 2000,drugs that inhibit the drug efflux pumps; further re-31():55- 67.search on the regulatory proteins involved in drug re-6. Slitenikof, C. P, Alex, L. Millr, T. K. et al, COS1, a puta-sistance and the corresponding regulatory mechanismstive two-component histidine kinase of Candida albicans, is anwill be necessary to address this possibility.vivo virulence factor, Med. Mycol, 2001, 39(1): 69- -74.7. Torosantucci, A., Chiani, P., De Bemardis. F. et al, Deletion ofIn summary, although superficial fungi are thethe two-component histidine kinase gene (CHKI) of Candida al-major human pathogenic fungus, our knowledge aboutbicans contributes to enhanced growth inhibition and klling bythis organism is still very limited. T. rubrum is a goodhuman neutrophils in vitro, Infect. Immun., 2002. 70(2): 985-model for the study of superficial fungi; our work in987.identifying ESTs in T. rubrum cNDA libraries will8. Samdani, A. J, Dykes, P. J, Marks, R.. The proteolytic activity ofstrains of T. mentagrophytes and T. rubrum isolated from tineafacilitate a better understanding of the molecularpedis and tineca unguium infections, J. Med. Vet. Mycol, 1995.mechanisms of its growth, metabolism, pathogenesis33(3): 167-170.and drug resistance. In addition, our work may aid in9. Apodaca, G, McKerrow. J. H. Expression of proteolytic activitythe identification of novel effective drug targets andby cultures of Trichrophyton rubrum. J. Med. Vet. Mycol, 1990.new antifungal agents. It is anticipated that this re-28: 159- -171.search will bring the study of superficial fungi, and10. Apodaca, G. McKerrow, J. H., Regulation of Trichophyton ru-brum proleolytic activity, Infect. Immun, 1989, 57(10): 3081-fungi as a whole, to a new level.3090.Acknowledgements This work was supported by The National High1I. Asahi, M. Lindquist, R., Fukuyama. K. et al.,. Purification andTechnology Research and Development Program of China (Grant No.characterization of major extracllular proteinases from Tricho-2001AA223021) and National Key Technologies R&D Programmephyton rubrum, Biochem. J..1985, 232(1): 139- -144.(Grant No. 2002BA711A14).2. Parkinson, T. Falconer, D. J. Hitchcock, C. A.. Fluconazole伦Referencessistance due l0 energy-dependent drug eflux in Candida glabrata,Antimicrob. Agent Chemother. 1995, 39(8): 1696- - 1699.， Fontaine, T.. Magnin, T. Melhert, A. et al, Structures of the gly-13. Denning, D. w., Venkaleswarlu, K, Oakley, K. L. et al, Ira-cosylphosphatidylinositol membrane anchors from Aspergillusfumigatus membrane proteins, Glycobiology, 2003,13(3): 169-conazole resistance in Aspergillus fumigaus, Antimicrob. Agent177.Chemother, 1997. 41(6): 1364- -1368.中国煤化工MYHCNMHG