The Preliminary Research Of Malate Dehydrogenase Of Candida Albicans

BackgroundWith the wide use of antibiotics, antitumor agent and glucocorticoid,especially in transplant patients or AIDS patients,the infections causedby fungus has dramatically increased.Candida albicans is the most commonhuman opportunistic pathogenic fungus and the most significant pathogeneof nosocomial infection.The report of resistance to azole antifungalagents became increased,it was severely challenge to control the fungusinfections effectively.The study of azole antifungal resistancemechanism and the development of new antifungal drug or vaccine areeffective way to solve problem.In a study of antifungal resistance mechanism,the Saccharomycescerevisiae, C.albicans is exquisitely sensitive to glucose,regulatingcentral metabolic genes even in response to0.01%glucose, increased theresistance to an azole antifungal agent.C.albicans is an intracellularpathogen which causes disseminated,progressive and life threateninginfections.During infection,C.albicans in macrophages is considered tobe in a poor environment in nutrients,which can’t employed tricarboxylicacid cycle carbon metabolism to maintain survival.In systematic screeningof differentially expressed proteins in an infection model withmacrophages during the infection process in candida albicans, theencoding gene of isocitrate lyase is up-regulated, but those forglycolysis is down-regulation.The glyoxylate was energy consumptionrequired for persisting in macrophages. Therefore, assessment of in candida albicans carbon metabolism shunt asglycolysis upregulation and glyoxylate down-regulation at the same timemay be related with the occurence of itraconazole resistance.Exploringthe differential of MDH expression between the Itraconazole resistancegroup and sensitive group in Candida albicans,to asses the relationbetween the glyoxylate key enzyme regulation and the occurence of theItraconazole resistance.The bioinformatics analysis predict a potentialdiagnosis and drug target domain. The positiv result with the anti-LDHbody identify the interaction point was highly conserved epitopes of CaMDHhomologous sequence and supply new methods for the vaccine, diagnosis anddrug target study of the CaMDH.Objective：Asses the relation between the glyoxylate key enzyme regulation and theoccurence of the Itraconazole resistance.Identify the interaction pointwas highly conserved epitopes of CaMDH homologous sequence and supply newmethods for the vaccine, diagnosis and drug target study of the CaMDH.Methods:1.Bioinformatics analysis the cDNA sequence of CaMDHThe structure, properties and function about encoding protein of malatedehydrogenase from candida albicans were analyzed and predicted bybioinformatics tool as NCBI、ExPASy and DNAstar in this study.CaMDHsequence with homologous sequence from other species of pathogenicmicroorganism are compared in phylogenetic analysis.2.Screening the Itraconazole-resistant and Itraconazole-sensitiveC.albicans strains and ELISA to measure the MDH expression.Select30strains including ten Itraconazole resistant strains, ten intermediary strains and ten sensitive strains,respectively measure andcompare the expression level of3groups by ELISA. Data was analyzed byanalysis of variance,p<0.05was statistically significant for thedifference.3.Identify CaMDH protein and Westernblot analysis with the homologoussequence recombinant protein immunne serum.Extracted the whole intracellular proteins of5strains Candiada albicansby P0013B RIPA lysis buffer,Westernblot analysis with the SjLDHrecombinant protein immunne serum.Results:Analysis show the amino acid sequence of schistosoma japomicum lacticdehydrogenase (SjLDH) had lowest identiy(18.2%) with CaMDH than that ofother pathogenic microorganism species(43%).CaMDH contain LDH conserveddomain and7main B cell epitopes. The MDH active site located at the NAD(P)binding domain.Determination of the MIC values of30candida albicans strains by the brothmicrodilution assay as NCCLS document M27-A described.The result of ELISA show combination with anti-LDH antibody was348.7143±34.3594in resistant Candiada albicans isolates, significantly lowerthan that in sensitive isolates(443.4286±35.6363) and the mediumisolates was327.0000±85.5990（F=8.516,P＝0.003）。CaMDH contain3epitopes display high levels of similarity with that ofSjLDH, and the Westernblot analysis with the SjLDH recombinant proteinimmunne serum show positive result.Conclusions:Bioinformatic analysis come out that CaMDH display high similarity withLDH of other pathogenic microorganism species. The occurrence of Itraconazole resistance in Candida albicans may berelatated with the decrease of expression of CaMDH.Identify the cross-reactive combination point was highly conservedepitopes of MDH/LDH homologous sequence.