The Study Of Differential Gene Expression Between Yeast And Mycelial Phase Of Malasesiza Furfur

Backgrounds The genus Malassezia has received considerable attentionin recent years from mycologists and clinicial doctors. This group oflipophilic yeasts, long known to be the pathogenic agents of pityriasis (tinea)versicolor and Malassezia folliculitis, is also increasingly being associatedwith the causation of balanitis, onychomycosis, papillomatosis, and invasivehuman infections, as well as potential immunogenic triggering of atopicdermatitis, seborrheic dermatitis, and dandruff. Otherwise, Malassezia canprobably irritate and deteriorate lesions in psoriasis; and inimmunocompromised patients and those of receiving intralipid therapy theycan induce severe systemic infection. Molecular pathogenic mechanism ofMalassezia is still unclear at present. Now genome project of Malasseziaisn't broungt to consideration. Very few reports about gene experession andthe virulence-associated genes of the fungi appeared so far, which results inthe slow development in the study of pathogenic mechanism of Malassezia. Malassezia is regularly present in its yeast phase in the skin of healthyadults, produces the clinical lesions of pityriasis versicolor when developingits mycelial phase. The conditions that induce such transformation remainunclear, although the available data at present indicate that it could be causedby changes in the local conditions (e.g. temperature, humidity, sebumcomposition) on an idiosyncratic basis. The morphological transformation ofMalassezia from a yeast phase to a mycelial phase is the key pathogenicmechanism of Malassezia that be commonly received. Malassezia furfur(M.furfur) is the only one species of Malassesia that can be induced toproduce its mycelial phase in vitro. To compare the difference of geneexpression between mycelial phase and yeast phase of M.furfur can help us toknow its molecular pathogenic mechanism.Since 1990s, to compare the difference of gene expression betweenhomospecific microorganism has become a focus of attention inmicrobiologic society, accompanied with total genome in manymicroorganism have been sequenced. The recently developed method ofsuppression subtractive hybridization (SSH) provided a powerful new way tostudy molecular pathogenic mechanism of pathogenic microorganism bycomparison with the difference of microorganism mRNA.Objectives To compare the difference of gene expression betweenmycelial phase of M. furfur and its yeast phase, and we try to found twodifferential cDNA libraries. To understand the molecular pathogenicmechanism of the fungi and its morphological switching mechanism from ayeast phase of growth to a mycelial phase.Methods①To isolate the total RNA both in mycelial and yeast phase ofM. furfur, then to isolate mRNA from them severally.②Reverse them to ds-cDNA and then digest them by RsaI.③Hybridize with mycelial phase ofM.furfur as tester and its yeast phase as driver with the method of suppressionsubtractive hybridization (SSH), a reverse process also to be performed at thesame time.④The resulted PCR mixture was connected to T vector toconstruct the subtractive library.⑤Dot blot techniques were employed toindentify those positive clones.⑥That positive differential gene expressionclone to be sequenced, then search similary genes and proteins of thatacquired gene sequences by BLAST (basic local alignment search tool,BLAST) in GenBank to predict their function, and analyse them by DNAsequence analysis softwares.Results 1. Succeed in culturing mycelial phase of M. furfur. 2. Succeed ingaining total RNA both in mycelial and yeast phase of M. furfur and isolatingtheir mRNA. 3. Two differential cDNA libraries had been found with M.furfur in mycelial phase and its yeast phase. 4. Twenty-two positive cloneswere acquired by subtraction in positive direction. Eight positive clones wereacquired by subtraction in reverse direction. Positive clones in both directionsand four suspicious positive clones were sent to sequencing. 5. Thirty-fourfragments were compared through BLASTn and BLASTx in GenBank.Thirteen fragments were either repeated or belonging to vector sequences;fifteen fragments acquired by subtraction in positive direction and sixfragments from reverse direction weren't repeated and also not belonging tovector sequences. 6. All of these 21 clones have more or less homologywith genes or proteins from other biological species. Sum up homologousgenes and proteins of these fragments, they can be clarified into fivecategories:①Fragments homologize to biochemical metabolic enzyme: A4,A16, A23, A38, A49, B4, B7, and B9.②Fragments homologize to transcription associated protein: A8, A13, A14, A40, and B 12.③Fragmentshomologize to growth factor: A10.④Fragments homologize to cell structureproteins: A12 and A22.⑤Others: A15, A21, B3, and B10.7. Open readingframeworks and lots of fungi motifs have been found existing in all of 21fragments by sequence analysis software. Initiation codons and terminationcodons exist in all of them too.Conclusions Succeed in founding two differential cDNA library with M.furfur in mycelial phase and yeast phase by suppressive subtractivehybridization techniques, and a series of fragments which possibly containedgenes related to virulence of M. furfur have been screened. These sequenceshomologize to genes of fungal virulence or molecular evolution such asmembrane protein, metabolic enzyme and cell structure, et cetra.