Profile And Cluster Analysis Of The Genes Expressed In Yeast And Hyphal Phase Of Candida Albicans

Candida albicans is an important pathogen, causing the majority of fungal infections in humans. These can range from relatively minor surface infections, such as thrush and vaginal yeast infections, to more serious and life-threatening systemic infections, particularly in immunocompromised individuals. C.albicans is usually a relatively benign commensal of humans,and the ability to become virulent is thus primarily determined by the immune state of the host. However, there are characteristics of C. albicans that contribute to its ability to cause disease in susceptible individuals. One of these is the ability to switch from a yeast phase of growth to a filamentous phase characterized either by pseudohyphae or true hyphae. Morphogenesis appears to be important for pathogenesis, because cells that are trapped in either the yeast or pseudohyphal states are less virulent in murine systemic infection models. Thus, the determinants of the morphological yeast to hyphal switching appear important for virulence. By several approaches, genes have been identified that are expressed exclusively or primarily in the hyphal state.Signal transduction cascades, modulated by elements such as cAMP, mitogen-activated protein kinases, and pH-responsive modules,appear to regulate this yeast to hyphal transition. Transcription factors important in the ability to filamentation have been identified. In this study, the genes expressed in yeast and hyphal phase induced from C.albicans were analyzed qualitatively and quantitatively with long serial analysis of gene expression (LongSAGE) protocol, and then the genes expression profiles were established. We try to find out the relationship of the expressed genes in yeast and hyphal cells to phase transition in C.albicans by the genes functional clustering analysis. ATCC-90028,a C.albicans standard strain,was induced to form germ tubes in the conditions that 1640 was adjucted to pH 7.0 and the temperature was 37℃.Total RNA was isolated from yeast and hyphal cells respectively only when the cells'purity was more than 95% by microscope examination. The RNA samples were amplified with HWP1 and caHSP90 primers by RT-PCR.Isolated RNA from yeast and hyphal cells produced caHSP90 bands.Ortherwise,only RNA isolated from hyphal cells produced HWP1 bands,negative from yeast.These showed that the RNA samples were derived from homogeneous cells and would keep the characteristics of the certain cells for construction of LongSAGE libraries,and the genes expression profile could represent the portrait of cellular molecules. LongSAGE library was constructed from total RNA corresponding to 5 X 107 cells. The mRNA was binded to biotylated Oligo-dT magnetic beads.The cDNA was synthesized on the beads containing mRNA using SuperScriptTM ⅡReverse Transcriptase,following the second-strand DNA synthesis using E. coli DNA polymerase and E. coli DNA ligase.After verifying the cDNA synthesis integrity with PCR amplifications with primer-sets of GAPDH and EF (glyceraldehyde-3-phosphate dehydrogenase (GAPDH),elongation factor-1α(EF)),the cDNA sample was digested with anchoring enzyme NlaⅢ,and the cleavage product was divided equally into two new tubes.Adapter A and adapter B was ligated to cDNA separately and then the tags were released with digestion of tagging enzyme Mme I.The tags were linked together to establish the 130bp ditags, which contained fragmants of expressed genes.Scale-up PCR tubes were amplified to obtain adequate 130bp ditags and the latter was purified from polyacrylamide gel.130bp ditags were digested with NlaⅢagain to produce 34bp ditags.The 34bp ditags were interlinked to yield concatemers.The concatemers were cloned into pZErO?-1 vector.By transforming competent E.coli cells with vectors,the transformants were screened with PCR to evaluate the transforming efficiency. Finally,the positive resistant transformants for the presence of insert were ready to sequence.The expressed tags were extracted from the raw sequencing files and put into database.Therefore,two LongSAGE libraries were constructed from yeast and hyphal cells induced from C.albicans. During the construction of LongSAGE libraries,four protocol procedures had been modified so that the libraries were achieved successfully.There were: (1) the micro-beads placed on a magnetic stand for double time as the kit protocol's recommendation,and the beads'lose were decreased;(2) the amount of adaptors was cut down and the 130 bp ditags production were higher than that of the protocol,and the 130 bp ditags became morespecific;(3) the polyacrylamide gel pieces were eluted twices,and the recycled purifying DNA was increased;(4) the eluted DNA solution from gel was extracted with phenol/chloroform to prevent the DNA degradation from nuclease,and more concatemers were obtained. 17,769 tags were obtained from yeast cells LongSAGE library with 7,715 unique tags,and 17,552 tags in hyphal cells LongSAGE library with 6,940 unique tags.The distribution of expressed genes in hyphal LongSAGE libraries showed a great deal of high-aboundance expressed genes with diversity and complexity.These indicated that hyphal cell could produce specific molecules or elevate some factors to elaborately regulate morphogenesis under environment cues. The high-aboundance genes in hyphal phase of C.albicans were divided into four types.(1),moleculers on cell surface,such as CYR1,HK1 and CMD1,which participated in signaling transduction of cell,could modulate the expression of genes or morphogenesis of cells.(2),some metabolic enzymes,such as AY445049,elevated expression in hyphal phase were helpful to the absorption of glucose.(3),structural genes,such as 25sRNA gene,HWP1 and CDC11,some expressed only in hyphal phase,were the consequence of phase transition of C.albicans.(4),moleculers with indistinct function,such as HIS4,SLA2,som1,AJ390523. Construction of genes expression profile in yeast and hyphal cells of C.albicans could play a role in the understanding of the phase transition mechanisms. Specific factors or low and over expressed molecules in yeast or hyphal phase of C.albicans could activate or repress hyphal formation.