Identification And Characteristics Of Genes ORF19.2734and ORF19.3222in Candida Albicans

Candida albicans is the most common clinical opportunistic pathogens. The incidence ofcandidiasis has increased significantly in recent years, due to the abuse of antibacterials, the risingnumbers of immunocompromised patients and so on. So the study in C. albicans is becoming more andmore important. The pathogenicity of C. albicans is related to its response to external stimuli, such asdrugs, metal ions and etc. Similar to the lysosomes in eukaryotes, vacuole is a kind of specificorganelles in fungi and plants which plays important roles in the fungal cell, such as protein transport,endocytosis, homeostasis in the cell, nutrients transporter, yeast-to-hyphae formation and virulence.Also, vacuoles play an important role in responding to the external stimuli. In our previous study, twoalleles of gene ORF19.3222which is supposed to be a sulfur transporter protein of the vacuolemembrane have been deleted. So we further investigated the potential function of ORF19.3222in C.albicans in this study.We also chose eight stress related genes, including ORF19.2734etc. in C. albicans, andconstructed the homozygous null mutants by the method of homologous recombination to study thefunction of these genes. The ORF19.2734null mutant has been successfully constructed. Then, thephenotypes of the null mutant were investigated by the spots assay, growth curve, laser confocalmicroscope, yeast-to-hyphae transition and so on. Furthermore, the integrity of the cell wall in theORF19.3222null mutant was investigated by the transmission electron microscopy (SEM), laserconfocal microscope and other methods. The rhodamine6G efflux experiments and the ergosterolcontent were measured to examine the potential mechanism of the reduced sensitivity to azole agents inthe ORF19.3222null mutant. We then examined whether the gene ORF19.3222participated in themetabolic pathway of cellular sulfur by investigating the stimulation of SO2, methionine and cysteineexpedition. And, we further analysed and speculated the biological function of the gene ORF19.3222using the whole genome expression profile.Our results showed that the ORF19.2734null mutant increased the sensitivity to azole drugs andslowed down the growthrate. But there was no change in the hyphae formation ability and themorphology of vacuole in the ORF19.2734null mutant. By the method of real-time quantitative PCR,we deduced that gene ORF19.2734may down-regulate the expression of MDR1, ERG11, and ERG2toincrease the intracellular drug concentration. We also deduced that the reduced Erg11p activitycontributed to the increased sensitivity to fluconazole.On the other hand, our results showed that the cell wall structures were changed in the ORF19.3222null mutant as indicted by the thinning mannose layer and the increasing exposure of theglucan layer, which made the null mutant much more easier to be identified by macrophage cells, andcaused the decline of virulence. The ORF19.3222null mutant increased the rhodamine6G efflux in20min, and reduced the intracellular ergosterol content. The ORF19.3222null mutant increased sensitivityto SO2, cysteine and methionine, but did not affect cell metabolism to produce H2S. The results of thewhole genome expression profile confirmed the previous results of the real time quantitative PCR, suchas the up-regulation of the sulfur transfer pathway gene MET16, GPI anchor cell surface protein PGA23,and the down-regulation of ALS9, etc. At the same time, the results of the whole genome expressionprofile suggested that the ORF19.3222gene might be related toV-ATPase activity. V-ATPase is one ofthe important protein, which maintains the steady-state of intracellular pH. The destruction of V-ATPaseactivity increases the vacuole pH value, and therefore reduces hyphae formation ability, etc.. V-ATPaseactivity can also be changed by the decreasing of the ergosterol content in the cell, and then increasesensitivity of azole drugs. The ORF19.3222null mutant showed the increasing acidification in vacuole,suggesting that V-ATPase activity was strengthened in the ORF19.3222null mutant with the treatmentof fluconazole. And without the treatment of fluconazole, there was no obvious difference in vacuole pHvalue. So we putative that the the reduced susceptibility to azoles might be caused by the enhancedV-ATPase activity. Thus we speculate that there could be a close connection between H+transporter andsulfur transporter on vacuole membrane in C. albicans.Conclusion: The ORF19.2734null mutant deceased the expression of MDR1, ERG11, ERG2andtherefore increased the sensitivity to fluconazole. ORF19.3222gene plays an important role inmaintaining normal cell wall structure and virulence. ORF19.3222gene is involved in cell sulfur andsulfur amino acid metabolism. Orf19.3222protein might be a sulfur transporter and affect the V-ATPaseactivity on the vacuole membrane.