Speaker ：曹欣瑜 Advisor ：藍清隆 老師 Date ： 2015.04.28 Identification of genes expressed by Cryptococcus gattii during iron deprivation Brazilian Journal of Microbiology.
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Presentation on theme: "Speaker ：曹欣瑜 Advisor ：藍清隆 老師 Date ： 2015.04.28 Identification of genes expressed by Cryptococcus gattii during iron deprivation Brazilian Journal of Microbiology."— Presentation transcript:
Speaker ：曹欣瑜 Advisor ：藍清隆 老師 Date ： 2015.04.28 Identification of genes expressed by Cryptococcus gattii during iron deprivation Brazilian Journal of Microbiology 45,3,813-820(2014)
3 Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii ) are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation.
前言 4 There are two main pathogenic species within the Cryptococcus genus, namely C. neoformans and C. gattii. C. neoformans var. grubii and C. neoformans var. neoformans have been isolated worldwide, and typically cause disease in hosts with impaired immunity.
前言 5 In mammalian hosts, the majority of iron is locked within iron-binding proteins, pathogens must therefore be equipped with competitive iron acquisition and uptake systems. C. neoformans possesses cell surface reductases that reduce ferric iron to its ferrous state (Cfo1), export reductants, such as 3-hydroxyanthranilic acid, and iron permease (Cft1) for transport into the cytosol as ferric ion. In addition, for C. neoformans, many studies have described genes related to iron regulation, including Cryptococcus iron regulator 1 (CIR1) and HAPX, genes that control the expression of iron-dependent genes.
前言 6 In C. gattii, little is known about iron metabolism and its implication in virulence. But there have observed a similar regulation of metabolic proteins in both the pathogenic yeasts, C. gattii and C. neoformans. However, certain proteins related to iron homeostasis in C. neoformans were not identified. In order to investigate the adaptive cellular responses when host-iron availability to pathogenic microorganisms is reduced, we attempted to identify the differential gene expression profile of the C. gattii reference strain R265 under conditions of iron deprivation, using representational difference analysis (RDA).
實驗項目 12 Table 1 - Summary of the computational analysis of the transcripts obtained from C. gattii grown in a low iron medium for 3 h at 37 °C Table 2 - Summary of the computational analysis of the transcripts obtained from C. gattii grown in a low iron medium for 12 h at 37 °C.
實驗流程 - 準備 Strain, culture conditions and RNA extraction 14 The C. gattii strain R265 was used for the RDA experiments and gene expression analysis. R265 was routinely grown in Yeast Peptone Dextrose (YPD) broth (yeast extract 1%, peptone 1% and glucose 2%) before cultivation in medium containing low levels of iron (limited-iron medium (LIM) and an iron-repleted medium (LIM+Fe, with the addition of 100MFeHEDTA) for 24 h.
實驗流程 - 準備 Strain, culture conditions and RNA extraction 15 To evaluate the effects of iron, 10 6 cells/mL of yeast were transferred to 50 mL of LIM or LIM-Fe. Cells were grown for two distinct periods of time (3 h or 12 h) in LIM or LIM-Fe at 37 °C. Cells were harvested by centrifugation and immediately frozen in liquid nitrogen before RNA extraction. Total RNA was isolated using the RNeasy mini kit, and cDNA was synthesized using the SMART PCR synthesis kit. First-strand cDNA synthesis was performed with reverse transcriptase from 500 ng of total RNA. An aliquot of 5 μL of first-strand cDNA was used as the template for second-strand synthesis.
實驗流程 - 步驟 Representational difference analysis (RDA) 16 cDNA from C. gattii grown for 3 h and 12 h in a low-level iron medium (LIM) as the “tester” and cDNA from C. gattii grown for 3 h and 12 h in iron-replete medium (LIM+Fe) as the “driver”. All cultures were grown at 37 °C with shaking. A ds-cDNA sample from each experimental condition was digested with Sau3AI and resulting products were purified using the illustra GFX PCR DNA and gel band purification kit. The RBam24/12 adapters were ligated to the digested cDNA in order to be used as a tester.
實驗流程 - 步驟 Representational difference analysis (RDA) 17 The first differential product (Dp1) was obtained by hybridization (20 h at 67 °C) of the driver and cDNA mixed at a 10:1 ratio, followed by PCR amplification with an RBam24 primer. In order to generate the second (Dp2) and third (Dp3) differential products, NBam and JBam adapters were ligated to the tester in each round of subtractive hybridization and the driver/tester ratio was increased to 100:1 and 1000:1, respectively.
實驗流程 - 步驟 Cloning and bioinformatics analysis of the RDA products 18