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英国利兹大学\University of Leeds:温度反应网络链接细胞形状和真菌毒力性状研究

论文价格: 免费 时间:2015-09-01 22:19:30 来源:www.ukassignment.org 作者:留学作业网

Introduction

 

Cells adapt to their environment by responding to specific environmental stimuli such as light, temperature, and nutrients. For microbial pathogens, mammalian body temperature can signal the induction of pathways required for host colonization and pathogenesis [1]. One such group of organisms is the thermally dimorphic fungal pathogens, which include Coccidioides, Paracoccidioides, Blastomyces, and Histoplasma species. These evolutionarily related fungi are notable among fungal pathogens in that they all cause disease in healthy individuals [2]. Each of these organisms grows in a mold form in the soil, forming long, connected filaments that produce vegetative spores [3]. When the soil is aerosolized, filamentous cells and spores can be inhaled by mammalian hosts and converted into a parasitic form within the host lung. Conversion entails a dramatic change in cell shape to a budding yeast form for the majority of these pathogens, as well as the transcriptional induction of virulence genes required to cause disease in the host [3]. For all thermally dimorphic fungi, host temperature is the key signal that triggers this developmental switch, but little is known about the coordinated induction of morphologic changes and virulence gene expression by temperature.
介绍
细胞适应环境,响应特定的环境刺激,如光,温度和营养物质。对于病原微生物,哺乳动物体内温度信号感应主机定植和发病机制[1]所需的途径。一个这样的一群生物热二态真菌病原体,其中包括的球孢子,Paracoccidioides,芽,组织胞浆菌物种。这些进化相关的真菌病原真菌中是有名望的,他们都引起疾病的健康人[2]。这些生物体中的每一个都在模具中的形式在土壤中生长,形成长,连接产​​生无性孢子丝[3]。当土壤被雾化,的丝状细胞和孢子可以吸入的哺乳动物宿主的寄生形式转换成主机内的肺。转换需要一个戏剧性的变化在芽殖酵母形式,为广大这些病原体,以及在主机[3]需要引起疾病致病基因的转录诱导细胞形状。对于所有热二态真菌,主机温度是关键的信号,触发这种发育开关,但协调的形态学变化及毒力基因表达诱导温度知之甚少。

 

Histoplasma capsulatum, which is endemic to the Ohio and Mississippi River Valleys of the United States, can cause life-threatening respiratory and/or systemic disease (histoplasmosis) [2],[4]. It is estimated that up to 25,000 people develop life-threatening infections in endemic regions each year, with at least 10-fold more mild or asymptomatic infections [2],[4]. Although the pathogen propagates as spores and in a filamentous form in the environment, H. capsulatum is found almost exclusively in the yeast form within mammalian hosts. Despite the prevalence of H. capsulatum and its threat to human health, we have a limited understanding of the transcriptional regulatory network that governs pathogenic yeast-phase growth. Previously, we identified three regulators, Ryp1, Ryp2, and Ryp3, and showed that they are required for yeast-phase growth [5],[6]. Whereas wild-type cells grow in the yeast form at 37°C, ryp1, ryp2, and ryp3 mutants grow constitutively in the filamentous form independent of temperature. In wild-type cells, RYP1, RYP2, and RYP3 transcripts and proteins accumulate preferentially at 37°C and each Ryp protein is required for the wild-type expression levels of the others [5],[6].
组织胞浆菌,这是特有的俄亥俄和密西西比河流域的美国,可能会导致危及生命的呼吸和/或全身性疾病(组织胞浆菌病)[2],[4]。据估计,多达25,000人的发展危及生命的感染流行地区,每年有至少10倍以上轻微或无症状的感染[2],[4]。虽然孢子和丝状的形式在环境中的病原体的传播,荚膜几乎只存在于哺乳动物宿主内的酵母形式。尽管荚膜的患病率及其对人体健康的威胁,我们有一个有限的转录调控网络的认识,管理致病酵母相生长。以前,我们确定了三个监管机构Ryp1,Ryp2,和Ryp3,表明他们所需要的酵母相生长[5],[6]。鉴于野生型细胞生长在37℃,ryp1,ryp2,和ryp3突变酵母形式组成独立温度丝状增长。在野生型细胞中,转录和蛋白质RYP1 RYP2,RYP3优先积累,在37℃下,每个RYP蛋白所需的其它野生型的表达水平.


RYP1 encodes a fungal-specific transcriptional regulator that is required for modifying the transcriptional program of H. capsulatum in response to temperature [5]. Ryp1 belongs to a conserved family of fungal proteins that regulate cellular differentiation in response to environmental signals. The best-studied member of this family of proteins is Wor1, which was identified as a master transcriptional regulator that controls a morphological switch required for mating in Candida albicans [7]–[9]. In the model yeast Saccharomyces cerevisiae, the Ryp1 ortholog, Mit1, is required for a morphologic switch that occurs under nutrient limitation [10]. Ryp1 orthologs in the plant pathogens Fusarium oxysporum (Sge1), Fusarium graminerium (Fgp1), and Botrytis cinerae (Reg1) are required for full pathogenicity and conidiation [11]–[13]. All of these observations signify the importance of Ryp1 orthologs for transduction of environmental cues to regulate cell morphology and virulence. Furthermore, it was recently demonstrated that Wor1 contains a DNA-binding domain that is conserved throughout the WOPR (Wor1, Pac2, Ryp1) family of proteins [14], suggesting that these regulators respond to specific signals by triggering a transcriptional program.



In contrast, Ryp2 and Ryp3 belong to the Velvet family of regulatory proteins [6], whose molecular function is unknown. This family is typified by Velvet A (VeA), which was initially characterized as a regulator of sexual spore production in Aspergillus nidulans [15],[16], but is now known to also regulate secondary metabolism and development in many fungi including Aspergillus species, Fusarium species, Neurospora crassa, and Acremonium chrysogenum (reviewed in [17]). In H. capsulatum, the VeA ortholog Vea1 has a role in sexual development but is dispensable for yeast-phase growth [18]. Additionally, many fungi have multiple Velvet family proteins that collaborate to serve regulatory functions. For example, in A. nidulans, three Velvet family proteins (the Ryp2 ortholog VosA, the Ryp3 ortholog VelB, and VeA itself) act together to regulate asexual and sexual development and secondary metabolism [19]. Notably, since Velvet family proteins do not contain canonical DNA binding domains or other domains of known function, their mechanistic role in regulation of developmental processes is unclear.  http://www.ukassignment.org/yylwdx/


As noted above, both WOPR and Velvet family proteins are widely distributed among fungi, although the Hemiascomycetes, including Saccharomyces and Candida species, lack Velvet family proteins. Since both families of proteins are required for yeast-phase growth in H. capsulatum, we explored if and how these two distinct classes of fungal regulators work together to govern temperature-responsive traits by dissecting the Ryp regulatory network in H. capsulatum. To this end, we performed whole-genome transcriptional profiling and chromatin immunoprecipitation experiments to determine the shared and unique roles of Ryp1, Ryp2, and Ryp3 in regulating yeast-phase growth. We show that 96% of yeast-phase enriched transcripts are dependent on Ryp1, Ryp2, and Ryp3 for their enhanced expression in response to temperature, whereas 66% of filamentous-phase enriched transcripts require Ryp1, Ryp2, and Ryp3 to prevent their inappropriate expression at 37°C. We demonstrate that all three Ryp factors physically interact and associate with the upstream regions of a core set of target genes, including those required for yeast-phase growth and virulence. Additionally, we identify a fourth transcriptional regulator, Ryp4, to be a component of the Ryp regulatory network required for temperature-responsive yeast-phase growth. Finally, the identification of two distinct cis-acting regulatory sequences that are bound and utilized by Ryp proteins provides the first evidence that highly conserved Velvet family proteins can directly bind to DNA and activate gene expression using a unique cis-acting element. Overall, our results provide a molecular understanding of how regulation of cell morphology and virulence gene expression is coordinated in response to temperature in H. capsulatum.

 

Results
Ryp1, Ryp2, and Ryp3 Are Required for the Expression of Genes Associated With Growth in the Pathogenic Yeast Form
Our previous studies showed that there are marked differences in the transcriptional profiles of wild-type yeast-form cells grown at 37°C and filamentous cells grown at room temperature [5],[20]. Cells lacking RYP1, RYP2, or RYP3 grow constitutively as filaments independent of temperature [5],[6], and Ryp1 is required for the expression of the majority of the transcripts enriched during yeast-phase growth at 37°C [5]. Here we sought to understand whether Ryp2 and Ryp3 are also involved in regulating expression of genes required for yeast-phase growth. To this end, we performed whole-genome expression profiling experiments comparing the transcriptional profiles of multiple biological replicates of ryp1, ryp2, ryp3 mutants and wild-type strains grown at room temperature (RT) and 37°C. We identified 388 genes with significantly increased transcript levels and 376 genes with significantly decreased transcript levels in wild-type yeast cells grown at 37°C compared to wild-type filaments grown at RT (Figure 1A and Table S1). These gene sets were referred to as yeast-phase–specific (YPS) and filamentous-phase–specific (FPS) genes, respectively.

 

EMSA
5′-IRDye800-labeled Motif A and Motif B probes were prepared by annealing 5′-IRDye800-CBP1-MotifA-Fwd and CBP1-MotifA-Rev, and 5′-IRDye800-CBP1-MotifB-Fwd and CBP1-MotifB-Rev, respectively, in 10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT. Nonlabeled competitor probes were prepared similarly with nonlabeled oligonucleotides (Table S7). Two µg of each purified protein (Ryp1-N-terminus, Ryp1, Ryp2, Ryp3, or control extract) and 1 nM of labeled probes were mixed in binding buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, 5% glycerol, 1 mM EDTA, 0.5 mM DTT, 100 ug/ml BSA, and 25 ug/ml poly(dI:dC)) and incubated for 30 min at room temperature. Reactions were separated on 6% DNA retardation gels (Invitrogen) in 0.5× TBE buffer. Mobility shifts were visualized and analyzed using the ODYSSEY imaging system (LI-COR Biosciences).


Co-immunoprecipitation
Wild-type (G217B) cells grown to late log phase at 37°C were harvested by filtration, and the pellet was frozen in liquid nitrogen. Whole cell extracts were made by cryogrinding the pellet in Retsch Mixer Mill MM 400. Co-immunoprecipitation experiments were performed using the Dynabeads Co-immunoprecipitation kit from Invitrogen following the manufacturer's instructions. Briefly, 100 ug of polyclonal α-Ryp2 (ID:387, SQSAGHMQSPSQVPPAWG) or α-Ryp3 (ID:356, SHGSKGQDGEGEDWENEG) antibodies were covalently linked to 5 mg of magnetic beads using Dynabeads Antibody Coupling kit. To prepare cell lysate, 2 g of ground samples were mixed with lysis buffer, vortexed, and spun down. Then, supernatant was incubated with 5 mg of antibody-coupled magnetic beads for 8 h at 4°C. After multiple washes, protein complexes that are bound to antibody-coupled beads were eluted in low pH buffer provided by the manufacturer. IPs with ryp2 and ryp3 mutants grown at 37°C and no antibody control were performed similarly. All fractions were separated by SDS-PAGE, visualized by silver staining, and analyzed by Western blotting using standard procedures. Polyclonal α-Ryp1 (ID:3873, ASSYQPGPPASMSWNTAATG), α-Ryp2 (ID:387, SQSAGHMQSPSQVPPAWG), or α-Ryp3 (ID:356, SHGSKGQDGEGEDWENEG) antibodies were used to detect Ryp proteins.


β-Galactosidase Assays
β-galactosidase assays were performed as previously described [61]. At least three independent isolates of each S. cerevisiae strain were grown to late log (for the yeast-two-hybrid assay) or stationary (for the in vivo transcriptional activation assay) phase. Each isolate was assayed in quadruplicate, and the results of representative isolates are shown in.

 

 

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