Characterisation of host transcriptional responses to Candida albicans infection

致病真菌每年导致约120万人死亡。在粘膜感染期间，真菌病原体白色念珠菌分泌溶念珠菌素1，这是一种破坏组织并激活表皮生长因子受体2以及p38和EGFR/ERK信号通路3的细胞溶解毒素。信号转导驱动下游转录网络的激活，这些转录网络是宿主对真菌感染的免疫应答的重要组成部分。先前的研究已经证明，AP-1家族的转录因子（例如，c-Fos、c-Jun和其他）有助于宿主上皮对白色念珠菌感染的应答4。然而，目前缺乏对白念珠菌和溶念珠菌素在宿主细胞中诱导的转录反应的全基因组概述。如果我们要详细了解粘膜对真菌感染的反应，这些知识是必不可少的。因此，本项目将研究口腔上皮细胞对白色念珠菌和念珠菌溶素感染的全基因组转录反应。该项目将使用染色质免疫沉淀测序（ChIp-Seq）来分析宿主转录反应并识别参与宿主免疫的新因素。染色质免疫沉淀（ChIP）将用于从感染C.白色念珠菌或用念珠菌毒素处理。随后将进行下一代DNA测序（NGS），以鉴定全基因组转录因子结合基序。将进行详细的二次验证，以进一步验证初始ChIp-Seq分析中鉴定的转录因子靶标。最后，选定的转录因子的作用将在白念珠菌感染的免疫防御的背景下进行研究。第1-2年：人类口腔上皮细胞将感染C。白色念珠菌和念珠菌溶解素在一定范围的MOI和浓度下。将确定真菌和念珠菌溶素的最佳MOI/浓度。使用市售试剂盒将选定的转录因子与哺乳动物基因组交联，提取基因组DNA并进行片段化，通过酶消化从交联样品中去除未结合的DNA。使用转录因子特异性抗体从该混合物中沉淀出被转录因子结合的基因组区域，并通过下一代测序鉴定结合区域的序列。仅溶剂（阴性）对照品将用作对照样品。转录因子结合将使用计算方法在整个基因组中作图，并用于鉴定由转录因子激活的靶基因组。类似地，计算方法将用于识别参与对C的粘膜反应的潜在/相关信号传导途径。白色念珠菌/念珠菌溶解素。2-3年级：为了确认/验证在初始Chip-Seq方法中获得的数据，将使用与初始Chip-Seq筛选中鉴定的序列对应的纯化寡核苷酸进行转录因子结合试验。这些方法将结合联合收割机测定法和测定法，在所述测定法中，上皮细胞暴露于念珠菌溶解素，在所述测定法中，细胞被不能产生念珠菌溶解素的白色念珠菌的野生型和突变株感染。将使用RT-qPCR验证选定靶基因的表达，并使用生物化学和遗传学方法（蛋白质印迹、小分子抑制剂、siRNA敲除）研究选定信号传导途径的参与。这将确定新的主机响应白色念珠菌和念珠菌溶解素。第4年：数据整理和项目撰写。

Pathogenic fungi kill approximately 1.2 million people every year. During mucosal infection, the fungal pathogen Candida albicans secretes candidalysin1, a cytolytic toxin that damages tissue and activates the epidermal growth factor receptor2 together with p38 and EGFR/ERK signalling pathways3. Signal transduction drives the activation of downstream transcriptional networks which are an essential component of host immune responses to fungal infection. Previous research has demonstrated that members of the AP-1 family of transcription factors (e.g., c-Fos, c-Jun, and others) contribute to the host epithelial response to Candida albicans infection4. However, a genome-wide overview of transcriptional responses that are induced in host cells by Candida albicans and candidalysin is currently lacking. The knowledge is essential if we are to have a detailed understanding of mucosal responses to fungal infection. Accordingly, this project will characterise the genome-wide transcriptional response of oral epithelial cells to infection with Candida albicans and candidalysin. This project will use chromatin immunoprecipitation sequencing (ChIp-Seq) to analyse host transcriptional responses and identify new factors involved in host immunity. Chromatin immunoprecipitation (ChIP) will be used to isolate DNA-bound protein from epithelial cells infected with C. albicans or treated with candidalysin toxin. This will be followed by Next Generation DNA Sequencing (NGS) to identify genome-wide transcription factor binding motifs. Detailed secondary validation will be performed to further characterise transcription factor targets identified in the initial ChIp-Seq analysis. Finally, the role of selected transcription factors will be investigated in the context of immune defence during Candida albicans infection. Year 1-2: Human oral epithelial cells will be infected with C. albicans and candidalysin at a range of MOIs and concentrations. The optimum MOI/concentration will be determined for both fungus and candidalysin. Selected transcription factors will be cross-linked to the mammalian genome using commercially available kits, the genomic DNA extracted and fragmented, and unbound DNA will be removed from the cross-linked sample by enzymatic digestion. Regions of the genome that are bound by transcription factors will be precipitated from this mixture using transcription factor-specific antibodies, and the sequences of the bound regions identified by next generation sequencing. Vehicle-only (negative) controls will be used as comparator samples. Transcription factor binding will be mapped across the genome using computational methods and used to identify panels of target genes that are activated by the transcription factor. Similarly, computational approaches will be used to identify potential/associated signalling pathways involved in the mucosal response to C. albicans/candidalysin. Year 2-3: To confirm/validate data obtained in the initial Chip-Seq approach, transcription factor binding assays will be performed using purified oligonucleotides corresponding to the sequences identified in the original Chip-Seq screen. These approaches will combine assays in which epithelial cells are exposed to candidalysin and assays in which cells are infected with wild type and mutant strains of Candida albicans unable to produce candidalysin. The expression of selected target genes will be validated using RT-qPCR, and the involvement of selected signalling pathways investigated using biochemical and genetic approaches (western blot, small molecular inhibitors, siRNA knockdown). This will identify novel host responses to Candida albicans and candidalysin. Year 4: Data collation and project write-up.