Characterisation of host transcriptional responses to Candida albicans infection

点击翻译按钮获取中文摘要

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.