The mediator of transcription in Candida albicans: roles in cell wall biogenesis, morphogenesis and host-pathogen interactions
thesisposted on 02.03.2017 by Uwamahoro, Nathalie
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
The fungal pathogen Candida albicans is the fourth leading cause of nosocomial infections and continues to cause life-threatening candidiasis. C. albicans virulence results from the intricate gene regulatory pathways that dictate morphogenesis, particularly cell wall remodelling, dimorphism and biofilm formation that allows the organism to evade the host. Studies in non pathogenic yeasts have demonstrated that gene regulatory networks rely on multi-subunit transcriptional complexes such as the Mediator of transcription to modulate gene activation and repression in association with RNA polymerase II. While Mediator is well-studied in non-pathogenic model yeasts, before the commencement of this project its roles in C. albicans were unknown. In this thesis work, I characterised the roles of three Mediator subunits in C. albicans, Med20 from the Head domain of the complex, Med31 from the Middle domain and Srb9 from the Kinase module. I identified global roles for Mediator in the genetic programs that regulate morphogenesis, including conserved roles with non-pathogenic yeasts in the regulation of components of the RAM (regulation of Ace2 and morphogenesis) cell wall remodelling pathway in association with the Ace2 transcription factor. Phenotypic and gene expression comparison using deletion mutants of the most evolutionarily conserved subunit Med31, provided evidence for both conserved and divergent roles, with differences observed in the regulation of cellular adhesion. Med31 controls the yeast to hyphal transition, and it affects the expression of genes coding for key morphogenesis regulators including transcription factors required in the nutrient-sensing cAMP filamentation signalling pathway, (e.g. EFG1, TEC1, CPH2 and RIM101). The downstream targets of the Mediator complex in C. albicans also include the adhesion genes ALS1, ALS3 and HWP1, as well as other cell surface molecules such as the immunologically relevant cell wall component 1,3 β-glucan. Collectively, the above mentioned effects of Mediator on cell wall regulation and cellular morphogenesis translate into cellular roles in biofilm formation by C. albicans, which is a key virulence attribute, as well as essential functions for Mediator in the ability of C. albicans to escape the innate immune response by evading macrophages. Overall, the Mediator complex was essential for C. albicans pathogenesis and my published work with collaborators showed the Mediator is also required for C. albicans virulence in vivo. I further used C. albicans Mediator mutants and a novel assay that I developed, which monitors in real time the killing of macrophages by C. albicans, to discover that macrophages are killed in a biphasic fashion by C. albicans hyphal cells. I demonstrated for the first time that C. albicans hyphal cells trigger the inflammatory suicide response in macrophages – pyroptosis. This work further demonstrated that the long-standing view that C. albicans hyphal cells mechanically damage macrophages has to be revised – instead, hyphal cells activate pyroptosis, which is lytic, and then hijack this processes to escape. I showed that hyphal morphogenesis is necessary for pyroptotic macrophage death, and have data that implicate hyphal cell wall 1,3 β-glucan in the mechanism. The pathogen and host factors and mechanisms of C. albicans morphogenesis and immune evasion identified and characterised in my thesis work add to the knowledge base and provide a platform for the identification of future strategies against human fungal infections.