An investigation into the stress response mechanisms and virulence of the human fungal pathogen, Candida albicans

Abstract

Candida albicans is a major fungal pathogen causing life threatening systemic infections in immunocompromised humans. While in the host C. albicans is exposed to a range of stresses during phagocytosis by host innate immune cells, including reactive oxygen species (ROS), cationic fluxes, and fluctuations in pH. The ability of C. albicans to adapt to such stresses is essential for survival and pathogenesis. Despite this, however, there is still much to be learnt regarding the stress responsive mechanisms mounted by this major pathogen. Hence, the overarching goal of this project was to provide novel insight into the cellular processes necessary to enable stress adaptation and virulence of C. albicans. To facilitate this, quantitative fitness analysis (QFA) of two C. albicans deletion libraries was performed using inducers of superoxide, cationic, and alkaline pH stresses. GO term analysis of sensitive genes highlighted distinct and overlapping biological processes, molecular functions, and cellular components enriched during adaptation to each stress. Notably, the importance of ion binding for resistance to cationic and superoxide stress was revealed, whereas cell wall biogenesis was enriched for alkaline pH stress. QFA also identified several regulatory genes not previously implicated in stress responses, including the Pho4 transcription factor. Cells lacking PHO4 were acutely sensitive to all three stresses tested and thus the role of Pho4 in mediating stress resistance was investigated further. Additional phenotypic testing revealed pho4Δ cells display impaired resistance to several organic and metal cations, and defects in morphogenic switching. Similar to Pho4 function in S. cerevisiae, deleting PHO4 in C. albicans completely abolished acquisition and accumulation of phosphate stored as polyphosphate (polyP) in the vacuole. Consistent with stress resistance and nutrient acquisition being important virulence determinants in C. albicans, cells lacking PHO4 were acutely sensitive to macrophage-mediated killing, and displayed attenuated virulence in Caenorhabditis elegans and murine models of infection. Further analysis of the role and regulation of Pho4 in stress adaptation in C. albicans revealed that in addition to the essential role of Pho4 in phosphate acquisition and storage, which enables survival in phosphate limiting and alkaline pH conditions, Pho4 function is also important for metal ion homeostasis which is essential for cationic and superoxide stress resistance. vi As C. albicans only causes systemic infections in immunocompromised hosts, the final objective of this study was to explore whether the immune status of the host dictated the importance of key stress regulators in promoting the virulence of this fungal pathogen. Although the Hog1 stress activated protein kinase and the Pho4 transcription factor were demonstrated to be essential for C. albicans virulence in the model mini host C. elegans, both were dispensable for virulence upon infection of immunocompromised worms. These findings infer that robust stress responses of C. albicans may only be required for virulence when immune responses are evoked in an immunocompetent host. Taken together, the data presented in this thesis highlight that metabolic adaptation is essential for the survival of C. albicans to host-imposed stresses, and that the immune status of the host may govern the importance of stress protective mechanisms in mediating the virulence of this major fungal pathogen.