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Title: The role and regulation of Pxl1 in the hyphal steering of Candida albicans
Author: Cruz e Almeida, Mariana
ISNI:       0000 0004 6349 2970
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2017
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Candida albicans lives as a commensal fungus in humans but can cause life-threatening bloodstream infections in susceptible patient groups. The transition between yeast and hyphal morphologies in C. albicans is an important virulence trait. However, as in many other polarised cells, such as neurons and pollen tubes, hyphal growth must be accompanied by the ability to steer and navigate, and is essential in this pathogen for the penetration of host tissue. Thus, investigation of the regulatory mechanisms of how hyphae sense environmental guidance cues and translate these signals to re-orient the direction of growth will further elucidate C. albicans pathogenesis mechanisms. The relatively recent discovery in fungi of a Paxillin-like protein, which is important for directional motility in mammalian cells, led to the hypothesis that this protein may be involved in directional growth in fungi. In Chapters 3 and 4, I identified in C. albicans homologs of the proteins Paxillin/Pxl1, FAK/Ptk2 and PAK1/Cst20 that, in mammalian cells, are required for directional cell motility. Deletion of these genes in C. albicans caused abnormal sinusoidal or straight hyphal trajectories, loss of steering responses, sensitivity to SDS and calcofluor white, and altered interactions with macrophages. Therefore, Pxl1, Ptk2 and Cst20 may be involved in a shared signalling pathway for directional responses to environmental cues such as stress, contact, electric fields and engulfment by host immune cells. In Chapter 5, localisation studies using fluorescently-tagged proteins, demonstrated that Pxl1- GFP and Cst20-GFP localised to the hyphal tip, although in different distributions (Pxl1 as a dot and Cst20 as a crescent). Pxl1-GFP localisation was independent of Cst20, Ptk2 and actin polymerization. Analysis by Transmission Electron Microscopy revealed cell separation defects in these mutants. Together with the observation that Pxl1-GFP localised to the yeast bud neck but not to hyphal septa, this suggested that Pxl1-complexes may be regulated by Ace2 and play an important role in cell abscission during cytokinesis. Yeast-2-hybrid methods to identify other Pxl1-interacting partners were unsuccessful due to technical difficulties. Although no conclusions could be drawn from these experiments, future work will focus on demonstrating interactions between Pxl1 and other putative regulators/effectors. Chapter 6 describes the development of a new genetically-encoded Ca2+ sensor in C. albicans, GCaMP6, which was transformed into wild-type and calcium-channel mutant strains. Livecell imaging experiments showed that this construct was functional in C. albicans and provided the first single-cell imaging tool for calcium flux in this fungus. A series of optimization experiments showed that production of the GCaMP6 signal requires an external pH of > 7 and [Ca2+] of over 1 mM. Cells were found to generate intermittent calcium flashes that increased in frequency and amplitude during stimulation with 5 mM Ca2+. Altogether, this thesis describes novel functions for two newly-identified genes in C. albicans, PXL1 and PTK2, and proposes a model for how Pxl1 is regulated. In addition, the generation of the first C. albicans-specific Ca2+ sensor, offers a new and informative method through which to examine calcium flux, channel function, homeostasis and signalling during responses to cell contact, cell stress and antifungal drugs.
Supervisor: Not available Sponsor: EU Marie Curie Initial Training Network ; Fungibrain
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Candida albicans