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The main aim of this project was to study Candida albicans cell wall biosynthesis in response to stress. The role of the MAPK, Ca2+/calcineurin and cAMP/PKA signal transduction pathways in regulating the C. albicans cell wall stress response was investigated. A library of mutants lacking receptors, signalling elements and transcription factors were screened for alterations in their ability to respond to a range of cell wall stressing agents, including CaCl2, Calcofluor White and caspofungin. Pretreatment of wild-type cells with CaCl2 and CFW, activates the Ca2+/calcineurin and PKC pathways, leading to an increase in chitin content, and reduced susceptibility to caspofungin. Although elevation of cell wall chitin content often resulted in decreased sensitivity to caspofungin, I show here that some strains with increased chitin levels remained sensitive to caspofungin. The results show that elevation of chitin is a common property of a range of mutants that are affected in coordinating cell wall stress pathways, but that multiple mechanisms are likely to operate in maintaining the robustness of the C. albicans cell wall. Some of the mutant strains of the MAPK, Ca2+/calcineurin and cAMP signalling pathways showed evidence of paradoxical growth, whereby less inhibition was achieved by higher concentrations of antifungal drug. The role of chitin-related genes and stress signalling pathways in regulating C. albicans paradoxical growth was also investigated. Based on these results, more detailed analyses were performed to investigate the correlations between sensitivity and resistance to caspofungin, in relation to paradoxical growth. The MAPK-Mkc1 and the calcineurin pathways played major roles in the paradoxical growth effect. There was a proportional relationship between echinocandin concentration and the chitin content of the cell wall although the chitin content did not continue to be upregulated by the highest echinocandin concentration. Different echinocandins, carbon source, cell morphology and medium composition influenced the extent of paradoxical growth effect. The existence of paradoxical growth in resistant strains such as Fks1 also highlights association of paradoxical growth with resistance mechanisms.

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published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

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Young developing fruits of nasturtium (Tropaeolum majus L.) accumulate large deposits of non-fucosylated "storage" XG in periplasmic spaces of cotyledon cells. The only XG that is clearly fucosylated in these fruits Is the structural fraction (approx. 1% total) integrated into growing primary walk. Storage XG can be fucosylated by a nasturtium transferase in vitro, but this does not happen in vivo, even as a transitory signal required for secretion which would subsequently be cleaved to produce mature non-fucosylated storage XG in the periplasmic space. The two fucosylated subunits that are formed in vitro are identical to those found in structural XG in vivo. A block appears to develop in the secretory machinery of young cotyledon cells resulting in extended galactosylation and diversion of XG traffic to the periplasm without fucosylation. The primary walls buried beneath accretions of storage XG eventually swell and lose cohesion, probably because they continue to extend without incorporating components like fucosylated XG that are needed for maintaining wall integrity.