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Fungal infections are a global threat, causing over 1.5 million deaths globally each year. Effective immunity from both innate and adaptive immune systems is essential for protection. Dendritic cells (DCs) are the bridge between innate and adaptive immune systems. They recognise fungal pathogens through C-type lectin receptors (CLRs), such as Dectin-1, then migrate to draining lymph nodes where they present antigen to T cells. The second signal required for successful T cell activation is from co-signalling molecules that are critical in shaping the T cell response. Loss of Dectin-1 on DCs during systemic Candida albicans infection in mice leads to increased fungal burden, and reduced CD4+ T cell activation and proliferation in the gut. The molecules on DCs that are involved in this aberrant T cell response were explored in this study. Using microarray and quantitative polymerase chain reaction analysis, a novel class of CLRs, were identified as downregulated on DCs from Dectin-1 knockout mice compared to wildtype mice. Furthermore, addition of Fc-CLR fusion proteins during in vitro T cell activation assays, showed that these CLRs have profound effects in modulating T cell activation. Additionally, the ligand for these CLRs was identified on all T helper cell subsets, including Th1, Th2, Th17 and Treg. Ligand expression was also identified on various cell types including epithelial cells, endothelial cells, stromal cells, CD4+ T cells and CD8+ T cells from multiple tissues including the gut, liver, pancreas, and lung, in both naïve and inflammatory conditions. Suggesting a broader role for these receptors in modulating the immune response. Finally, several methods were optimised to identify unknown ligands for these CLRs. Together these findings represent the novel discovery of CLRs as DC receptors, and their impact on T cells supports the identification of a novel class of co-signalling molecules. This has huge implications for development of therapeutics and vaccines for fungal infections and beyond.

Candida albicans is an opportunistic pathogen that lives as part of the mammalian commensal flora within mucosal passages, such as the gastrointestinal tract. C. albicans is a polymorphic fungus that can switch between yeast, true hyphal and pseudohyphal morphologies. Hyphae are associated with C. albicans virulence and possess hypha-specific proteins, such as Als3p, a multifunctional adhesin that is able to manipulate its own uptake by host cells and aids in the progression of C. albicans infections. C. albicans infections can range from superficial infections such as thrush to life threatening nosocomial bloodstream infections in immunocompromised patients, known as systemic candidiasis. There are more than 250,000 cases of systemic candidiasis worldwide each year with a mortality rate of 40%, even following administration of antifungal treatment. Haemostatic proteins have been demonstrated to bind to the surface of C. albicans. However, the implications of these binding interactions in respect to bloodstream infections, remain to be understood. Coagulation protein factor XII (FXII) is one of the main proteins which comprise the contact activation system. Once activated, FXII (FXIIa) is able to initiate several downstream pathways including the intrinsic coagulation cascade, fibrinolysis, the bradykinin pathway and the complement pathway. FXII is dispensable for haemostasis but contributes to the development of thrombosis. Research from the last decade has shown that FXII can increase immune responses including increasing monocyte and macrophage activity and phagocytosis as well as neutrophil migration. However, it is not known if FXII retains these attributes when bound to C. albicans. The current study aimed to identify the effect of FXII binding to C. albicans in the progression of systemic candidiasis. I have demonstrated that FXII binds specifically to hyphal morphologies, but not to yeast cells in both purified and plasma conditions. One of the main risk factors for mortality in systemic candidiasis is biofilm formation on catheters, giving a constant route for C. albicans escape into the bloodstream. I have demonstrated that FXII promotes biofilm formation on catheter-like surfaces and in contrast to this, biofilm formation was reduced in the presence of FXII deficient plasma. This suggests a role for FXII in biofilm formation. My investigations using C. albicans mutant strains has identified Als3p as a likely FXII binding ligand. Interestingly, FXII does not require zinc to bind to the hyphal surface despite it being well understood that FXII requires zinc for its activation. FXII also becomes locked in its zymogen form upon binding to the hyphal surface, even when in the presence of a number of its known activators. FXIIa also binds C. albicans hyphae, which retains its activity on the hyphal surface and can still be inhibited once bound. Collectively these data suggest that the hyphal surface is unable to mediate FXII activity or act as a constant 'on' switch for activation of FXII's downstream pathways. This differs from our understanding of bacterial interactions with FXII, suggesting FXII plays different roles between fungal and bacterial infections. Around 46% of plasma FXII binds hyphae but does not affect plasma clot lysis time, suggesting that FXII binding to hyphae does not impact normal clot degradation. Furthermore, this interaction augments macrophage engulfment time of C. albicans preincubated with FXII and FXIIa, in comparison to C. albicans without preincubation. Data from this project has shown that platelets do not aggregate on the hyphal surface, conversely, hyphae preincubated with FXIIa induced platelet aggregation. The immune data gathered in this study indicate a potential role for FXII and FXIIa in the immune response against C. albicans. I have identified several crucial points during the progression of systemic candidiasis where FXII could potentially impact the progression of C. albicans infection. Overall, this study has not only increased our current knowledge on the interactions of plasma proteins and C. albicans, but also highlighted an urgency to understand more on these binding interactions. I have shown that FXII binding to catheters can be beneficial to C. albicans in respect to the promotion of biofilms and therefore being detrimental to the host. In contrast, I have demonstrated that FXII may be increasing the immune response once bound to the hyphal surface, being favourable to the host. This study has identified a putative link between haemostasis and C. albicans bloodstream infections, influencing not only immune recognition but also biofilm formation and platelet aggregation, highlighting an urgent need to continue this field of investigation.

"Adaptive prediction" can evolve in microorganisms that repeatedly encounter the same two environmental stimuli in a conserved temporal order. Over an evolutionary timeframe, exposure to the first environmental condition can become associated with, and prime a response to, the second anticipated condition, thereby providing a fitness advantage. The opportunistic fungal pathogen Candida albicans has evolved anticipatory adaptive behaviours that increase virulence, immune evasion, and survival in the host. These behaviours reflect the environments where C. albicans has recently evolved, and examination of the pathways which regulate these adaptive responses have the potential to inform therapeutic strategies. Therefore, to help with the dissection of anticipatory responses, a fluorescence reporter system called the 'Rainbow System' was constructed. This system is designed to verify the order in which environmental conditions are temporally encountered by C. albicans in vivo. However, as discussed, further improvements are required before the system can be implemented. This study also examined one specific example of adaptive prediction in C. albicans. When this pathogen transits from the colon into the bloodstream it moves from a glucosefree niche to a glucose-containing environment where is likely to be attacked by circulating host phagocytes. Physiological levels of glucose enhance the resistance of C. albicans to oxidative stress and phagocytic killing. This glucose-enhanced oxidative stress resistance (GEXSR) is a likely example of adaptive prediction. C. albicans was shown to achieve this by accumulating the disaccharide stress protectant, trehalose, in response to glucose. This increase in trehalose accumulation, which appears to be controlled by the protein kinase A and glucose repression (AMPK) pathways, provides a significant fitness advantage during exposure to hydrogen peroxide in vitro and human neutrophils ex vivo. GEXSR might also promote host colonisation during systemic infection.

The fungal-pathogen Candida albicans causes invasive systemic infections and life-threatening biofilms associated with increasing mortality rates. Due to rapid emergence of resistance to anti-fungal drugs, in combination with a paucity of effective drug targets, treatment options for Candida-related infections are alarmingly inadequate. Heat shock protein 90 (Hsp90), a highly conserved regulator of virulence, morphogenesis, biofilm development, and drug resistance, holds potential as an indirect therapeutic target to enhance antifungal drug efficacy. Hsp90 is modulated via a network of less conserved cochaperones, which could provide potential targets for drug development if implicated in C. albicans virulence. When measuring expression of Hsp90 and five cochaperones at the transcriptional and post-transcriptional levels, Hsp90 and Sti1-TAP were differentially expressed between biofilm and planktonic filamentous conditions. Significant SBA1 upregulation was observed in biofilm, but not planktonic filamentous conditions which suggested involvement in either maintenance of mature biofilms or stress responses. Microscopy showed filamentation was not dependent on expression of Aha1, Sba1, or Sti1. Comparative analyses revealed TPR-containing cochaperones CPR6, CNS1, and CPR7 are differentially coregulated under various hyphal-inducing conditions. Furthermore, Hsp90 cochaperones exhibited differential regulation with regard to environmental stimuli, even in yeast-locked morphological forms. When larval models M. sexta and G. mellonella were challenged with cochaperone deletion mutants, no difference in mortality compared to wild-type-challenged larvae was observed. Thus, these studies present first evidence that C. albicans Hsp90 cochaperone transcriptional regulation is independent of morphogenesis but governed by environmental stimuli. Although Hsp90 modulates virulence and morphogenesis in C. albicans, Hsp90 cochaperones are not implicated in these processes. These findings reveal the transcriptional and post-transcriptional landscape of Hsp90 cochaperones under two virulence-associated conditions, report filamentation phenotypes of cochaperone deletion mutants, elucidate the impact of Hsp90 cochaperones on fungal virulence, and contribute to the promising development of Hsp90-inhibiting antifungal drug therapies to treat Candida-related infections.

Fungal disease remains a significant threat to human health. A coordinated and tailored immune response requires intact and controlled innate and adaptive immune defences. Phagocytosis is an integral part of the crucial first line defence of the innate immune response against fungi. This receptor-mediated process consists of various stages, including phagocyte migration towards sites of infection, internalisation of the pathogen via sequential PAMP-PRR interactions into an endocytic, membrane-bound compartment called a phagosome, phagosome maturation to form the microbicidal phagolysosome, and killing of pathogen or host cells by a combination of oxidative and nitrosative stresses. Traditionally, phagocytosis was studied using fixed time point experiments which failed to recognise the contribution of spatiotemporal dynamics to this host-pathogen interaction. Subsequent live cell imaging work did identify morphology, cell wall composition and spatial orientation as key determinants in the dynamics of Candida albicans phagocytosis. However, little is known about how the engagement of specific receptors influences these dynamics. Dectin-1 is a major non-opsonic phagocytic receptor on myeloid cells and has been shown to bind to ß-glucan in the fungal cell wall to mediate immune responses. This project dissects the spatiotemporal dynamics of C. albicans phagocytosis by peritoneal macrophages in the context of dectin-1 deficiency and altered ß-glucan exposure on the fungal cell wall. In dectin-1 knock-out macrophages, migration and phagosome maturation were delayed when phagocytosing wild type C. albicans yeast cells. The dectin-1 knockout population exhibited slower uptake of yeast during the initial stages of phagocytosis, but this was restored by 6 h leading to comparable final uptake and macrophage killing rates. Increased ß-glucan exposure arising from heat- and UV-killing of fungal cells or mutational disruption of the outer mannoprotein layer, led to enhanced migration and quicker phagosome maturation. However, engulfment was delayed in mannosylation mutants. Delayed phagosome maturation during the phagocytosis of glycosylation mutants in dectin-1 deficient macrophages, corresponded with an increased ability to lyse the host cell. Dectin-1-ß-glucan interactions therefore significantly influence the spatiotemporal dynamics of C. albicans phagocytosis in macrophages, affecting multiple stages in the pathogen-phagocyte interaction. Future work should further elucidate the underlying mechanisms and thereby identify targets that could be potentially exploited for therapeutic purposes in clearance of infection.

Fungal skin infections caused by dermatophytes or yeasts (e.g. Candida species) are one of the most common causes of human diseases. Dermatophytosis is caused by fungi that can degrade keratin, infecting the keratinised tissues such as skin, hair and nails. Although the infections are mild and rarely life-threatening, they are highly recurrent, and their incidence has increased continuously. Simultaneously, there is a growing resistance to antifungals, exposing, in particular, immunocompromised patients to higher levels of risk. This thesis is devoted to the study of polymers as physical barriers that can prevent and treat the early stages of skin infections and avoid the development of antifungal resistance and cross-contamination. To do this, a series of studies were undertaken to identify the ideal antifungal polymer. The first part of the thesis aimed to develop an ex vivo porcine skin model to study dermatophyte infections. The explanted porcine skin was sterilised by chlorine gas to remove contamination with other microbes. To characterise the model, adherence of Trichophyton rubrum was examined via adherence assays to the ex vivo skin, and this was compared to an in vitro model using HaCaT cells. Subsequently, the pathophysiology investigated by electron microscopy analysis. It was shown that the ex vivo model is particularly appropriate to the study the early stages of the fungal skin infection and to test novel antifungal regimes. In the second part of the thesis, two cationic polymers, ABIL T Quat 60 and Eudragit E100 were identified for their potential as barriers to skin infections caused by T. rubrum, Trichophyton interdigitale and Candida albicans. Both polymers were found to be fungistatic agents that can prevent and treat fungal skin infections in the porcine skin model. The results suggest that the mechanism of action of ABIL T Quat 60 is based on its ability to bind to the negatively charged fungal cell wall, followed by impeding the fungi to acquire crucial nutrients such as essential metal ions and carbohydrates. As for Eudragit E100, this polymer might bind to the fungal cell wall as well, but its antifungal activity was affected by the environmental pH. The third section aimed to evaluate the performance of these two polymers to form films in vivo, and the patient acceptability regarding these films. Following application as sprays to the arms of healthy subjects, the washability, drying time and stickiness of the films were tested for topical use. The same in vivo study characterised the tolerability of the films, in terms of the transepidermal water loss and skin redness. The results suggested the films to be well-tolerated and non-toxic, with no signs of skin irritation, barrier damage or skin occlusion. Apart from the antifungal activity of polymers, their antibacterial activities against methicillin-sensitive/-resistant Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were examined, to determine potential use to prevent wound infection. Only ABIL T Quat 60 showed an antibiofilm activity against S. aureus and E. coli but, surprisingly, stimulated the biofilm formation of P. aeruginosa. In addition to the two polymers, the antifungal activities against the T. rubrum of three common formulation ingredients, ethylenediaminetetraacetic acid, cetrimonium chloride and polyoxyethylene (10) tridecyl ether were established through a novel phenol red viability assay. Cetrimonium chloride was identified as fungicidal, whereas the other two compounds were fungistatic. The phenol red viability assay was further developed and enabled the study of the antifungal susceptibility in a mildly acidic environment optimised for dermatophytes growth, and to resolve technical challenges caused by the absorption of stains used in other types of assays by polymers.

Oral mucosal models are valuable tools for studying oral infection. Existing models have been implemented to study pathogen tissue invasion and resultant damage. These models fail to incorporate an innate immune component such as macrophages, essential for study of host response to infection. Macrophages exist within a regulatory to inflammatory polarisation spectrum, whereby the local microenvironment determines their activity, contributing to the overall behaviour of a tissue. Three-dimensional oral mucosal models were formed using primary (POK) or immortalized (HaCaT) keratinocytes seeded upon a human gingival fibroblast (HGF) embedded type-1 collagen matrix. Later, HaCaT based models (3DOMMs) were further developed to incorporate THP-1 pro-monocyte cells into the model matrix producing a novel immunocompetent oral mucosal model (IC3DOMM). Models were characterised using a variety of histological, biochemical, and molecular techniques to detail the model microenvironment and assess their capability to detect and respond to Staphylococcus aureus and/or Candida albicans infection. The HaCaT based 3DOMM was selected to further develop into an IC3DOMM, due to its superior epithelial barrier, and ability to upregulate both IL-6 and IL-8 in response to infection, when compared to the to the POK based P3DOMM. Model-incorporated THP-1 cells increased in size and altered morphology, suggesting differentiation into macrophages. The polarisation of THP-1-derived macrophages was not fully elucidated, as selected markers CD206 and CD163 did not sufficiently discriminate between THP-1 cells, PMA-derived, and VD3-derived macrophages. Newly highlighted, was the inflammatory environment of the 3DOMM at early and late stage culture. High levels of IL-6 and IL-8 production strongly correlated with LDH release, hence cell death. However this did not detract from the models’ ability to upregulate pro-inflammatory cytokine production in response to infection, and it is proposed that models are used at day 14 post production. Individual model cell types modulated IL-6, IL-8, and/or TNFα production upon stimulation with supernatant, killed cells, collagen, and differentiation reagents (PMA/VD3), demonstrating the complexity of the tissue-engineered environment. Despite this, it is necessary to consider the model as a whole, not as a sum of individual interactions, as the collective local microenvironment may modulate all cell types contained within the model. Due to the 3DOMMs intact epithelial barrier, ability to upregulate pro-inflammatory cytokine production in response to infection, and confirmed potential to incorporate THP-1-derived macrophages, it is proposed that the novel IC3DOMM is a suitable model for the study of host response to infection.

Candida albicans is an opportunistic pathogen that resides as a commensal colonising the mouth, gastrointestinal tract, moist areas of the skin and genital areas. It is kept under control by local and systemic immune defence mechanisms, by microbial control, and by restricting its access to deeper tissues by epithelial barriers. If this control is lost Candida becomes an opportunistic pathogen and cause superficial and invasive infections including candidaemia with high mortality rate. There are five antifungal drug classes used to treat Candida infections but the emergence of resistance is an increasing problem in patient management and treatment outcomes. Methods used to diagnose invasive infection usually rely on culture based methods with low sensitivity and long turn-around times. Therefore, more rapid and accurate molecular tools are needed to guide the choice of appropriate antifungal drugs and improve patient outcomes. This thesis addressed these issues by focusing on the epidemiology of Candida infections at a UK tertiary referral hospital, and next-generation sequencing (NGS) and pyrosequencing as two molecular tools for rapid detection of polymorphisms associated with host adaptation and drug resistance in C. albicans isolates. A Candida database for a 12- year period, obtained from Wythenshawe Hospital, was analysed. The main finding was the reduction of candidaemia cases with no emergence of echinocandin resistance. Then, genome sequences obtained from NGS of 14 isolates from autoimmune polyendocrinopathy candidosis ectodermal dystrophy (APECED) were analysed and variants associated with APECED were identified. These included single nucleotide polymorphisms (SNPs) in cell wall and DNA repair genes that could enhance their adaptation in APECED patients. Seven APECED isolates were pyrosequenced for 27 SNPs found in the multi-drug resistance regulator MRR1 gene and the results were comparable to the results obtained from NGS for the same isolates. Pyrosequencing of three (L171P, V341E and L592F) of these 27 SNPs was used to screen 100 isolates with variable azole susceptibility from the Mycology Reference Centre Manchester depository. This method was shown to be accurate and rapid to detect polymorphisms in C. albicans.

Candida albicans colonizes the gastrointestinal tract of up to 75 % healthy individuals. It usually cohabits the gut as an innocuous commensal. But in critically ill patients whose gut barrier, immune system and normal gut microbiota are compromised, C. albicans often transmigrates the gut barrier, transforms into an invasive pathogen and causes fatal systemic infections. The genetic transitions that drive this transformation in C. albicans have been a major focus of research and have led to the identification of key transcription factors that regulate this commensal-to-pathogen transition. However, the current challenge lies in identifying the downstream pathways and effectors that bring this transition into effect. This thesis addressed this challenge by developing 11 new bioinformatics tools, including 6 comprehensive databases, 4 novel software packages and 1 analysis framework. These databases included a comprehensive topological map of the mammalian gut biogeography, a C. albicans microarray database comprising of 3,091 publically available microarray transcript profiles, C. albicans RNA-seq gene expression and small variant databases extracted from 1,177 publically available RNA-seq samples, a C. albicans gene alias database comprising of 113,297 gene aliases representing the 6,735 open reading frames of C. albicans, and a C. albicans gene ontology slim comprising of 1,194 C. albicans-specific gene ontology terms. These databases were accompanied by a robust analysis framework which brought together these resources for quality control, batch correction and weighted gene co-expression network analysis. All these tools were finally employed in a pilot exploration of the C. albicans gut commensal-to-pathogen transition, which demonstrated the effectiveness of these bioinformatics resources. The analysis unveiled known regulators, uncharacterized gene networks, pathways and effectors potentially crucial for the C. albicans gut commensal-topathogen transition. These resources are a step towards a better understanding of this transition and can also be utilized for examining various other aspects of C. albicans biology.

Candida species are amongst the most frequent causes of life-threatening fungal infections worldwide. Current knowledge of anti-Candida immunity has been gleaned almost exclusively from studies of Candida albicans, the most commonly isolated pathogenic Candida species. It is becoming clear that C. albicans is a moving target for the immune system as it adapts to the microenvironments in human niches. To survive and grow in these niches the fungus adapts to changes in local nutrients and stress imposed by the host. This thesis addresses this adaptation and the resultant effects upon fungus-host interactions. C. albicans mounts robust oxidative stress responses in an attempt to protect itself against environmental challenges that include phagocytic attack. These responses involve the induction of CAT1, which encodes catalase, an abundant ferro-enzyme that detoxifies hydrogen peroxide. Constitutive CAT1 expression has been shown to enhance oxidative stress resistance and killing by phagocytic cells, and yet C. albicans has not evolved to express the high levels of catalase that mediate this protection. To investigate why, the fitness of isogenic barcoded C. albicans mutants expressing different levels of catalase was compared in the presence and absence of oxidative stress. As expected, C. albicans mutants overexpressing CAT1 were more resistant to high doses of oxidative stress. Also, clinical C. albicans isolates that are relatively resistant to oxidative stress generally displayed high basal CAT1 expression levels. However, in the absence of oxidative stress, CAT1 overexpression reduced the fitness of C. albicans. CAT1 overexpression was shown to perturb the expression of iron homeostasis genes, and the fitness defect was suppressible by iron supplementation. I also showed that CAT1 is not required for C. albicans virulence in murine models of systemic candidiasis. I concluded that, while catalase overexpression protects C. albicans from host-imposed stresses, it places demands on iron scavenging from the host. Changes in nutrients, thermal fluctuations and morphogenetic signals have been shown to trigger alterations in the C. albicans cell wall, which contains important immune stimulatory pathogenassociated molecular patterns (PAMPs) such as β-glucan. Exposure to host-derived lactate, for example, is known to induce β-glucan masking at the C. albicans cell surface, which attenuates the phagocytic response. Therefore, the impact of iron depletion upon C. albicans-host interactions was investigated using a combination of flow cytometry, phagocytosis assays and fluorescence and transmission electron microscopy. Iron-deprivation was found to trigger an increase in cell wall thickness and a reduction in β-glucan exposure. These cell wall changes appeared to be mediated by parallel signalling pathways: one involved the iron regulators, Ftr1 and Sef1, and the other required protein kinase A (Tpk1, Tpk2). Both pathways were essential for β-glucan masking in response to iron deprivation. These cell wall changes affected C. albicans-host interactions as they correlated with reduced fungal phagocytosis by murine bone-marrow derived macrophages (BMDMs) and less inflammatory responses from human peripheral blood mononuclear cells (PBMCs). C. albicans must also adapt to oxygen poor niches in the host, such as the gut, and hypoxia has been reported to affect the cell wall proteome. Therefore, I tested whether hypoxia affects cell wall architecture and PAMP exposure. Hypoxic C. albicans cells were found to have thinner cell walls than normoxic cells, and to expose less β-glucan at their cell surface. This hypoxia-induced β-glucan masking was dependent on mitochondrial signalling, which apparently involved the production of reactive oxygen species in the intramembrane space, and also upon downstream cAMP-PKA signalling. Hypoxia-induced β-glucan masking correlated with decreased cytokine responses from human PBMCs and reduced rates of phagocytosis by murine BMDMs. Taken together, the data suggest that C. albicans has evolved to exploit a range of host-derived signals to modulate the exposure of a major PAMP on its cell surface and thereby attempt to evade phagocytic attack. This is likely to contribute to the success of C. albicans as a commensal fungus and as an opportunistic pathogen.

Chitin synthesis and septum formation are highly regulated processes, fundamental for cell viability, taking place during cell division. Understanding fungal septation may lead to the identification of novel drug targets for future antifungal chemotherapeutic strategies. In the pathogenic fungus Candida albicans, a primary septum consisting of chitin is formed between two dividing cells. In C. albicans, chitin is synthesised by four chitin synthases: Chs1, Chs3, Chs2 and Chs8 and all four enzymes localise to sites of septation. Strains expressing pairs of fluorescently tagged chitin synthases were generated and assessed by live-cell fluorescence microscopy to elucidate the temporal and spatial distribution of the chitin synthases in relation to each other. It was observed that the timing and pattern of configuration of the chitin synthases during septation is different in yeasts and hyphae with Chs3 potentially playing a role in secondary septum formation. This information was used to generate a new model for septal chitin synthesis during septation in C. albicans yeasts and hyphae. Proteomic analyses of GFP pull down assays identified further proteins that may be part of the septation complex. Through Chs3-GFP immunoprecipitation, it was shown that Chs3 may interact with different sets of proteins in yeasts versus hyphae. Moreover, the localisation of the chitin synthases in the absence of CaCdc10 and CaBni4 whose homologues are involved in tethering of ScChs3 at the septation site of S. cerevisiae was assessed. This study demonstrates that the septation model regarding the regulation and tethering of the chitin synthases to septation site is different between C. albicans and S. cerevisiae yeast cells, as well as between C. albicans yeasts and hyphae. This study has extended our knowledge of how chitin synthesis is orchestrated during septation and identified proteins that might be involved in septum formation.

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis in developed countries. In the majority of cases, GBS is vertically transmitted from the mother during or preceding birth. Candida albicans is an opportunistic fungal pathogen of the female GU tract, causing vaginal thrush, for which pregnancy is a risk factor. As C. albicans is known to synergistically interact with Streptococcus bacteria within the oral cavity, it was hypothesised that C. albicans and GBS may interact within the vaginal tract. Using a vaginal epithelial cell association assay, it was shown that C. albicans significantly promoted GBS association with vaginal epithelial cells (VECs) and, likewise, GBS significantly promoted C. albicans. The AgI/II family surface-expressed adhesins of GBS, designated Bsp proteins, were found to contribute to GBS interactions with VECs and with C. albicans, while the C. albicans cell surface protein Als3 was pivotal for the coassociation between these species. Investigations into the VEC response to GBS and C. albicans implied that coassociation may reduce neutrophil chemotaxis, despite enhanced transcription of proinflammatory cytokine genes. Proteomics studies revealed that extracellular matrix (ECM) components, or proteins that modulate ECM components, were significantly elevated in dual-species-infected VECs, while apoptosis related proteins and proteins involved in MAPK signalling were largely downregulated. Taken together, these data suggest that GBS and C. albicans synergistically interact in a manner that could promote GU tract colonisation and persistence, and that this coassociation is dependent on C. albicans Als3 and partially dependent on GBS Bsp proteins.

Phagosome maturation is a dynamic process involving the engulfment and degradation of pathogens by phagocytic cells. However, several pathogens have employed mechanisms that facilitate their survival and escape from the phagosome. The fungal pathogen, Candida albicans, is capable of switching from yeast to hyphal form to facilitate its pathogenicity and escape from the phagosome. Rab GTPases are key regulators in phagosome maturation by mediating interactions with the endocytic pathway and leading to biogenesis of the phagolysosome. The temporal localisation dynamics of Rab2 on maturing phagosomes containing live C. albicans was investigated. Live-cell imaging revealed green fluorescent protein (GFP)-tagged Rab2 was recruited to C. albicans-containing phagosomes. Rab2 appeared on phagosomes within 2 min following complete engulfment of C. albicans. Rab2 persisted transiently on macrophage phagosomes and this correlated with the length of C. albicans hyphae at the time of uptake, suggesting C. albicans morphology modulates Rab2 localisation dynamics. Expression of dominant negative or dominant active Rab2 did not affect macrophage migration, the rate of engulfment or phagosome acidification during the early stages of phagosome maturation. Furthermore, altered expression of Rab2 did not interfere with C. albicans ability to escape from and kill macrophages, suggesting Rab2 is not involved in the outcome of the host-pathogen interaction. However, altered expression of Rab2 reduced the acquisition of the late-stage phagosome maturation markers, cathepsin B and LAMP1, suggesting Rab2 impacts upon phagosome-lysosome fusion. Finally, the uptake of other particles by macrophages revealed Rab2 recruitment, as well as localisation dynamics on phagosomes, may be cargo-dependent. Through the use of live-cell imaging, real-time dynamics of phagosome biogenesis in live cells was examined, offering unique insight at the host-pathogen interface.

Candida albicans is an opportunistic fungal pathogen that can cause systemic infections in immunocompromised patients, where the formation of invasive hyphal filaments contributes to fatal levels of sepsis and organ failure. Hypha-mediated tissue damage depends on directional growth responses that are regulated by the small NRas-like GTPase, Rsr1. In S. cerevisiae, Rsr1 is K-Ras-like and specifies the yeast bud site where it is activated by its guanine-nucleotide exchange factor (GEF), Bud5, and de-activated by its GTPase activating protein (GAP), Bud2. However, its role in C. albicans hyphae is less understood. To elucidate the role of Rsr1 in hyphae, we generated mutant strains expressing a single copy of Rsr1 in a GDP-locked (Rsr1K16N) or GTP-locked (Rsr1G12V) form and compared their phenotypes with those of the rsr1?/?, bud5?/? and bud2?/? null strains. Deletion or dysregulation of Rsr1 caused large, rounded yeast cells and shorter hyphae that were unable to steer round obstacles or penetrate agar. Strikingly, these defects were seen in the strain expressing Rsr1K16N but not in the bud5?/? null, suggesting that Rsr1 may be regulated by a second GEF. Deletion and mis-regulation of Rsr1 caused abnormal intracellular organization in yeast and hyphae, and cells were multinucleate and exhibited vacuolar fusion defects. The site of Rsr1 activity is thought to be regulated by the localization of the Bud5 and Bud2 regulators at the tip and subapical region of hyphae, respectively. Here we show that YFP-Rsr1K16N and YFP-Rsr1G12V were differentially localized. YFPRsr1K16N appeared as patches on the plasma membrane whereas YFP-Rsr1G12V was uniformly localized to the plasma membrane, similar to WT. Proteomics results using heterologously-expressed GST-tagged mutant proteins with yeast and hyphal lysates showed that activity state and cell morphology differentially determined the binding partners of Rsr1. These findings suggest a more complex role for the N-Ras Rsr1 GTPase in C. albicans than has been described for the K-Ras Rsr1 GTPase in the yeast, S. cerevisiae.

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.

Candida albicans is a human commensal organism which can cause superficial, systemic and deep tissue infections in otherwise healthy individuals. It is well-known that eukaryotic cells including C. albicans regulate essential gene expression programs during stress and infection. Although several studies have investigated the transcriptional and proteomic changes during stress conditions in C. albicans, few studies have assessed the role of protein synthesis, which is the focus of this thesis. This study has examined the regulation of translation initiation mediated by the Gcn2 and Caf20 translation factors during amino acid starvation and oxidative stress conditions. Gcn2 is the sole C. albicans kinase that phosphorylates eIF2alpha, which is an essential translation initiation factor. Caf20 is a putative eIF4E-binding protein (BP) that can competitively inhibit the interaction of eIF4E with eI4G and reduces eIF4F complex formation. We show that amino acid starvation induced by 3-amino-1,2,4-triazole (3AT) and oxidative stress (H2O2, cadmium and diamide) cause an inhibition of translation initiation in C. albicans. Translation initiation is predominantly blocked via the action of GCN2 during 3AT, H2O2 and cadmium stresses, whereas, diamide inhibits initiation in a Gcn2-independent manner. During this global inhibition of protein synthesis, a major transcriptional activator, CaGcn4, is activated. We mapped the transcriptional start site of GCN4 and identified three upstream open reading frames (uORFs) present in the 5'-untranslated region (5'-UTR) of GCN4. The 5'-UTR of the GCN4 mRNA was cloned upstream of a Renilla luciferase reporter gene and used to study translational control of GCN4 under stress conditions. During amino acid starvation conditions, GCN4 is regulated both at the transcriptional and translational levels. Transcriptional regulation is mediated in a Gcn2-independent manner, whilst translational regulation is activated via Gcn2-mediated phosphorylation of eIF2alpha. A single uORF3 is shown to be necessary and sufficient for translational regulation of GCN4. This novel translational control mechanism involves leaky scanning past uORF3 under stress conditions. During hydrogen peroxide and cadmium stress conditions, GCN4 is also predominately regulated at the translational level in a Gcn2-dependent manner. Additionally Gcn4 is required for tolerance and adaptation to H2O2 stress. In contrast, Gcn4 and Gcn2 are not required during diamide stress and this correlates with a low level of translation regulation of GCN4. We discuss the similarities and differences in oxidant-specific regulation of translation initiation between S. cerevisiae and C. albicans. In the final part of the thesis, we studied the single putative eIF4E-BP identified by the C. albicans genome project, namely CAF20. CAF20 was successfully deleted in C. albicans and wild-type CAF20 and its 4E-BP mutant (CAF20m) reintegrated in the mutant. Caf20 was directly confirmed to be a 4E-BP using cap affinity chromatography. Caf20 expression was also shown to be elevated in response to an increase in growth temperature. Despite a number of experimental approaches being used, including growth analysis, polysome analysis, stress sensitivity tests and hyphal growth studies, no phenotypic differences were detected in the caf20 mutants compared with a wild-type strain. Thus, it is unclear, what if any functional roles Caf20 plays in C. albicans. We suggest that future studies could be performed to assess the levels of eIF4F complex formation, and to investigate stress sensitivity and hyphal formation during diverse stress conditions such as nutritional limitations, osmotic stress, endoplasmic stress and pH alterations.

Candida albicans is a polymorphic yeast that can cause life threatening systemic infections in immuno-compromised individuals. One key attribute of C. albicans that enhances its pathogenicity is the ability to switch morphologies between filamentous and vegetative modes in response to specific environmental conditions. Stressful changes in such cellular conditions commonly cause a rapid inhibition of global protein synthesis leading to altered programmes of gene expression. In Saccharomyces cerevisiae, fusel alcohols signal nitrogen scarcity and induce pseudohyphal growth enabling yeast colonies to spread towards nutrient replete areas. These alcohols also inhibit protein synthesis by targeting the translation initiation factor, eIF2B. eIF2B is the guanine nucleotide exchange factor for eIF2, which supports eIF2-GTP production and represents a key regulated step in translation initiation. eIF2-GTP interacts with Met-tRNAiMet to form the ternary complex which is essential for translation initiation. Fusel alcohols target eIF2B leading to reduced levels of ternary complex and reduced protein synthesis. In Candida albicans, a variety of cell biological and genetic assays suggest that fusel alcohols and ethanol inhibit protein synthesis by targeting the translation initiation factor, eIF2B, and they also induce hyphal/pseudohyphal growth, a process that is associated with pathogenesis in C. albicans. In contrast to fusel alcohols, farnesol, aquorum sensing alcohol, does not appear to impact upon eIF2B activity. Rather, biochemical and mass spectrometric analysis suggest farnesol affects the interaction of the mRNA with the small ribosomal subunit during translation initiation. Further elucidation of the effect of farnesol on C. albicans transcript levels and ribosome association by next generation sequencing gave insight into the genes that are differentially expressed following farnesol treatment. While genes involved inmorphological differentiation were generally repressed, those involved in protein synthesis were upregulated, possibly as an adaptive response to inhibition of protein synthesis by farnesol. Intriguingly, the regulation of these functional categories of genes occurred in a co-ordinated manner at either the transcript level or at the level of ribosome association, but rarely was gene expression regulated at both transcriptional and post-transcriptional levels for the same gene.

One of the major aims of this thesis was to identify novel regulators that drive C. albicans adaptation during growth under different nutrient and temperature conditions. The classical stress response cascades have been previously characterised under standardized, but physiologically irrelevant growth conditions (YPD at 30°C). In this study these pathways and other regulators were examined under more physiologically relevant inputs because metabolic plasticity and thermo-tolerance have been shown to affect stress adaptation (Arguelles et al., 1999; Brown et al., 2014; Cowen, 2009; Diezmann et al., 2014). In this study, we characterized 18.5% of the functional C. albicans ORFeome under 144 different stress conditions by employing a standardized system of robotic screening (Chapter 3). These screens highlighted extensive carbon and temperature-conditional regulators in C. albicans. We identified carbon-conditional contributions of the transcriptional regulators Sfp1 and Rtg3 to stress adaptation in this pathogenic fungus (Chapter 4). Sfp1 was found to regulate the expression of key stress regulators during growth on glucose, whereas Rtg3 induced the expression of these stress genes during growth on lactate. Our screens also revealed a distinct set of transcription factors, Hap43, Swi4, Sfp1, Cap1 and Zcf31, that control regulators of cell wall integrity and that promote antifungal drug resistance in a temperature dependent and yet Hsp90- independent manner. The screens also provided new information about a relatively obscure group of transcriptional regulators in C. albicans; the zinc cluster proteins with focus on Zcf3 and Zcf18 which we further pursued with RNA-sequencing to establish them as modulators of cell cycle, stress resistance and virulence in C. albicans. Lastly, our screens reveal a network of regulators that are homologous to human oncogenes and control fungal growth via modulation of TOR signaling. In conclusion, this thesis has revealed many novel targets for possible antifungal drug development and highlighted the extensive and intricate cross-talk between stress response modules facilitated by physiologically relevant nutrient sources and ambient temperatures.

Allelic expression imbalance, or AEI, is the term given to differences in the expression levels of the two alleles of a gene. AEI has been previously identified in a number of species using various techniques. Here, the genome-wide extent of allelic expression imbalance in the pathogenic yeast species, Candida albicans, was examined through use of RNA sequencing in combination with a novel computational pipeline based around the diploid reference genome. Techniques for validating these results were investigated, and the difficulties surrounding specificity and quantification are discussed. As C. albicans is a highly heterozygous species, it was hypothesised that polymorphisms within alleles lead to differences in allele expression, which are further linked to differences in allele function. The functional consequences of AEI were therefore interrogated through investigation of Gene Ontology, identification of condition specific responses in AEI, and targeted construction and phenotypic screening of heterozygous knockout strains. Together, these results strongly suggest that divergence in allele expression is not linked to differences in allele function. Investigations of the possible control mechanisms behind the differences in allele expression were considered, with a focus upon structural factors such as chromosomal location, GC content, allele length and codon usage. However, issues with establishing causality are present, and difficulties lie in distinguishing between functional differences and consequences of bias in sequencing technologies. This piece of research has advanced the understanding of gene expression mechanisms within a medically important pathogen, paving the way for further investigations into the functional consequences of allelic expression imbalance in Candida albicans.

The virulence of the opportunistic fungal pathogen Candida albicans is in part due to its ability to switch between a yeast and hyphal form; permitting physical rupture and escape from macrophages after phagocytosis. This interesting feature makes C. albicans a good model organism to study in the context of phagosome maturation and in particular, the role of the small GTPase Rab14 in this process. Rab14 is recruited to bacterial phagosomes containing Chlamydia trachomatis (Capmany & Damiani, 2010) and Mycobacterium bovis BCG (Kyei et al, 2006) and aids their survival in macrophages but its role in phagosomes containing fungal pathogens is not known. Here, an important role for Rab14 in protecting macrophages against hyphal mediated lysis by C. albicans is demonstrated. Macrophages were transfected to express eGFP-Rab14, or dominant negative variants (eGFP-Rab14 S25N and eGFP-Rab14N124I); or were transfected with anti-Rab14 siRNA; then infected with live C. albicans and observed using sophisticated live cell imaging and analysis tools. Phagosomes containing live C. albicans became transiently Rab14-positive within 2 minutes following engulfment. Interestingly, a prolonged retention of Rab14 on phagosomes depended on C. albicans morphology. Phagosomes containing hyphal forms retained Rab14 for twice as long as for phagosomes containing the yeast form. Depletion of endogenous Rab14 did not affect macrophage migration towards C. albicans, the rate of engulfment or acquisition of markers of early phagosome maturation to phagosomes containing C. albicans. Furthermore, reduced Rab14 expression did not influence the kinetics of Rab5 localisation to phagosomes in macrophages that were co-transfected. Importantly, partially depleting Rab14 delayed the appearance of late phagosome maturation indicators LAMP1 and activated cathepsin in phagosomes containing live C. albicans.Rab14 knockdown was associated with a significant increase in macrophage killing by C. alibica The data presented in this thesis demonstrate the dynamic relationship between host and pathogen which can be visualised in real time at the level of individual phagosomes. Rab14 plays an important role during phagosome maturation which impacts on the later stages of phagosome maturation and is important for phagocyte survival after phagocytosis of C. albicans.

During the infection process Candida albicans has to respond to various stresses imposed by host environment including oxidative and osmolarity stress generated by phagocytic cells such as macrophages and neutrophils. Exposure to caspofungin and other antifungal antibiotics also imposes stress on the C. albicans cell wall. These various stress responses are orchestrated through the activation of multiple stress pathways including the cAMP-PKA, several MAPK pathways and the Ca2+-calcineurin pathway which influence cell wall shape and composition. Such changes were predicted to influence recognition of C. albicans by innate immune cells. During my Ph.D. studies I primarily investigated the effect of the activation or inhibition of these pathways on the interaction with the innate immune cells by examining phagocytosis, the cytokine profile induced by mononuclear and polynuclear cells of the innate immune system. I found that the activation and inhibition of these pathways plays an important role in remodeling of cell wall and hence the immunological profile. Inactivation of cAMP, Calcium signaling pathway by the deletion of TPK1 and CNA1 resulted in marked reduction in pro-inflammatory cytokine production. Inactivation of MAPK pathway by deletion of HOG1 altered major pro-inflammatory cytokine secretion. Cytokine production was also affected by exposure of C. albicans signaling mutants to Calcofluor White, caspofungin, oxidative and osmotic inducing stresses. Cytokine stimulation was also affected by deletion of URA3, exposure of C. albicans to rifmapicin and antimycin A. These results suggest that stress signaling pathways act to regulate collateral changes in the cell wall, which in turn affects the immune reactivity. Pro and anti-inflammatory cytokine and antifungal profiles of Candida haemulonii was also found to be highly variable. Thus regulation and exposure to different microenvironments significantly modifies immunological signature of fungal cells, suggesting that responses to local stresses make the fungal cell surface a moving target for immunological surveillance.

Candida albicans is a member of the human microbiota and normally maintains a commensal relationship with the host. However, C. albicans is an opportunistic pathogen and can cause both mucosal and life threatening systemic infections. Dectin-1 is a pattern recognition receptor and part of the innate immune system. Although Dectin-1 is essential in controlling a number of fungal infections its exact role during C. albicans infections is controversial due to two conflicting reports from the Iwakura and Brown groups. This thesis demonstrates that this controversy is due, at least in part, to strain specific adaptions to the host, resulting in the modulation of cell wall content and structure, in particular chitin levels. Having resolved this controversy the role of Dectin-1 in controlling C. albicans growth within the gastrointestinal tract following systemic and oral infection is determined. Following systemic infection Dectin-1 plays an essential role in the innate immune response controlling fungal growth, with Dectin-1 deficiency leading to increased fungal burdens and aberrant cytokine responses. Surprisingly, no requirement for Dectin-1 in the control of C. albicans colonization of the GI tract was demonstrated following oral infection. Thus in murine models Dectin-1 is essential for controlling systemic C. albicans infection, but appears to be redundant in controlling C. albicans colonisation of the GI tract following infection by the oral route. How C. albicans morphology affects colonisation of the GI tract and host immune responses are poorly understood. Here it is demonstrated that filamentous morphologies are less able to colonise the GI tract than yeast forms, with a yeast favouring environment existing within the GI tract. However, limitations of the models used here cloud the exact immune responses to C. albicans morphological forms but give valuable insights into the complexity of host pathogen interactions. The results presented here provide substantial new insights into C. albicans adaptions to the host and have important implications for understanding susceptibility to and treatment of C. albicans infections.

Chitin biosynthesis in the fungal cell wall is essential for cell viability. Chitin play roles in maintaining cellular integrity and up regulation of chitin synthesis helps protect the cell from cell wall stresses or cell wall damaging antifungal agents such as caspofungin. In Candida albicans chitin is synthesised by four chitin synthases (Chs), Chs1, Chs2, Chs3, and Chs8. The biological function of Chs2 and Chs8 (class I chitin synthases) is less well understood. The major objective of this thesis was to understand the biological function of the class I chitin synthases. Chs2-YFP and Chs8-YFP showed a dynamic localisation at septation sites, which were first visualised as a bar which then contracted to a spot. This spot then separated into two spots, one on each sides of the septum. These two spots remained there until yeast cell separation, and remained at this location throughout several subsequent hyphal cell cycles. Chs2-YFP also localised to hyphal tips. The phenotype of a chs2Δchs8Δ double mutant was re-investigated using the propidium iodide. Intact dead germ tubes and hyphal tip lysis was observed in a chs2Δchs8Δ mutant cells. This suggested that Chs2 and Chs8 play a major role in the maintenance of hyphal tip integrity and polarised growth and perhaps a minor role in septum formation. Studies were also performed to assess whether phosphorylation regulated the localisation of Chs2-YFP. It was shown that the localisation of Chs2-YFP to septation sites was regulated by phosphorylation on S222. A version of Chs2-YFP that could not be phosphorylated (Chs2S222A-YFP) localised at the septa in lower amounts than the Chs2-YFP, and a version of Chs2-YFP that mimicked constitutive phosphorylation (Chs2S222E-YFP) localised normally. This suggested that phosphorylation of Chs2 on S222 facilitates the localisation of Chs2-YFP at septation sites, and that dephosphorylation is not required for this cellular localisation. In the presence of cell wall stresses (CaCl2/CFW) and caspofungin, more Chs2-YFP was observed and the average intensity of fluorescence of Chs2-YFP was higher in the presence of these stresses than in untreated cells.

Diseases caused by fungi are increasing worldwide and are often associated with high mortality rates. In particular, the normally harmless commensal Candida albicans can cause serious disease if immunological and physiological barriers are perturbed, leading to systemic infection, which is fatal in up to 45% of cases. The adaptive immune response is believed to be important in protection against systemic candidiasis, however, the roles of different helper T (Th) cell subsets, particularly Foxp3+ regulatory T (Treg) cells, remain largely unexplored. The aims of this study were to adapt a mouse model of systemic C. albicans infection to test whether the numbers of Th1, Th2, Th17 and Foxp3+ Treg cells increase in mice with systemic C. albicans infection, and determine their contribution to disease. C. albicans drove the expansion of Th1, Th2 and Th17 cells, as well as multiple Foxp3+ populations that displayed characteristics of natural Treg, induced Treg, Th17 and Th1 cells in vitro and in vivo. The expanded Foxp3+ T cells inhibited Th1 and Th2, but promoted Th17, responses to C. albicans antigens in vitro and exacerbated disease, since their depletion in vivo reduced kidney fungal burden and inflammatory lesions. Furthermore, systemic infection with a weakly virulent C. albicans strain was associated with reduced Treg responses compared to those induced during lethal systemic infection. These data lead to a model for systemic candidiasis whereby Treg expansion promotes Th17 responses that drive pathology, and have implications for future immunotherapy.

Candida albicans is a human commensal organism resident on mucosal surfaces, but also can be an opportunistic pathogen when the host's immune system is impaired causing superficial and systemic infections. As a pleomorphic organism, C. albicans can switch between different forms including yeast, pseudohyphae and true hyphae and this morphological plasticity is considered to be an important virulence factor. It has been proposed that yeast cells were adapted for systemic spreading while hyphal forms may be required for tissue invasion and escape from immune cells. Studies using C. albicans monomorphic mutants showed that both yeast and filament (pseudohyphae and true hyphae) forms are needed for full virulence as the inability to switch from a yeast to a filament cell (or vice versa) resulted in a reduced virulence or avirulence of monomorphic mutant strains. In addition, the population of C. albicans found at the sites of infection is a mixture of all three forms. These observations confirmed the importance of both yeast and filament forms, however the individual role of each form in C. albicans pathogenesis is not well known. Although present at sites of infection with yeast and hyphal cells, the role of pseudohyphae in C. albicans virulence and immune recognition has not been carefully examined. This is a significant gap in our understanding of C. albicans pathogenesis and immune response stimulation. The aim of this project was to investigate the relevance of yeast to filament (pseudohypha or hypha) switching in the recognition of C. albicans by human innate immune system. In this project I addressed two main questions: (1) what differences exist in innate immune response to C. albicans cells of different shape? and (2) if so, what is the basis of such differential immune responses? To investigate the innate immune response to C. albicans cells of different forms, cytokine production by human peripheral blood mononuclear cells (PBMCs) was stimulated by coincubating PBMCs with either yeast, pseudohyphal or hyphal cells and comparing cytokine responses to different C. albicans cells. Cytokine responses to a mixed population of C. albicans cells were also analysed by co-incubating PBMCs with mixtures of yeast with pseudohyphae or yeast with hyphae. The results showed that C. albicans yeast cells induced a significantly higher TNF-a, IL-10, IL-1ß, IL-1a and IL-6 responses than hyphal cells and these responses were neither strain nor growth condition dependent. C. albicans pseudohyphae stimulated intermediate cytokine responses between those stimulated by yeast cells and hyphae. C. albicans cell wall integrity and mannosylation were found to be important for the induction of cytokine responses. In addition, the results from this project showed that C. albicans filaments (pseudohyphae and hyphae), when co-incubated with yeast cells, had the capacity to suppress the immune response associated with the normal recognition of yeast cells by immune cells. The cell wall is the external component of the C. albicans cell and is the immediate point of contact between C. albicans and host cells. To determine the basis of differential immune response to yeast, pseudohyphal and hyphal cells, the cell wall composition of these three forms of C. albicans was analysed. Yeast, pseudohyphal and hyphal cell wall carbohydrate contents were analysed by HPLC and the porosity of cell wall was used to assess differences in cell wall permeability. The cell walls of all three forms of C. albicans were composed of glucan, mannan and chitin in different proportions. Yeast cell walls contained more mannan, less glucan and less chitin than hyphal cell walls. Pseudohyphal cell walls contained intermediate mannan and chitin content between those of yeast cells and hyphae. The results from this project also demonstrated that mutations in cell wall glycosylation and cell wall proteins differentialy affected C. albicans yeast, pseudohyphal and hyphal cells. In conclusion, results from this project confirmed that there is a differential innate immune response to C. albicans cells of different forms (yeast, pseudohypha and hypha). These differences in immune recognition of C. albicans maybe related to differences in cell wall such as gross cell wall composition, porosity and permeability and hence accessibility to pattern recognition receptors of myeloid cells. This project established that morphogenesis plays an important role in the recognition of C. albicans by cells of human innate immune system. Therefore C. albicans may use the ability to switch between different forms (recognised differently or not at all) to avoid detection by the host immune system.

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.

Fungal nitrosative stress responses are understudied and poorly understood. Also, while the responses of the pathogen Candida albicans to individual oxidative and cationic stresses have been characterised relatively well, the responses of this pathogen to simultaneous combinations of these and nitrosative stresses have not been elaborated. Yet, C. albicans is exposed to these combinatorial stresses during disease progression. Therefore, the overall aim of this project was to characterise the "Nitrosative and Combinatorial Stress Responses in Candida albicans". The first objective was to elucidate nitrosative stress response mechanisms in C. albicans at a cellular, transcriptional and post-translational level. Genetic screens revealed that the Hog1 and Mkc1 signalling pathways contribute to nitrosative stress adaptation in addition to Cta4 signalling. The next objective was to characterise responses to combinatorial nitrosative, oxidative and/or cationic stresses with respect to the relevant signal transduction pathways. Unexpectedly, combinatorial stresses did not activate classical nitrosative (Cta4) and oxidative (Cap1) signalling mechanisms. Instead, Hog1 signalling was found to play a significant role in the combinatorial stress adaptation. Dramatic changes in the GSH redox potential were observed in response to both single and combinatorial stresses. Thus, GSH redox regulatory mechanisms were explored in more detail. BLAST searches of the C. albicans genome were performed, revealing genes encoding a putative NADPH-dependent glutathione reductase (Glr1) and a GSH-dependent S-nitrosoglutathione reductase (Fdh3). We confirmed that both conserved enzymes are involved in the maintenance of redox homeostasis and hence are crucial for C. albicans adaptation during host-pathogen interactions. This was achieved by showing that inactivation of Glr1 or Fdh3 disturbs GSH redox homeostasis, confers oxidative and formaldehyde stress sensitivity and attenuates virulence.

Candida albicans, a successful human pathogen, displays the phenomenon of glucoseenhanced oxidative stress resistance (Rodaki et al., 2009), which is not observed in other yeast species tested. The molecular bases of the phenomenon are not clear. It was suggested that glucose signalling might play a major role. Therefore, the impact of specific C. albicans mutations was tested to determine which of three known major glucose signalling pathways are required for glucose-enhanced oxidative stress resistance. Two major glucose signalling pathways were found to contribute to the phenomenon (the glucose repression pathway and the cAMP pathway), and a third pathway (the SRR pathway) is not essential for this response. The next step was to identify targets of these pathways that might contribute to the phenotype. First, it was tested whether known oxidative stress systems contribute to the GEXSR. The selected targets represented almost all main systems involved in redox control and ROS detoxification (catalase, superoxide dismutases, thiredoxins, and glutathione peroxidases) which seemed to contribute not significantly to the GEXSR. The exceptions to this were glutathione reductase (Glr1) and glutaredoxin (Grx2/Ttr1), inactivation of which affected manifestation of the phenomenon. This reinforced the view that the GSH/GSSG balance plays a key role in the GEXSR. Second, comparative analyses of transcriptomic profiles of C. albicans glucose- and lactategrown cells in response to oxidative stress and glucose treatment correspondingly revealed a small set of commonly up-regulated genes: UCF1, RNR22, MOH1, orf19.3302, and HSP21/orf19.822 (Enjalbert et al., 2006; Rodaki et al., 2009). Each potential GEXSR-specific gene appeared to be regulated in a distinct manner by the major glucose signalling pathways. The ectopic expression of potential GEXSR targets did not provide any experimental evidence to support their roles in this response. That might be related to inefficient expression from ACT1 promoter under the experimental conditions tested, and also caused by other effects.

Candida albicans is the most common systemic fungal pathogen of humans. Virulence factors, fitness attributes and the host immunity ultimately determine the ability of C. albicans to reside as a commensal organism or an opportunistic pathogen in its human host. C. albicans displays a high level of flexibility in adapting to and infecting diverse niches in its human host. Generally occupying glucose-poor niches, C. albicans often depends upon alternative carbon sources to grow and establish infections. However, most cellular and molecular investigations of this pathogen have been performed on glucose-grown cells, and the relationship between physiological nutrients and drug resistance or virulence has not been studied. The main objective of this thesis was to study the effects of host carbon sources upon C. albicans cell biology and how this in turn affects the stress and drug resistance, immune recognition and virulence of this fungus. Growth on this physiologically relevant alternative carbon source was shown to influence the cell wall architecture of C. albicans and alter its resistance to a variety of stresses and antifungal drugs. Differences in stress resistance were less dependent on the key stress signalling pathways than on major architectural changes in the C. albicans cell wall that influenced its biochemical and biophysical properties. It was also shown that carbon source has a major impact upon C. albicans pathogenicity, altering cell adhesion and host virulence, in both systemic and vaginal infections. Considering the different niches that Candida can colonise in the body, these findings have the potential to change our views about the interactions that occur between the fungus and its human host. Taken together, the results of this project demonstrate that differential nutrient availability within diverse host niches influences the ability of pathogenic Candida species to counteract local chemical insults and pharmacological interventions.

The human pathogen Candida albicans is polymorphic, and its ability to switch growth forms is thought to play an important role in virulence. The primary research aim of this thesis was to understand the role the mitotic exit network plays in C. albicans with particular focus on the Tem1 GTPase protein. This aim was split into three specific goals; to study the role of Tem1 through the construction of a regulatable tem1 mutant, to understand the regulation of Tem1 through localisation and protein interaction studies, and to construct new molecular tools utilising the NAT1 positive selection marker in order to achieve two previous goals. In this thesis we demonstrated that TEM1 is an essential gene in C. albicans, and its essential function is signalled through the Cdc15 protein. Surprisingly, Tem1p depleted cells arrested as hyper-polarised filaments containing one or two nuclei and ultimately lost viability. These filaments formed from budding yeast cells, suggestive of a blockage late in the cell cycle. Ultimately the failure of these filaments to undergo cytokinesis was linked to a defect in septin ring dynamics and the formation of actomyosin ring. To understand the regulation of Tem1 we localised both the Tem1 and Lte1 proteins and found that Tem1 localised to spindle pole bodies in a cell-cycle dependent fashion by recruited at the onset of S phase. In contrast, the Lte1 homolog localised to the daughter cell cortex prior to release into the cytoplasm at the end of the cell cycle. A yeast 2-hybrid analysis of the MEN components demonstrated the potential of Bfa1/Bub2 and Tem1 to form a complex and the ability of Tem1 to homodimerise which may play a role in its self-activation. In order to carry out various aspects of this work we constructed a fully functional set of cassettes, including the constitutively active ENO1 promoter, V5-6xHIS epitope tag and various fluorescent protein genes fused to the NAT1 positive selection marker. When considered together, these results indicate that Tem1 is required for timely mitotic exit and cytokinesis in C. albicans, similar to S. cerevisiae, but the final output of the pathway must have diverged.

Programmed cell death in animals, plants and protists is in part regulated by a variety of proteases, including cysteine aspartyl proteases, (caspases, paracaspases and metacaspases), cathepsins, subtilisin-like serine proteases, vacuolar processing enzymes and the proteasome. The role of different proteases in the cell death responses of the fungi is however largely unknown. A greater understanding of the fungal cell death machinery may provide new insights into the mechanisms and evolution of PCD and potentially reveal novel targets for a new generation of antifungal drugs. The role of a metacaspase encoding gene, MCA1, in the cell death response of the human pathogen Candida albicans pathogen has been investigated by functional analysis. MCA1 deletion not only alters the sensitivity of cells to a number of cell death stimuli, it also enhances virulence in an insect model. C. albicans shows altered cell and colony morphology on Lee’s medium. Evidence is presented to suggest that these functions appear to be dependent upon active mitochondria. In this study it has also been shown that key caspase substrates may be conserved between humans and the yeasts Saccharomyces cerevisiae and Candida albicans. Many substrates, particularly those which are essential, have retained their caspase cleavage motifs. 14 protease mutants displayed altered activity against caspase 1, 3, 6 or 8 substrates during acetic acid-induced PCD and caspase 1-like activity appeared to be particularly associated with PCD. Using a novel bioinformatic analysis of experimental LC-MS/MS data, changes in the degradation patterns of the proteome (destructome) following acetic acid-induced cell death have been investigated in wild-type yeast. In addition, potential native substrates of the yeast Mca1 have also been identified. The future challenge is to characterise the destructome of different proteases under a range of cell death conditions. In this way it may be possible to identify key components of the cell death machinery and their substrates and so reveal the most promising targets for future therapeutics.

The main objective of this thesis was to study the effects of glucose on the regulation central carbon metabolic functions in the human fungal pathogen Candida albicans (C. albicans). The virulence of C. albicans is dependent upon fitness attributes as well as virulence factors. These attributes include robust stress responses and metabolic flexibility (Brown, 2005). The assimilation of carbon sources is fundamentally important for growth in all organisms and essential for the establishment of infections by pathogens, such as C. albicans in their human host. The C. albicans PCK1 and ICL1 genes, which encode the gluconeogenic and glyoxylate cycle enzymes phosphoenolpyruvate carboxykinase and isocitrate lyase are required for growth on non-fermentable carbon sources such as lactate and oleic acid. This thesis examines the impact of glucose upon the assimilation of secondary carbon sources such as lactate and oleic acid by C. albicans. The addition of 2% glucose repressed the CaPCK1 and CaICL1 genes. However, the enzymes CaIcl1 and CaPck1 were not destabilised by glucose and they retained at high levels following glucose addition. As a result, C. albicans cells continued to assimilate lactate and oleic acid in the presence of glucose. In contrast, the ScPck1 and ScIcl1 proteins were degraded rapidly in S. cerevisiae, and lactate and oleic acid assimilation was repressed in response to glucose. Therefore, while C. albicans and S. cerevisiae display similar responses to glucose at the transcriptional level, their responses at post-transcriptional and metabolic level diverge significantly. As a result, C. albicans can assimilate both glucose and alternative carbon sources at the same time. Next, the molecular apparatus that triggers the destabilisation of target proteins in response to glucose in C. albicans was studied. The expression of C. albicans the ICLI ORF in S. cerevisiae suggested that CaIcl1 has lost the molecular signal that triggers destabilisation in response to glucose, as CaIcl1 was not degraded in response to glucose in S. cerevisiae. However, when ScIcl1 was expressed in C. albicans ScIcl1 was rapidly degraded in response to glucose indicating that C. albicans has retained the molecular apparatus for glucose-accelerated degradation of target proteins. ScIcl1 degradation was slowed in C. albicans ubi4/ubi4 mutants. Furthermore, the addition of a putative of S. cerevisiae ubiquitination site carboxy terminus of CaIcl1 led to glucose-accelerated degradation of this protein in C. albicans cells. Therefore, glucose triggers accelerated degradation of target proteins in C. albicans via a ubiquitin-dependent process.

Organisms exist in complex and dynamic environments. Facing numerous challenges, microorganisms must continuously monitor environmental changes and adapt to these if they are to survive. For example, the major fungal pathogen of humans, Candida albicans experiences diverse stresses, including temperature fluctuations, oxidative stress and enzymic processes, that cause molecular damage. Post-translational modifications such as phosphorylation play major roles in stress adaptation, for example through activation of MAP kinase pathways and transcription factors such as the heat shock transcription factor, Hsf1. However, other post-translational modifications such as ubiquitination and sumoylation have been relatively understudied. Nevertheless, they are believed to play crucial regulatory roles in many cellular processes including stress adaptation. Therefore, in this study the roles of ubiquitin and SUMO (small ubiquitin-like modifier) have been investigated in C. albicans. Proteomics was used to identify ubiquitination and sumoylation targets, and this was combined with molecular analyses of the UBI4 and SMT3 genes, which encode polyubiquitin and SUMO, respectively. Both ubiquitination and sumoylation were shown to play important roles in morphogenesis, cell division and stress adaptation in C. albicans, including adaptation to heat and oxidative stresses. In addition, the dynamics of heat shock adaptation were examined in C. albicans using a systems biology approach. Hsf1 is known to activate HSP90. In this study, Hsf1 was found to be transiently phosphorylated in response to heat shock, and Hsp90 was found to down-regulate this Hsf1 phosphorylation. This led to the identification of an autoregulatory loop that controls thermal adaptation in C. albicans. A mathematical model of heat shock regulation (constructed in collaboration with Katarzyna Tyc and Edda Klipp) provided novel insights into the regulation of this evolutionarily conserved environmental response and the significance of thermal adaptation during systemic Candida infection.

Disruption of N-mannosylation and O-mannosylation on the C. albicans outer cell wall increased the rate by which C. albicans is ingested by macrophages. Conversely, disruption of phosphomannosylation reduced the rate of C. albicans is phagocytosis. Alterations to the outer cell wall and genetic or chemical inhibition of hyphal morphogenesis in C. albicans resulted in significantly abrogated macrophage killing in vitro. Disruption of C. albicans ability to tolerate oxidative stresses also perturbed its ability to escape from and kill macrophages. The engagement of specific receptors on the macrophage surface is an essential component of C. albicans recognition and clearance. In the presence of serum, blocking pattern recognition receptors associated with specific fungal cell wall epitopes (Mannose Receptor, Dectin 1 and CD16/32) resulted in an initial decrease in phagocytosis and decreased macrophage killing. Blocking macrophage pattern recognition receptors using soluble components of the C. albicans cell wall resulted in decreased phagocytosis under serum free conditions of O-linked mannans only, and reduced macrophage killing for macrophages pre-exposed to N-mannan and laminarin. The presence of serum increased the rate of uptake for macrophages pre-exposed to N-mannan and laminarin, and had no affect upon macrophage killing. The interaction of C. albicans cell wall epitopes with macrophage pattern recognition receptors, coupled with C. albicans ability to respond to stresses encountered after ingestion are critical determinants of the macrophage’s ability to ingest and process C. albicans.

The main aim of this project was to determine the potential of increased chitin content as a mechanism of resistance to caspofungin in different fungal pathogens. C. albicans wild-type cells were pre-grown with a combination of CaCl2 and CFW prior to caspofungin treatment. This result sin a three-fold increase in cell wall chitin. Wild-type cells, which had elevated chitin content, were less susceptible to caspofungin. Priming cells to activated chitin synthesis was also able to compensate for the loss of the normally essential CaCHS1, through formation of three novel forms of salvage septa. In the absence of both CaChs1 and CaChs3, which are typically involved in septum formation, the class I chitin synthases, CaChs2 and CaChs8, could be stimulated to synthesise a proximally offset salvage septum. When CaChs3 was the only remaining chitin synthase, treatment with CaCl2 and CFW, led to the formation of thick chitin-rich salvage septa. CaChs2 and CaChs3 could be stimulated by treatment with CaCl2 and CFW to synthesise a thin salvage septum similar to the septum of wild-type cells. All three salvage septa were capable of restoring viability and cell division in C. albicans. The compensatory increase in chitin content in response to caspofungin treatment was not specific to C. albicans because clinical isolates of C. tropicalis, C. parapsilosis and C. guilliermondii and the filamentous fungus, A. fumigatus, also demonstrated an increase in chitin content after treatment with caspofungin. Isolates of C. glabrata and C. krusei showed no change in chitin content when exposed to caspofungin. The results of this thesis highlight the potential for using chitin synthase inhibitors in combination therapy with the echinocandins.

Candida albicans is the major fungal pathogen of humans and its cell surface mannoproteins play important roles in adhesion, interactions with the host and signal transduction. Most of the covalently attached mannoproteins are linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Over 100 putative GPI-proteins have been identified in silico in the C. albicans genome, yet the majority remain uncharacterised with regards to their function and regulation. This study uses different approaches to define the role and regulation of selected GPI-protein genes. Expression analyses demonstrated transcriptional regulation of these genes in response to membrane and wall stresses including antifungal drugs. We propose that these genes encode proteins with important roles in cell wall remodelling. One protein, Pga54 was selected for further analysis. Null mutant and reintegrant strains were constructed and phenotypically analysed. The mutant phenotype was for the greatest parts inconclusive, but it showed a minor virulence defect in a mouse model of systemic infection. Using a tagged version of Pga54 the protein could be detected in the GPI fraction of the cell wall in wild type C. albicans cells and was shown to be higher expressed in cells subjected to stress. To establish whether they play a role in adhesion to host cells selected uncharacterised GPI proteins were heterologously expressed in the non-adherent yeast Saccharomyces cerevisiae. The newly generated S. cerevisiae strains did not show any differences in adhesion to human buccal epithelial cells. Further, the examination of tandem repeat variation in genes encoding GPI anchored proteins in different C. albicans strains indicates that this is could be a further means of creating diversity and heterogeneity at the cell surface. This study employs different methods to study the role and the dynamics of cell surface proteins. In future studies these approaches could be applied to all identified GPI proteins to obtain a comprehensive picture of cell surface diversity in Candida albicans.

Microscopical examination of Candida albicans grown on contoured artificial surfaces provided evidence that hyphae responded thigmotropically to features of the growth substrate. Hyphae of C. albicans followed grooves and ridges on various artificial membranes and penetrated pores of Nucleopore filters. The thigmotropic response in C. albicans was attenuated by gadolinium ions and by verapamil suggesting that calcium uptake may be involved in thigmotropic regulation. Thigmotropism was also observed for the first time in three genera of dermatophytic fungi (Epidermophyton, Trichophyton and Microsporum) and two saprophytic fungi (Mucor mucedo and Neurospora crassa). Therefore thigmotropism may be a general feature of fungal hyphae that must forage for nutrients on surfaces and within solid materials. Since Ca2+ appears to be involved in the regulation of thigmotropism attempts were made to construct strains expressing the Ca2+ sensitive photoprotein aequorin. The apoaequorin d gene was cloned in to C. albicans and S. cerevisiae using the YPB-ADHpt expression vector. Southern analysis indicated low copy number of the plasmid in C. albicans as compared with S. cerevisiae. Aequorin was reconstituted in protein extracts of C. albicans and S. cerevisiae by supplementing them with coelenterazine. The level for C. albicans was ten times higher than for Neurospora crassa, the only filamentous fungus to be transformed with this gene so far. Aequorin was successfully reconstituted in transformed living cells, and the luminescence levels were sufficiently high to be detected when external Ca2+ was added to the growth medium. Transformed C. albicans cells undergoing the dimorphic transition from yeast-to-hyphal form exhibited higher resting levels of luminescence indicating that cells induced to form hyphae have higher [Ca2+] than yeast cells. The work presented in this thesis presents first evidence of construction of strains expressing the luminescence photoprotein aequorin in a pathogenic fungus. This method provides a non-toxic, non-invasive method for monitoring [Ca2+] in C. albicans.

It has been envisaged that lytic enzymes may be crucial in determining the morphology and growth of fungi, and may therefore represent a target for antifungal agents. The chitinolytic system of Candida albicans was investigated using a range of 4-methylumbelliferyl glycosides as model substrates, and its potential as a target for antibiotics has been assessed. Maximum hydrolysis was obtained with substrates having monomer and tetramer chain lengths, being attributed to N-acetylglucosaminidase and endochitinase respectively. Activities were investigated in cell fractions, vacuoles, and also in whole cell preparations. The characteristics of both chitinase and N-acetylglucosaminidase were examined, including pH and temperature optima and Km values. Chitinase was semi-purified on Fast Protein Liquid chromatography system and activity could be located after native polyacrylamide gel electrophoresis. Analysis of chitinase activity during the growth of the yeast morphology of C.albicans revealed maximal activities during the logarithmic phase, suggesting a relationship of chitinase levels to active growth of the pathogen. It was found that the antibiotic allosamidin was a potent inhibitor of chitinase activity of C.albicans, but not of N-acetylglucosaminidase. Conversely, an analogue of N-acetylglucosamine was found to be a potent inhibitor of N-acetylglucosaminidase but not of chitinase. Treatment of yeast suspensions with allosamidin resulted in an increased chain length. No cell death, or discernible pattern of change in the radiolabelling was observed in the presence of either allosamidin or N-acetylglucosamine analogue. Similarly no consistent change in the optical density of cultures was observed in the presence of either inhibitor. Even in the presence of the membrane permeabilising agent amphotericin no effects were observed above those achieved with amphotericin alone. Comparative studies were carried out upon the chitinolytic activity of Kluyveromyces lactis toxin and bovine serum. The chitin synthetic system of Benjaminiella poitrasii was compared to that of C.albicans.

Advances in DNA sequencing technologies have galvanized research efforts into understanding the effects of host-associated microbiomes on host health. While a staggering amount of data has been generated to describe taxonomic differences between “healthy” and “dysbiotic” microbiomes, the functions performed by microbiomes during homeostasis remain incompletely understood. This in turn makes understanding dysbiosis in the context of different etiologies difficult. Thus, it is paramount to develop new frameworks to expand our knowledge on the beneficial functions of host-associated microbiomes. To accomplish that goal, we created metabolic footprinting, a methodological framework which utilizes gnotobiotics, comparative-untargeted metabolomics, and growth assays to determine what microbes consume in vivo. Using metabolic footprinting, we determined that commensal Clostridia perform an important digestive function for the mammalian host by consuming sugar alcohols in the large bowel. Sugar alcohol intolerance is commonly reported by patients with irritable bowel syndrome or inflammatory bowel disease, suggesting the loss of Clostridia may be an important factor in these etiologies. Additionally, we used metabolic footprinting to discover that the human fungal pathogen Candida albicans consumes small sugars in the gut. Interestingly, C. albicans required oxygen to utilize these sugars in vitro, and we found that the gut microbiota prevented the expansion of C. albicans in the gut by restricting access to oxygen in vivo. The finding that oxygen is a critical resource for C. albicans in the large bowel could improve prophylaxis aimed at preventing invasive candidiasis in susceptible patients. Collectively, the results in this thesis demonstrate that metabolic footprinting is a useful tool for elucidating the functions performed by host-associated microbiomes.

Candida albicans is the most common cause of life-threatening disseminated fungalinfections. It is capable of undergoing phenotypic switching between two distinct cell types, named ‘white’ and ‘opaque’. The white-opaque switch is controlled by a complex genetic regulatory network (GRN) that consists of transcriptional regulators (TRs). The advent of methodologies for profiling mRNA transcript levels and specific protein-DNA interactions at a genome-wide level has greatly expanded our ability to determine the structure and output of genetic regulatory networks, however uncovering the logic of how these networks function remains a challenging endeavor. The field of genetic regulatory network inference aims to meet this challenge by using computational modeling to derive the structure and logic of GRNs based on the experimental data provided by these genome-wide approaches. Boolean, probabilistic, ODE-based, and other models have been developed to infer GRNs. However, most existing models do not incorporate dynamic transcriptional data, since it has historically been less widely available in comparison to “static” transcriptional data. In this work, a novel evolutionary algorithm-based ODE model that integrates kinetic data and considers “static” transcriptional profiles as attractors has been developed to infer GRN structure. The model performed well on both in-silico and real-life datasets, and it was able to predict unknown transcription profiles produced upon genetic perturbations of the Candida albicans white-opaque GRN. Those genetic perturbations can be engineered in vivo and the result can be utilized to either support or further refine the model. Therefore, the model allows for an iterative refinement strategy to decipher GRN and verify its reliability: the model facilitates GRN candidate selection for experimentation and the experimental result in turn provides validation or improvement for the model.

Gene expression is one of the most fundamental processes in a cell, allowinggenetic information to be processed into functional products. Regulation of gene expression is determined by the underlying gene regulatory networks (GRNs). We can think of a GRN as a directed network with hub nodes denoting master regulators, which are DNA-binding proteins and target nodes denoting downstream genes whose expression is modulated. The directed network edges denote strength of the interaction between the master regulators and downstream target genes. High-throughput genomic and epigenomic data is used to construct GRNs and to estimate the robustness of these networks. In this study, we used the GRN underlying the formation of complex multicellular structures called biofilms, in the yeast species Candida albicans, to understand the mechanisms of GRN divergence between species and variation within species. Biofilm formation has evolved multiple times in the fungal tree of life and the ability to form biofilms is highly varied across species and strains. To study the effects of these variations on components of the GRN, we developed three standalone bioinformatic tools. The first tool was developed to infer the structure of GRNs across Candida species by identifying and annotating binding loci of master regulators. The second was used to identify mutations in the C. albicans population and the effect of these mutations on the network components. The third tool was used to estimate the evolutionary forces acting on the components of the biofilm GRN. Using these tools, we inferred the sources of genetic variations in the biofilm regulatory network components. We found that the motif preferences of the DNA-binding proteins are conserved across large evolutionary distances but their interaction with target genes is highly divergent. This is driven in part by mutations resulting in gains and losses of genomic regions where the DNA-binding proteins preferentially bind. Furthermore, mutations accumulate in segments of DNA-binding proteins that are required to interact with other hub proteins in the network. This affects the overall structure of the network both within and between species.