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. <i>C. albicans</i> 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 <i>CaCHS1</i>, through formation of three novel forms of salvage septa. In the absence of both <i>Ca</i>Chs1 and <i>Ca</i>Chs3, which are typically involved in septum formation, the class I chitin synthases, <i>Ca</i>Chs2 and <i>Ca</i>Chs8, could be stimulated to synthesise a proximally offset salvage septum. When <i>Ca</i>Chs3 was the only remaining chitin synthase, treatment with CaCl₂ and CFW, led to the formation of thick chitin-rich salvage septa. <i>Ca</i>Chs2<i> </i>and <i>Ca</i>Chs3 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 <i>C. albicans.</i> The compensatory increase in chitin content in response to caspofungin treatment was not specific to <i>C. albicans</i> because clinical isolates of <i>C. tropicalis, C. parapsilosis </i>and <i>C. guilliermondii</i> and the filamentous fungus, <i>A. fumigatus</i>, also demonstrated an increase in chitin content after treatment with caspofungin. Isolates of <i>C. glabrata</i> and <i>C. krusei</i> 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.

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.

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.

Mini-dissertation submitted in partial compliance with the requirements of the Master's Degree in Technology: Homoeopathy, Durban Institute of Technology, 2002. / The purpose of this study was to determine the efficacy of certain Compositae species herbal extracts (Arctium lappa, Calendula officinalis and Echinacea purpurea) in the inhibition of in vitro growth of Candida albicans as compared to nystatin in terms of the disc diffusion test. Candida albicans was obtained from the Department of Biotechnology (Technikon Natal). Three components were tested on Candida albicans: namely the herbal extracts which were the experimental group, nystatin which was the allopathic component serving as a positive control and the 62% ethanol which was the negative control. Commercially available herbal extracts of Arctium lappa, Calendula officinalis and Echinacea purpurea respectively, with an ethanol concentration of 62% v/v, were purchased from Parceval (Pty) Ltd. The herbal extracts were prepared according to the German Herbal Pharmacopoeia (1991) standards. Commercially available nystatin suspension was obtained from \ Bristol-Myers Squibb (Pty) Ltd, 62% (vlv) ethanol was prepared according to the German Homoeopathic Pharmacopoeia (1991) standards. In carrying out the experiment, 5mm filter paper discs were placed on Sabouraud's dextrose agar plates which were streaked with Candida albicans. Thereafter 0.7 microlitres of the test and control substances were pipetted onto / M

El propósito del presente fue de identificar las especies de Candida implicadas en estomatitis subprotésica en pacientes del Departamento de Odontoestomatología del Centro Médico Naval “CMST” – 2007 Se analizaron los 30 primeros pacientes con diagnóstico de estomatitis subprotésica que acudían al Departamento, a los cuales se les realizó cuatro frotises, dos para el examen directo microscópico (con coloración Gram) para confirmar la presencia de levaduras, y dos para el cultivo en Agar Sabouraud+Cloranfenicol, del crecimiento en este agar, se hizo la prueba del tubo germinal para determinar la presencia de Candida albicans, de salir negativo esta prueba, se llevaba a cabo la identificación de la especie mediante el sistema Api Candida. Se obtuvieron entre otros resultados que Candida albicans fue la especie más implicada en la estomatitis subprotésica con un 96.66% seguido de Candida tropicalis con un 3.33%. / -- The purpose of this was to identify the species of Candida involved in stomatitis subprotésica in patients of the Department of Dental Naval Medical Center "CMST" - 2007 We analyzed the first 30 patients diagnosed with stomatitis subprotésica who went to the Department, to which were conducted four frotises, two for the direct microscopic examination (colouring Gram) to confirm the presence of yeast, and two to be planted in Agar Sabouraud + Chloramphenicol, growth in the agar, it was the germ tube test to determine the presence of Candida albicans, to leave this negative test was carried out to identify the species by the API system Candida. Among other results were obtained that Candida albicans was the most involved in stomatitis subprotésica with a 96.66% followed by Candida tropicalis with a 3.33%.

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.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / Introducción El vertiginoso aumento de la población adulta mayor en Chile, hace relevante el estudio de las patologías más comunes en este grupo. La estomatitits protésica (EP) es una de las lesiones orales más prevalentes en sujetos portadores de prótesis removible (PR) y se define como un proceso inflamatorio crónico de la mucosa adyacente al aparato protésico. La etiología de la EP es multifactorial, pero se asocia en un alto porcentaje a levaduras del género Candida, denominándose en estos casos como candidiasis oral. Otros de los factores que han sido relacionados con la enfermedad, son alteraciones en parámetros salivales como velocidad de flujo salival (VFS), pH y concentración total de proteínas en la saliva, además de la presencia de xerostomía. La evidencia sostiene que éstos podrían jugar un rol en la patogenia de la EP asociada a candidiasis. El propósito de este estudio fue caracterizar dichos parámetros salivales en sujetos adultos mayores portadores de PR con EP asociada a candidiasis y compararlos con un grupo de sujetos portadores de PR sin la enfermedad. Metodología El presente estudio incluyó 70 voluntarios mayores de 60 años pertenecientes a un establecimiento de larga estadía para adultos mayores (ELEAM) y a la clínica de prótesis totales, de la Facultad de Odontología de la Universidad de Chile. La muestra se dividió en 2 grupos, el experimental, constituído por pacientes con EP asociada a candidiasis, y el control, quienes no presentaban dicha enfermedad, ambos portadores de PR. Previa firma del consentimiento informado, la recolección de datos se realizó mediante una ficha clínica en donde se registró la presencia de xerostomía, y se obtuvieron muestras salivales, las cuales fueron analizadas para determinar VFS, pH y concentración total de proteínas en la saliva. Resultados No hubo diferencias estadísticas entre la VFS, prevalencia de xerostomía y concentración total de proteínas en la saliva entre ambos grupos de estudio. En cuanto al pH, éste fue menor en el grupo experimental comparado con el grupo control, con diferencia estadística. Además la VFS fue menor en el grupo con xerostomía que en el grupo que no presentó el síntoma. 2 Conclusiones Existe poca evidencia respecto a los parámetros salivales y su relación con la EP asociada a candidiasis. En este estudio solo se encontró diferencia significativa en el pH de los pacientes con y sin EP, sin embargo, son necesarios más estudios que aíslen las variables confundentes para poder dilucidar el rol de la saliva en la patogenia de la enfermedad, y así, conocer sus implicancias en el diagnóstico y terapéutica de la EP asociada a candidiasis.

It is estimated that fungal infections kill greater than 1.6 million people annually, a number that is comparable to the number of deaths associated with tuberculosis. Candida species are the fourth leading cause of hospital-acquired blood infections and Candida albicans is the most common cause of these fungal blood infections (known as candidemia). Immunocompromised individuals, such as those who have HIV/AIDS, those undergoing chemotherapy treatments, or those on broad-spectrum antibiotics, are most likely to develop candidemia. Candidemia is associated with a 20-40% mortality rate. However, when patient treatment for candidemia is delayed for over 48 hours, associated mortality rates increase to 78%. Blood infections can disseminate Candida albicans throughout the body, eventually leading to infection in vital organs like the liver, kidney and brain. Optimal patient outcomes are achieved if antifungal therapy is given within 12 hours after a blood sample is obtained for culture and testing. However, current blood tests cannot reliably detect Candida this early and thus antifungals are not routinely given to patients in this time frame. Counterintuitively, it is believed that some fungi, like many bacteria, are non-harmful residents in small intestines of most adults and this hypothesis is supported by the fact that the most common fungal species in the human gut is Candida albicans. However, intestinal overgrowth of C. albicans is linked to Crohn's disease, and disease-causing forms of C. albicans can arise from commensal strains that once resided in the patient’s gastrointestinal tract. The specific molecular mechanisms by which C. albicans interacts with host immune cells versus intestinal cells, and those that trigger Candida pathogenicity remain unknown. Many strains of Candida albicans have developed resistance to azoles, the major class of drugs used to treat both superficial and systemic infections. In order to develop new treatments, we must better understand host-fungal pathogen biology to determine novel antifungal targets or therapeutics to fight fungal infections at the early stages of infection. In this work, we developed a novel tool that allows us to measure which genes are important to both the host and Candida albicans simultaneously, in specific infection states. We have applied this tool to measure gene expression in Candida albicans interacting with mammalian macrophages and small intestine epithelial cells– at both the population and single cell levels. When examining populations of sorted infection samples, we found that host immune cells both exposed to and infected with fungal cells exhibit similar expression patterns. In contrast, phagocytosed C. albicans exhibit unique expression patterns compared to those merely exposed to macrophages. We found that immune response genes in single, Candida infected macrophages exhibited bimodal expression patterns for some immune response genes. We also observed examples of expression bimodality in live Candida inside of single macrophages. Both Candida albicans and host small intestine epithelial cells demonstrate distinct patterns of expression when exposed to each other at the population level, compared to unexposed controls. However, the magnitude of these differences is dependent on the multiplicity of infection. Some expression programs overlapped with those observed in populations of Candida cells interacting with macrophages, with key differences. We also observed expression bimodality among epithelial cells infected with C. albicans. We believe the information obtained using this technique could be used when considering new antifungal or therapeutics targets; if uniform and high expression of particular in genes in Candida populations phagocytosed by macrophages or invading epithelial cells leads to high and early protein production, these proteins may be effective antifungal targets. Similarly, if some host immune response genes are not expressed in a population of Candida infected macrophages as uniformly and highly as expected, these genes or proteins could be target of a therapeutic for patients with Candida infections that are resistant to azoles.

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2012 / Oral candidiasis is an infection prevalent in immunocompromised individuals. The main causative agent is Candida albicans. Many antifungal agents are available and are effectively used. However, due to the development of drug resistance, toxicity and poor solubility resulting in poor absorption; medicinal plants have been investigated. Dodonaea viscosa var. angustifolia, an indigenous South African plant has shown to have an antifungal effect including inhibition of adherence of C. albicans to oral epithelial cells; which is the crucial first step of infection. This study investigated the effect of the crude extract on the ultrastructure of C. albicans cell wall, which might be responsible for the reduced adherence to oral epithelial cells. The effect of the plant extract on C. albicans germ tube and biofilm formation was also studied since biofilm structure allows for high resistance to antifungal agents and host defense mechanisms. Crude plant extracts were prepared using dried leaves and acetone. Three C. albicans strains were used throughout the study. Minimal fungicidal concentrations of plant extract were determined using a microdilution technique. Three subinhibitory concentrations 3.125, 1.562 and 0.781 mg/ml were selected for further studies. The effect of these subinhibitory concentrations of plant extract on the C. albicans cell wall structure, cell membrane, germ tube formation, biofilm formation and cell wall proteins were studied using transmission electron microscopy, light microscopy, scanning electron microscopy and SDS-PAGE respectively. v The subinhibitory concentrations of crude plant extract rendered C. albicans cell wall thinner and at some places caused cell wall breakage and disruption. This effect increased with a decrease in plant extract concentration. The cell membrane was also damaged by the plant extract showing increased undulation. This effect was not concentration dependent. The subinhibitory concentrations decreased C. albicans germ tube formation and the effect increased with an increase in concentration. Biofilm formation was reduced by the plant extract and in addition, hyphal formation by cells within the biofilm was also reduced. However, SDS-PAGE showed that on a molecular level, the plant extract did not remove any specific adhesin proteins from the cell wall. The crude plant extract of D. viscosa var. angustifolia at high concentrations, kills C. albicans and at low concentrations, renders the surviving cells avirulent. Therefore it has the potential to be developed into an effective therapeutic agent to treat and prevent oral candidiasis. However, further research is required to identify the mode of action of the extract, the specific chemicals responsible for the effect, and the cytotoxicity.

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.