No description available.

Azole-resistance in Candida albicans is becoming common and is associated with the widespread prophylactic use of azoles. Resistance to one azole is usually associated with resistance to other structurally dissimilar azoles. C.albicans is also inherently resistant to a wide range of eukaryotic inhibitors such as cycloheximide and gentamycin. Certain studies have shown that azole-resistance in some strains of C.albicans is associated with alterations in the cell membrane. This project has sought to determine whether azole-resistance in C.albicans strain 3302 was due, at least in part, to a multidrug resistance mechanism. An assay was developed using the fluorescent dye Rh123 to measure P-glycoprotein like activity. Active efflux of Rh123 has been shown to correlate with P-glycoprotein activity in a number of organisms. Results from this assay suggest that an energy-dependent efflux mechanism for Rh123 is present in azole-resistant strain 3302 but not in azole-sensitive strain 3153. The P-glycoprotein inhibitor, reserpine, inhibited Rh123 efflux. However, azoles did not appear to compete with Rh123 for efflux in the azole-resistant strain 3302, suggesting that azole-resistance in this strain is not mediated by a P-glycoprotein like mechanism. Southern analysis showed that sequences homologous to MDR genes existed in C.albicans. A PCR strategy was used to clone gene fragments containing the Walker motif which is found in MDR genes.

Little is known of the biology and importance of the ectomycorrhizal symbiosis in the Dipterocarpaceae. This project was undertaken: 1) to follow dynamics of mycorrhizal infection of dipterocarp seedlings at different sites in the forest, to characterise the major fungal associations involved; 2) to follow mycorrhizal infection of dipterocarp seedlings under laboratory conditions with different inoculum sources; 3) to determine whether dipterocarp ectomycorrhizas function in a manner similar to temperature ectomycorrhizas in the uptake of specific nutrients. Twenty-four different ectomycorrhizal types were described from roots of newly germinated seedlings, two to seven month-old seedlings and wildings of <i>Shorea leprosula</i> (Miq.), and approximately 20 year-old <i>S. acuminata</i> Dyer, <i>S. dasyphylla</i> Foxw., <i>S. leprosula</i> and <i>S. parvifolia</i> Dyer trees. Seventeen types were tentatively identified to family level. Vesicular-arbuscular mycorrhizas were not found. In the forest, some 14 day-old <i>S. leprosula</i> seedlings were already ectomycorrhizal but infection could be absent up to seven months after germination. The results implied that hyphal connections were important in early infection of seedlings in the vicinity of parent trees. Mycorrhizal infection of sequentially sampled two to seven month-old seedlings declined over the sampling period at two sites in Gombak, Selangor and one in Ulu Langat, Selangor. Five to six ectomycorrhizal types were dominant on seedlings at each site and a succession of types was observed on seedling roots. At final harvest, increased plant growth was significantly correlated with ectomycorrhizal infection only at one site in Gombak where infection by 'dominant' types exceeded 30%. Non-mycorrhizal seedlings of <i>S. acuminata, S. leprosula, Hopea helferi</i> and <i>H. odorata</i> were able to grow normally in sterile soil under non-competitive situations. Seedlings were able to form ectomycorrhizas even with inoculum present in grassland soils or with inoculum from different host species in the case of <i>H. helferi</i>. Increased phosphorus uptake by ectomycorrhizal seedlings of <i>S. acuminata, S. leprosula</i> and <i>H. odorata</i> was demonstrated.

It was of interest to clone key genes involved in O-glycosylation with a view to using reverse genetics to establish their function. The Candida homolog of the S. cerevisiae MNT1 gene (Hausler and Robbins, 1992) was cloned by heterologous probing of a genomic DNA library. The CaMNT1 gene was found to be regulated differentially in response to the environment and exhibited a transitory increase in the level of transcription during early germ tube formation. Low stringency Southern analysis of C. albicans genomic DNA identified several CaMNT1 homologs suggesting CaMNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases. In order to demonstrate that specific glycosyl residues were actively involved in the host-fungus interaction, the CaMNT1 gene was disrupted in two strains using the ura-blaster technique. Disruption at the CaMNT1 locus led to a 90% reduction in -1,2-mannosyltransferase activity when -methyl mannoside was used as an acceptor, but had no obvious influence on viability, growth rate, germ tube formation or proteinase production. CaMnt1 appears to be involved in O-glycosylation since the Camnt1 null mutant strain accumulated intracellularly the O-glycosylated enzyme chitinase. Mannosyltransferase-deficient Camnt1 mutants were significantly reduced in their ability to adhere to human buccal epithelial cells in vitro and were attenuated in virulence in systemic models of candidosis. O-linked mannan may therefore be important for direct interactions with epithelial surfaces or for the stabilization and function of cell surface adhesins. The low virulence potential displayed by Camnt1 mutants clearly demonstrates the important role glycosylation plays in the virulence of C. albicans. Given that O-glycosylation differs significantly between yeast and man, this protein modification may constitute a novel target for antifungal agents.

No description available.

Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2016-03-03T17:04:26Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Carolina Maria da Silva.pdf: 976111 bytes, checksum: 727eabad7b0e248858d85fdda9638e23 (MD5) / Made available in DSpace on 2016-03-03T17:04:26Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Carolina Maria da Silva.pdf: 976111 bytes, checksum: 727eabad7b0e248858d85fdda9638e23 (MD5) Previous issue date: 2011-11-29 / CNPQ / Infecções fúngicas invasivas têm se tornado cada vez mais freqüentes em neonatos, principalmente devido ao aumento da sobrevivência de prematuros e a deficiência do sistema imune. Dessa forma, torna-se relevante o conhecimento dos fatores epidemiológicos e susceptibilidade aos antifúngicos, uma vez que permitem o melhor conhecimento dos fatores associados à doença e a resistência dos agentes etiológicos, além da concentração ideal do medicamento a ser administrado para inibir e /ou matar o agente causal da infecção. Nesse contexto, os objetivos deste estudo foram diagnosticar candidemia em neonatos, associando os fatores epidemiológicos predisponentes e o perfil de susceptibilidade às drogas antifúngicas dos agentes etiológicos. No período de março de 2010 a julho de 2011, foram feitas coletas das amostras clínicas em neonatos de Unidades de Terapia Intensiva Neonatal do Hospital Agamenon Magalhães e do Instituto de Medicina Integral Professor Fernando Figueira. O diagnóstico micológico foi realizado através do exame direto, cultura e identificação dos agentes etiológicos. Foram coletadas amostras de sangue de 301 pacientes e isoladas 30 culturas, sendo identificadas Candida albicans (11), C. parapsilosis (11), C. pelliculosa (5), C. glabrata (1), C. guilliermondii (1) e C. tropicalis (1). Dos 30 pacientes com hemoculturas positivas para fungos, 90% eram pré-termos, 60% do sexo masculino, 93,4% possuíam peso ao nascer inferior a 2,5kg e as condições clínicas mais associadas foram icterícia e síndrome do desconforto respiratório. A grande maioria dos pacientes fazia uso de dispositivo terapêutico invasivo, destacando-se nutrição parenteral (96,7%) e cateterismo umbilical (73,3%). Quanto à susceptibilidade antifúngica todos os isolados de levedura foram sensíveis a anfotericina B, porém foi observada resistência ao fluconazol e voriconazol, principalmente por C. albicans, e 7 dos 11 isolados de C. parapsilosis foram resistentes a anidulafungina. As infecções fúngicas invasivas são frequentes em neonatos, permanecendo as espécies de Candida como as mais isoladas. Pacientes prematuros de baixo peso e que fazem uso de dispositivos invasivos são os mais acometidos, o conhecimento destes dados aliados aos resultados de susceptibilidade antifúngica in vitro possibilitam a prevenção e o tratamento mais adequado destas infecções. / Invasive fungal infections have become increasingly frequent in neonates, due to the increased survival of premature and disability of the immune system. The knowledge of epidemiological factors of these infections, as well as testing susceptibility to antifungal agents is relevant in this group of patients, because they allow a better understanding of the factors associated with the disease, the evaluation of the occurrence of fungal resistance, and the optimal concentration of the drug to be administered to inhibit and / or kill the agent of infection. In this context, the objectives of our study were to detect candidemia in neonates, the epidemiological factors associated with these infections and determine the antifungal susceptibility profile of the isolates. The samples were collected in the Neonatal Intensive Care Units from Agamenon Magalhães Hospital and Institute of Integrative Medicine Fernando Figueira, according to the medical request, from March 2010 to July 2011. The samples were manipulated to perform the direct examination and culture and then purified and identified. Samples were collected from 301 patients and we had isolated yeasts in 30 samples of blood , they were identified as Candida albicans (11), C. parapsilosis (11), C. pelliculosa (5), C. glabrata (1), C. guilliermondii (1), C. tropicalis (1). Of the 30 patients with positive blood cultures for fungi, 90% were preterm, 60% male, 93.4% had birth weight below 2.5 kg and the more usual conditions associated were clinical jaundice and respiratory distress syndrome. The vast majority of patients used invasive therapeutic device, especially parenteral nutrition (96.7%) and umbilical catheterization (73.3%). The antifungal susceptibility showed that all isolates were sensible to amphotericin B but some were resistente to fluconazole and voriconazole, mainly species of C. albicans, and 7 of 11 isolates of C. parapsilosis were resistant to anidulafungin. Invasive fungal infections are common in neonates, remaining Candida species as the most isolated. Preterm infants with low birth weight and use of invasive devices are the most affected and this knowledge combined with the in vitro antifungal susceptibility results enables a better prevention and treatment of these infections.

Fungal infections are ignored by social and political communities. However, they are estimated to affect more than a billion people, resulting in approximately 11.5 million life-threatening infections in the 'at risk' population and more than 1.5 million deaths annually. Though there have been huge advances in diagnostics and antifungal drug development over the past two decades, however, resource limited settings have not benefited from these advances. The aim of this research was to determine the burden of serious fungal infections in Nigerians with the appropriate underlying diseases. This epidemiological research was conducted across four study populations. Study 1; HIV-infected patients with CD4+ counts < 250 cells/mm³, irrespective of their ART status, a CrAg lateral flow assay was used for detecting cryptococcal antigenaemia (n=214). Study 2; a cross-sectional multicentre survey of TB patients being managed for smear negative or treatment failure TB irrespective of their HIV status (n=208). Study 3; a multicentre histoplasmin skin sensitivity survey amongst healthy HIV-infected and non-HIV infected participants; intradermally; induration ≥ 5 mm was considered to be histoplasmin positive (n=750). Study 4; a prospective cohort study of critically ill patients in a Nigerian ICU (n=71). Two retrospective studies to analyse the clinical picture of serious fungal infections in two at risk populations (HIV/AIDS and neonatal intensive care babies) in Nigerians was also conducted (n=7034; n=2712 respectively). Results revealed an overall seroprevalence of cryptococcal antigenemia of 8.9% with 6 (9.8%) in those with CD4+ cell counts < 100cells/mm³, 4 (5.0%) in the 100-200 group and 9 (12.3%) in 200-250 cells/mm³ group; a CPA prevalence of 8.7% (6.5% had HIV infection and 14.5% were HIV-negative) and a prior subclinical histoplasmosis of 4.4%. The ICU study revealed a 45% healthcare associated infection rate representing an incidence rate of 79/1000 patient-days in the ICU. The retrospective studies revealed a 2.3% rate of neonatal ICI with a case fatality rate of 18.5%. In the 12 years retrospective study 18% had a fungal OI with 88% of patients having initiated ART. In conclusion, serious fungal infections do occur in the at risk population in Nigeria and they constitute a significant public health challenge. Our findings demonstrate that there has been an underestimation of the burden of the problem in Nigerians. There is a dire need to design guidelines for the management of fungal infections in at risk population.

Background. As a result of the AIDS pandemic, many people areimmuno compromised andopportunistic fungal infections (OFIs) such as candidiasis are common. Despite the widespread use of medicinal plants in South Africa, there is a dearth of knowledge regarding the use of such plants in the management of these infections. This study evaluates three South African medicinal plants (Arctotis arctotoides, Pittosporum viridiflorum, and Gasteria bicolor) traditionally used in the treatment of OFIs in HIV/AIDS patients, in the Eastern Cape Province, South Africa. Materials and methods. A six-stage process of documentation, evaluation and analysis of results was conducted: (1) Selection of medicinal plants most frequently used in the treatment of OFIs through ethnomedical studies and the survey of specialised literature; (2) Collection and preparation of the extract of each plant; (3) Antifungal evaluation of the crude plant extracts. (4) Phytochemical and antioxidant evaluation of the active crude plant extracts; (5) Cytotoxicity evaluation of the bioactive extracts using the Chang liver cell line, and (6) Statistical analysis of the results. Ethnobotanical information was obtained through interviews with traditional healers and AIDS patients with the aid of semi-structured questionnaires, direct observations and by reviewing studies reported in the literature. Following the approval from the University of Fort Hare‘s Ethics Committee, 101 HIV/AIDS patients were recruited through convenience sampling into an anonymous cross-sectional questionnaire study. The agar diffusion and micro-dilution methods were used to determine the antifungal activities of the hexane, acetone and aqueous extracts of A. arctotoides, G. bicolor and P. viridiflorum against 10 opportunistic fungi.

L'objectif de cette thèse a été de contribuer à la compréhension des stratégies d'infection du pathogène opportuniste Aspergillus fumigatus. Ce pathogène est une cause émergente de morbidité et de mortalité chez les patients dont l'immunité est compromise et dans les milieux hospitaliers. Une infection avec Aspergillus est en général appelée une Aspergillose et ellepeut se développer dans un certain nombre d'organes, le plus fréquemment dansl'appareil respiratoire (poumons et sinus). Outre les infections (Aspergillosis invasive), la colonisation par ce champignon peut causer des réactions allergiques (Aspergilloses broncho pulmonaire allergique) et de l'asthme. Le nombre de patients immunodéprimés augmente régulièrement à cause des avancées des traitements du SIDA, du cancer, de la mucoviscidose ainsi que par le nombre grandissant de transplantation d'organes. De nouveaux médicaments antifongiques et des médicaments préventifs sont nécessaires pour venir en soutienaux soins médicaux des patients. Bien que plusieurs fongicides existent déjà sur le marché, l'Aspergillosisinvasive reste souvent fatale. Ceci est lié d'une part à la difficulté d'établir le diagnostic, et d'autre part au fait que des résistances émergent rapidement. La motivation de cette thèse est de comprendre les mécanismes impliqués dans le premier contact entre les conidies et le tissu de l'hôte. Ces mécanismes d'adhésion initiaux sont souvent réalisés par des liaisons entre les lectines et les carbohydrates. Le tissu épithélial et la surface muqueuse du système respiratoire sont couverts de structures possédants des carbohydratestels que les glycoprotéines, les glycolipides et les glycosaminoglycanes. L'identification des lectines d'A. fumigatus et leurs caractérisations devraient dorénavant contribuer à la compréhension de la glycostratégie de ce pathogène opportuniste ainsi que des mécanismes impliqués dans l'adhésion et l'infection. L'analyse détaillée de la structure des lectines permettra d'établir le rôle de cesprotéinesdans la virulence et de guider la conception de glycomimétiques, afin d'inhiber le phénomène d'adhésion. Cettenouvelle approche consistant à bloquer l'adhésion de l'agent pathogène plutôt que sa prolifération, vise à diminuer les résistances par une réduction de la pression évolutive. Deuxstratégies différentes ont été utilisées pouridentifier de nouvelleslectines. Tout d'abord une purification des lectinesà partir d'extraits fongiques brutsa été tentée et d'autre part un criblage pour trouver des séquences similaires avec les protéines codées par A. fumigatusa été réalisé parmi une banque de lectines fongiques connues. / The aim of this thesis was to contribute to the understanding of infection strategies of the opportunistic pathogen Aspergillusfumigatus. This pathogenic mould is an emerging cause of morbidity and mortality in immuno-compromised patients and hospital environments. An infection with Aspergillus is generally referred to as Aspergillosis; it can develop in a variety of organs but the most common sites are the respiratory apparatus i.e. lungs and sinuses. Besides infections (invasive aspergillosis), colonization with the fungus can cause allergic reactions (allergic broncho pulmonary aspergillosis) and asthma. The number of immuno-suppressed patients is steadily increasing due to advancement in the HIV, cancer and cystic fibrosis medical care, as well as an increasing number of organ transplantations. Needless to say that new antifungal drugs and preventive medication is desperately needed to support medical care for those patients. Even though several fungicides already exist on the market, invasive aspergillosis remains to be often fatal. On one hand, this is due to difficulties in diagnosis and on the other hand, resistances are emerging rapidly. The motivation behind this thesis is to understand the underlying mechanisms that are involved in the first contact between conidial spores and host tissues. Initial adhesion steps often involve carbohydrate binding proteins, called lectins. They recognize glycoconjugates such as glycoproteins, glycolipids and glycosaminoglycans which cover the epithelial tissue and mucosal surface of the respiratory tract.. Identification and characterization of the lectins from A. fumigatus will therefore contribute to the understanding of the glycostrategy of this opportunistic pathogen and of the mechanisms involved in adhesion and infection. Detailed structural analysis of the carbohydrate-protein interactions will allow ascertaining the lectins role in virulence and guide the design of glycomimetics, as adhesion inhibitors. With this novel approach of targeting the pathogen adhesion rather than its proliferation, resistances are believed to be less frequent due to the lack of evolutionary pressure. In this work, two different strategies were employed to obtain novel lectins. Firstly, lectins were purified from crude fungal extracts and secondly the A. fumigatus genome was screened for encoded proteins showing sequence similarity with known fungal lectins. While lectin purification from the crude extracts was inconclusive due to low lectin activity in the starting material, genome screening showed that several putative lectins were present. One of these lectins, named AFL6, belonged to the cyanovirin-N homolog (CVNH) family and it was recombinantly expressed and purified. Glycan array and micro calorimetry techniques were carried out to investigate its carbohydrate binding specificityand the three dimensional structure was determined using X-ray crystallography. The structure showed an overall similarity with other CVNHs with slight differences in the presumed carbohydrate binding sites. Unlike other family members, it shows a low affinity for mannosides and an apparent affinity for lactosamine containing glycan structures.

Several Fungal species have the potential to cause a broad spectrum of diseases in humans, ranging from mild superficial to disseminated invasive infections that involve the bloodstream and vital organs. Invasive fungal infections are severe, life-threatening diseases that result in the deaths of 1.5 million patients each year. The most common fungal species responsible for the majority of invasive fungal infections include Candida, Cryptococcus, and Aspergillus. The current treatment options for invasive fungal infections are restricted to three classes of antifungals: Azoles, polyenes, and echinocandins. The emergence of new fungal species, especially C. auris, marked by high resistance profiles and increased mortality rates (30-60%), has further exacerbated the limitations in its therapeutic options. This emphasizes the urgent need for effective alternatives to combat these deadly pathogens. C. auris isolates exhibited high resistance capability especially against azole (fluconazole) and polyene (amphotericin B) antifungals. Here, we utilized the combinatorial strategy to screen ~3400 FDA-approved drugs and clinical compounds to identify hits that can enhance/restore the antifungal activity of azoles and amphotericin B against resistant C. auris. The HIV protease inhibitors (lopinavir and ritonavir) were identified as potent enhancers to the antifungal activity of azole drugs (fluconazole, voriconazole and itraconazole). We confirmed that lopinavir and ritonavir have the capability to interfere with fungal efflux pump machinery. The in vivo efficacy of the combination of azole antifungals and HIV protease inhibitors was also evaluated to discover the best combination of itraconazole, lopinavir and ritonavir. Three drugs (lansoprazole, rolapitant and idebenone) were identified to effectively enhance the antifungal effects of amphotericin B and overcome its resistance in C. auris. Furthermore, the synergistic interactions of these combinations were applied on other medically important Candida, Cryptococcus, and Aspergillus species. In a comprehensive mechanistic study, we discovered that lansoprazole interferes with an essential target in the fungal mitochondrial cytochrome system, cytochrome bc1. This interference induces oxidative stress in fungal cells and subsequently enhances the antifungal activity of amphotericin B. For rolapitant, a transcriptomic analysis along with ATP luminescence assays confirmed that rolapitant at sub-inhibitory concentrations significantly interferes with ATP production in C. auris. For idebenone, checkerboard assays confirmed the synergistic interactions between amphotericin B and idebenone against a diversity of medically important fungal species. This combination exhibited a rapid fungicidal activity within 4 hours. Additionally, the cytotoxicity of this combination was assessed in a cell line model of kidney cells. Based on the potent in vitro and in vivo synergistic relationships observed for the identified combinations, it can be concluded that our approach offers a new hope to restore the antifungal activity of the existing antifungal drugs, even against resistant fungal infections. Additionally, it provides valuable insights into identifying novel targets to overcome resistance in multidrug-resistant fungal pathogens. / Doctor of Philosophy / Fungi comprise a diverse group of organisms that interact with humans in many good and bad aspects. Candida auris, a recently identified fungus, poses a significant threat to patients with weak immune systems. Infections with C. auris can be associated with mortality rates of up to 60%. Notably, this fungus is characterized by its powerful spreading capability and displays extraordinary resistance to antifungal agents, rendering many existing antifungal drugs ineffective. As a result, there is an unmet need to find efficient treatments for such deadly fungal infections. In this study, several drugs were identified with the potential to restore the activity of traditional antifungal drugs. The study identified four promising drugs (lopinavir, lansoprazole, rolapitant, and idebenone) with the potential to enhance the activity of the antifungal drugs against C. auris. lopinavir showed great potential to enhance the activity of azole antifungals, including fluconazole, voriconazole, and itraconazole. Furthermore, three other drugs (lansoprazole, rolapitant, and idebenone) were identified for their potential to enhance the activity of amphotericin B, which is considered a last-line antifungal therapy. We clarified the mechanisms by which these drugs could restore the activity of antifungal agents. Finally, we confirmed the effectiveness of these combinations in animal models, providing valuable insights into their potential for clinical applications. In summary, our research has opened promising avenues to overcome resistance and develop new treatments for hard-to-treat fungal infections.