Candida albicans is the most common human commensal fungus, able to colonize host niches such as skin, mouth and gastrointestinal tract. Colonization of diverse microenvironments requires the ability to evade or overcome innate host protection and adapt to rapid transitions between environments with different stresses and nutrient availability. Colonization of the gastrointestinal tract requires passage through the stomach containing toxic levels of nitric oxide, generated from acidification of nitrite in the low pH of the stomach. Although resistance of C. albicans to nitric oxide is mediated by the flavohemoglobin Yhb1, little is known about the physiologically relevant ligands that regulate YHB1 expression. Here I propose the hypothesis that nontoxic saliva chemicals induce YHB1 expression and promote resistance to nitric oxide generated in the stomach. Supporting this hypothesis is the observation that two ions actively concentrated in the saliva – nitrate and thiocyanate – induce YHB1 expression. Indeed, whole-genome transcriptional analysis of C. albicans treated with nitrate or thiocyanate produce gene expression profiles nearly identical to cells treated with nitrite or nitric oxide. Pretreatment of C. albicans with either of these two nontoxic compounds increases resistance of the yeast to nitric oxide. I propose that this is an evolved response in which C. albicans anticipates nitric oxide stress generated in the stomach. C. albicans thus upregulates nitric oxide stress response genes in response to saliva signals that precede nitric oxide formation further on in the gut. Only a few examples of anticipatory signaling have so far been identified and it is not known how common this type of regulation is among microbes. Expression of the YHB1 gene in response to nitric oxide is regulated by the transcription factor Cta4. I show that Cta4 binds to the YHB1 promoter in vivo as a homodimer and is necessary, but not sufficient, for nitric oxide, nitrate and thiocyanate induced expression of YHB1. Based on these data I propose a model in which Cta4 transcriptional activation is inhibited under non-inducing conditions by a negative regulator. Understanding the mechanism by which C. albicans senses and responds to nitric oxide, nitrate and thiocyanate remains a question for future research.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 203-238).

The number of superficial candidal infections has grown due to an increase in the elderly cohort and ever-increasing immunocompromised population, and the increased prevalence of antifungal drug resistance. The aim of this research was to investigate the antifungal efficacy of a citrus fruit extract, Biosecur c320c (B320), against two strains of Candida albicans (nystatin sensitive and resistant). The viability of C. albicans strain treated with 10% B320 was reduced by 90-99% depending on biofilm age. A B320/nystatin combination demonstrated even greater efficacy at killing biofilm cells in either strain. The nystatin sensitive strain did not develop resistance to B320 while resistance was developed following long-term exposure to nystatin. Scanning electron micrographs of C. albicans biofilms revealed cellular debris after treatment with combined B320/nystatin. Citrus fruit extracts containing polyphenols might contribute to the development of novel therapeutics for the treatment of oral candidiasis, particularly in those refractory to nystatin therapy.

The number of superficial candidal infections has grown due to an increase in the elderly cohort and ever-increasing immunocompromised population, and the increased prevalence of antifungal drug resistance. The aim of this research was to investigate the antifungal efficacy of a citrus fruit extract, Biosecur c320c (B320), against two strains of Candida albicans (nystatin sensitive and resistant). The viability of C. albicans strain treated with 10% B320 was reduced by 90-99% depending on biofilm age. A B320/nystatin combination demonstrated even greater efficacy at killing biofilm cells in either strain. The nystatin sensitive strain did not develop resistance to B320 while resistance was developed following long-term exposure to nystatin. Scanning electron micrographs of C. albicans biofilms revealed cellular debris after treatment with combined B320/nystatin. Citrus fruit extracts containing polyphenols might contribute to the development of novel therapeutics for the treatment of oral candidiasis, particularly in those refractory to nystatin therapy.

Neutrophils are among the first white blood cells recruited to the site of infection once microbial pathogens enter the host organism. At site, they perform a well-orchestrated chain of processes that aims to kill the microbial invader. Most prominent, neutrophils engulf microbes to inactivate them intracellularly, a process called phagocytosis. Alternatively, neutrophils can release neutrophil extracellular traps (NETs). NETs consist of chromatin decorated with antimicrobial effector proteins – a structure that can entangle bacteria and fungi. Neutrophils are crucial during fungal infections. This is reflected in the increased risk of fungal infections resulting of neutropenia. The concept of nutritional immunity describes every infection as a battle for resources. Those are mostly metal trace elements. For a long time, neutrophils were seen as powerful, but “mindless”, killers with a limited set of actions and no transcriptional capacity, but this view is in the flux. In the presented thesis, it was my goal to gain new insights into the interplay of neutrophils and fungi – with special attention to metal-nutritional aspects. We compared human neutrophils lacking the ability to undergo NETosis, due to a non-functional NADPH complex, and neutrophils from the same person that were “cured” by gene therapy. We investigated those NETs and found that their inhibitory activity towards the mold A. nidulans depends on calprotectin, a known zinc-chelator. Considering the high influx of neutrophils, we wanted to unravel the neutrophils’ contribution to the metal milieu at the site of infection and trace element changes resulting from NETosis. By combining synchrotron radiation XRF and ICP-MS, we analyzed the neutrophil metallome and the spatial element distribution in activated neutrophils and NETs. Most strikingly, we found neutrophils to be exceptionally high in Fe and the process of NETosis to be reducing available Zn in the surrounding and the early phagosome, possibly by the formation of Zn-rich vesicles. Using RNA-sequencing, we analyzed the interplay of the C. albicans and neutrophils face-to-face. We dissected their transcriptional profile and revealed a manifold response in neutrophils that include cytokine induction and cellular rearrangement. We further were the firsts to explore the transcriptional response of C. albicans to NETs. Our data indicates a distinct response compared to intact neutrophils or other known stress triggers. Metal homeostasis was affected in Candida in both set-ups. In summary, this thesis provides new insights into the interaction of fungal pathogens with neutrophils and emphasizes the impact of nutritional aspects on this interplay. A deeper understanding of the nutritional immunity during fungal infection might open up new strategies to tackle fungal infections – a growing threat worldwide.

La poly(A) polymérase (PAP) est une enzyme essentielle pour le métabolisme de la cellule qui catalyse l'ajout d'une longue séquence de polyadénosines à l'extrémité 3' des ARNm. Cet ajout, effectué par un large complexe multi-protéique de clivage et de polyadénylation est nécessaire pour la stabilité, le transport et la traduction des ARNm. L'ATP est la molécule qui sert de source d'énergie à la PAP et représente aussi son unique substrat pour la réaction de polyadénylation. La PAP est une enzyme hautement conservée de la levure à l'humain et est même encodée dans le génome de certains virus. En effet, elle est aussi retrouvée chez les eucaryotes inférieurs, comme les levures, et même certains virus à ADN encodent une enzyme jouant le même rôle. Le champignon Candida albicans , source d'infections sévères surtout chez les patients immunodéprimés, encode sa propre PAP. L'étude de cet organisme tantôt levure unicellulaire, tantôt champignon mycellaire demeure essentielle en raison de la perte d'efficacité des antibiotiques actuellement disponibles découlant de l'émergence de souches multi-résistantes. La PAP représente une nouvelle cible thérapeutique fort intéressante due à son implication directe dans les étapes de maturation des ARNm, processus essentiel à tout organisme eucaryote. L'étude présentée dans ce mémoire se concentre sur l'étude du site actif de la PAP de C. albicans , plus précisément au niveau des interactions moléculaires associées à la sélection du substrat par l'enzyme. Le but final est de déterminer les groupements fonctionnels favorisant la reconnaissance de l'ATP au niveau du substrat nucléotidique, mais aussi de déterminer les acides aminés de la protéine qui sont potentiellement impliqués dans cette sélection moléculaire. Pour ce faire, une gamme d'analogues de nucléotides, principalement de purines, a été testée pour évaluer la capacité de ceux-ci à compétitionner avec le substrat pour le site actif de l'enzyme. Du côté de la protéine, plusieurs mutants ponctuels ont été produits afin d'évaluer la discrimination de ces mutants envers les différents nucléotides et/ou analogues de nucléotides. La sélection des acides aminés mutés s'est basée sur le modèle bioinformatique de la PAP de C. albicans généré à partir de la radiocristallographie de la PAP d'un autre organisme eucaryote effectué en présence d'ATP et d'ARN, Saccharomyces cerevisiae . Les résultats ont en effet démontré que la présence d'un groupement donneur de pont hydrogène en position 6 d'une purine permet sa liaison au site actif de l'enzyme et son transfert subséquent sur le brin d'ARN. En effet, ceci explique en grande partie la discrimination entre l'ATP et le GTP par la protéine. Aussi, l'apport de certains acides aminés tels Asn-222, Met-306 et Cys-307 dans ce mécanisme de distinction des nucléotides a été démontré. De manière insoupçonnée, certains analogues se sont avérés de bons substrats pour la PAP malgré leur modification chimique, donnant lieu à une queue poly(A) modifiée chimiquement. L'impact de cette modification sur la stabilité et l'efficacité de traduction d'ARNm portant divers types de modification sur la queue poly(A) a donc été évalué en cellules. Il s'avère que la stabilité semble négativement affectée de manière générale alors que l'efficacité de traduction s'est vu être dépendante du type de modification chimique.

Candida species are ubiquitous, ranging from pure saprobes through endo-symbionts of animals, to pathogens in many animals including humans. Some of the pathogenic species are of medical importance, especially Candida albicans. However, the prevalence of other non-albicans Candida species as human pathogens has been increasing worldwide. The aim of this study was to use conventional phenotypic tests and molecular methods to isolate, identify and characterise 600 Candida isolates from three teaching hospitals in Ghana, namely Korle Bu, Komfo Anokye and Tamale from mid-January to April, 2012. The prevalence of these species in cases of genitourinary candidiasis and pelvic inflammatory disease was investigated. Preliminary identification and characterisation of Candida isolates using four conventional phenotypic tests showed that C. albicans was the most common species, which constituted 41% of the isolates whereas non-albicans Candida species constituted 59% of the total number of Candida isolates. In patients presenting with vulvovaginal candidiasis (VVC) for at least two or more times, chi-square analysis indicates that the frequency of Candida species isolated were not statistically different from patients presenting for the first time with VVC. Candida albicans was the most common species in vaginal swabs from patients presenting with vulvovaginal candidiasis (VVC) for the first time in each of the three locations, present in 53.4% of the total swabs. The other species that were present were C. glabrata (21.6%), C. parapsilosis (15.5%), C. tropicalis (4.7%) and C. krusei (4.7%). Similar Candida species distributions were found in swabs taken from patients presenting with suspected pelvic inflammatory disease (PID). Across the three locations, however, there was a significant difference in the frequency of C. albicans, which was present in 68 and 69.6% of patients from Komfo Anokye and Tamale, but only 26.7% of patients from Korle Bu. Urine samples were taken in two of the locations, Korle Bu and Tamale, from female patients presenting with candiduria. Statistical analysis indicated a significant difference in the frequency of Candida isolates in cases of candiduria between the two locations. In Korle Bu, C. glabrata was the most prominent species (37.8%) followed by C. albicans (22.4%), C. parapsilosis (21.7%), C. tropicalis (10.5%), C. krusei (7%) and C. lusitaniae (0.7%). In Tamale, the species distribution was C. albicans (60.9%), C. glabrata (21.7%), C. parapsilosis (13%) and C. krusei (4.3%). The data highlight the prevalence of species other than C. albicans in case of candidiasis in Ghana. Delineation of C. albicans strains using the 25S rDNA to investigate the genotypic variation among C. albicans isolates showed that genotype A constituted about 95% of the Ghanaian C. albicans isolates, genotypes B and C constituted 2.5% each respectively. The general-purpose genotype (GPG) which corresponds to clade 1 among C. albicans was also investigated to know the prevalence of clade 1 among the C. albicans isolates investigated. The presence or absence of general-purpose genotype (GPG) gene was used to categorise the 240 C. albicans to clade 1 or other clade. The test revealed that 64.2% had the GPG genotype which corresponds to clade 1 and the remaining 35.8% were of non-GPG genotype; thus belongs to other clades. The population structure of C. albicans from the three teaching hospitals indicates a mainly clonal and homogeneous population across the three sampling locations from Ghana. Molecular analyses of the transposable group 1 intron in the ITS1-5.8S-ITS2 region using universal primer pair ITS1 and ITS4 revealed the presence of two rare Candida species; Candida rugosa and Candida mesorugosa. To the best of my knowledge this is the first report of either of these in Africa. Antifungal susceptibility tests among Candida isolates recovered from patients presenting with clinically suspected or symptomatic candidal vaginitis for the first time and patients presenting with candidal vaginitis on two or more occasions revealed a high percentage of Fluconazole-resistant C. albicans. This study highlights the prevalence of species other than C. albicans in cases of candidiasis in Ghana. Furthermore, this study has also demonstrated that no single conventional phenotypic test has been highly efficient to delineate Candida species into their respective species type. Thus, development of an identification scheme, which can discriminate between Candida isolates both at species and strain levels, will have prognostic and therapeutic significance for effective patient management.

S-Adenosylmethionine (SAM) synthetases isolated from both the yeast and hyphal-phase of the dimorphic fungus, C. albicans, were partially purified using DEAE cellulose ion-exchange column chromatography. Further characterization was accomplished using enzyme kinetics and specific enzyme effectors. SAM synthetase is the enzyme responsible for synthesis of SAM which is the major methyl group donor in the methylation of macromolecules. Kinetic studies on column samples, from both phases, were performed. The yeast-phase enzyme had apparent Km ranges for L-methionine and ATP of 1.06-1.42mM and 1.11-1.69mM, respectively. The hyphal-phase enzyme had apparent Km ranges for L-methionine and ATP of 1.34-2.66mM and 3.29-6.28mM, respectively. Effector studies (in vitro) indicate that 10% (v/v) dimethyl sulfoxide (DMSO) and 5mM cycloleucine inhibit SAM Synthetase from both phases, 24% and 46%, respectively. The methionine analogues DLmethionine sulfone, DL-methionine-DL-sulfoxide and L-methioninesulfoximine and sinefungin, an analog of SAM, had no effect on SAM synthetase activity. Although the data is inconclusive with respect to the existence of isozymes, the observed Km's of the yeast and hyphal-phases are different suggesting that isozymes may exist. Additionally, the yeast-phase DEAE column profile has a shoulder prior to the main peak of activity indicating that more then one form of the enzyme may be present. / Department of Biology

Diss. Umeå : Umeå universitet, 2005.

Thesis (M.S.)--Georgia State University, 2008. / Title from file title page. Keith Pascoe, committee chair; Alfons Baumstark, Jerry Smith, Davon Kennedy, committee members. Electronic text (46 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed July 8, 2008. Includes bibliographical references (p. 45-46).