Magister Scientiae Dentium - MSc(Dent) / Candida albicans is a clinical fungal isolate that is most frequently isolated from different host niches, and is implicated in the pathogenesis of several fungal infections, including oral candidiasis. The pathogenesis and antifungal resistance mechanisms of Candida species are complex and involve several pathways and genes. Oral candidiasis incidence rates are rapidly increasing, and the increase in resistance to conventional antifungals has led to the need to develop innocuous and more efficacious treatment modalities. The purpose of this study was to explore a single pot process for phytosynthesis of zinc oxide nanoparticles (GZnO NPs) and to assess their antifungal potential.

This study deals with the identification of 114 yeast isolates from clinical material collected at Dameron Hospital, Stockton, California between August 15, 1990 and September 16, 1991 . The isolates were identified biochemically using the MicroScan Yeast Identification Panel. All isolates were also tested by four conventional cultural methods: germ tube formation, cycloheximide resistance, chlamydospore production, and spidery colony formation. Biochemically, the yeast isolates were identified to nine species of Candida: C. albicans (ll) , C. tropicalis (8), c. stellatoidea (3), c. guilliermondii (2), C. parapsilosis (2), and one of each species of c. catenulata, C. krusei, C. lusitaniae, and C. pseudotropicalis; one species of Torulopsis: T. glabrata (11) and one species of Kluyveromyces, K. lactis (2). Other than those identified as C. albicans, all the other yeasts identified to the species level were negative for all the four conventional tests except for one of two strains of C. guilliermondii which was positive for germ tube, cycloheximide resistance and chlamydospore production. Based on these three tests this isolate of C. guilliermondii would be identified as C. albicans. Five isolates could not be biochemically identified to species. One of the five was positive for the germ tube, resistance to cycloheximide and chlamydospores. Based solely on these three conventional cultural methods this isolate would be considered c. albicans. This isolate and one of the two strains of C. guilliermondii referred to above raise the question as to a possible shortcoming in the Microscan YIP . An interesting observation was the finding that all nine strains of C. albicans recovered from stools of different patients belong to the same biotype.

Orientador: Antonio Olavo Cardoso Jorge / Banca: Sheila Cavalca Cortelli / Banca: Elizabete Brasil dos Santos / Banca: Luiz Eduardo Blumer Rosa / Banca: Yasmin Rodarte Carvalho / Resumo: O objetivo deste trabalho foi avaliar o desenvolvimento de candidose e a recuperação de Candida albicans na cavidade bucal de ratas controles e ovariectomizadas. Foram utilizadas 124 ratas não portadoras do gênero Candida na cavidade bucal, divididas em dois grupos: controle e ovariectomizado. Em cada grupo, quatro ratas foram utilizadas para avaliação morfológica do dorso da língua por microscopia de luz e microscopia eletrônica de varredura; e 58 ratas receberam inoculações de C. albicans para estudo da candidose e recuperação de leveduras. O desenvolvimento de candidose no dorso da língua foi observado em microscopia de luz e microscopia eletrônica de varredura nos períodos de seis horas, 24 horas, sete e 15 dias após a última inoculação. A recuperação de C. albicans foi realizada através de coletas de amostras bucais em intervalos de tempo de um, dois, cinco, sete dias, e progressivamente, a cada 15 dias até a obtenção de culturas negativas para leveduras. Os resultados foram analisados estatisticamente pelos testes t de Student e Mann-Whitney. A morfologia do dorso da língua das ratas do grupo ovariectomizado não infectado por Candida foi semelhante ao controle. Entre as ratas infectadas, o grupo ovariectomizado exibiu menor quantidade de lesões de candidose em relação ao controle. A recuperação de C. albicans da cavidade bucal dos animais ovariectomizados foi inferior aos controles em todos os períodos de observação. Concluiu-se que a candidose e a recuperação de C. albicans na cavidade bucal das ratas foram menos freqüentes no grupo ovariectomizado em relação ao controle / Abstract: The purpose of this work was to observe the development of candidosis and the recovery of C. albicans from the oral cavity of control and ovariectomized rats. Hundred twenty-four rats originally negative for the Candida spp. in the oral cavity were divided into two groups: control and ovariectomized. ln each group, four rats were used for tongue dorsum morphologic analysis by optical microscope and scanning electron microscope; and 58 rats received inoculations of C. albicans for the study of candidasis and recovery of yeasts. The development of candidosis on the tongue dorsum was observed in optical microscope and scanning electron microscope in the periods of six hours, 24 hours, seven days and 15 days after the last inoculation. The recovery of C. albicans was performed through oral samples collected at one, two, tive, seven days and progressively at each 15 days until negative cultures for the yeasts were obtained. The results were statistically analyzed by the t-student and Mann-Whitney tests. The tongue dorsum of the control and ovariectomized rats, not infected by Candida, presented normal aspect. Among lhe infected rats, the ovariectomized group showed less quantity of candidosis lesions in relation to control. The recovery of C. albicans from the oral cavity of ovariectomized animals was lower in relation to control in all periods of observation. lt could be concluded lhat candidosis and recovery of C. albicans ln the oral cavity of rats were less frequent in the ovariectomized group in relation to control / Doutor

Microorganisms that colonize the human body face large fluctuations in their surroundings. Therefore, those microbes developed sophisticated mechanisms that allow them to adapt their cell biology and maintain cellular homeostasis. One organelle vital to preserve cell physiology is the vacuole. The vacuole exhibits a wide range of functions and is able to adjust itself in response to both external and internal stimuli. Moreover, it plays an important role in host interaction and virulence in fungi such as Candida albicans. Despite this connection, only a few regulatory proteins have been described to modulate vacuolar biology in fungal pathogens. Furthermore, whether such regulation alters fungus-host interplay remains largely unknown. This thesis focuses on the characterization of ZCF8, a fungus-specific transcription regulator in the human-associated yeast C. albicans. To this end, I combined genome-wide protein-DNA interaction assays and gene expression analysis that identified genes regulated by Zcf8p. Fluorescence microscopy uncovered that several top targets of Zcf8p localize to the fungal vacuole. Moreover, deletion and overexpression of ZCF8 resulted in alterations in vacuolar morphology and in luminal pH and rendered the fungus resistant or susceptible to a vacuole-disturbing drug. Finally, in vitro adherence assays showed that Zcf8p modulates the attachment of C. albicans to human epithelial cells in a vacuole-dependent manner. Given those findings, I posit that the previously uncharacterized transcription regulator Zcf8p modulates fungal attachment to epithelial cells in a manner that depends on the status of the fungal vacuole. Furthermore, the results highlight that vacuolar physiology is a substantial factor influencing the physical interaction between Candida cells and mammalian mucosal surfaces. / Mikroorganismen, die den Menschen besiedeln, sind großen Schwankungen in ihrer Umgebung ausgesetzt. Daher haben sie ausgeklügelte Mechanismen entwickelt, die es ihnen ermöglichen, ihre Zellbiologie anzupassen und die zelluläre Homöostase aufrechtzuerhalten. Eine für die Aufrechterhaltung der Zellphysiologie wichtige Organelle ist die Vakuole. Sie verfügt über ein breites Spektrum an Funktionen und ist in der Lage, auf externe und interne Stimuli zu reagieren. Außerdem spielt dieses Organell eine wichtige Rolle bei der Pilz-Wirt-Interaktion und somit für die Pathogenität von Pilzen wie Candida albicans. Trotz dieses Zusammenhangs wurden bisher nur wenige regulatorische Proteine beschrieben, welche die Biologie der Vakuolen in pathogenen Pilzen modulieren. Zudem ist weitgehend unbekannt, ob eine solche Regulierung das Zusammenspiel von Pilz und Wirt verändert. Diese Arbeit konzentriert sich auf die Charakterisierung von ZCF8, einem pilzspezifischen Transkriptionsregulator in der pathogenen Hefe C. albicans. Zu diesem Zweck wurden Protein-DNA-Interaktionstests und Genexpressionsanalysen kombiniert, um Gene zu identifizieren, die direkt von Zcf8p reguliert werden. Fluoreszenzmikroskopie zeigte zudem, dass mehrere der wichtigsten Ziele von Zcf8p in der Pilzvakuole lokalisiert sind. Darüber hinaus führte die Deletion und Überexpression von ZCF8 zu Veränderungen der Morphologie und des luminalen pH-Werts der Vakuole, und veränderte die Sensitivität des Pilzes gegenüber Stoffen, welche Funktionen der Vakuole beeinträchtigen. Schließlich deuteten In-vitro-Adhärenztests daraufhin, dass Zcf8p die Anheftung von C. albicans an menschliche Epithelzellen auf eine Weise moduliert, die abhängig von der Vakuole ist. Angesichts dieser Ergebnisse kann davon ausgegangen werden, dass der bisher unbekannte Transkriptionsregulator ZCF8 die Interaktion zwischen Pilz- und Epithelzellen des Wirts kontrolliert, und das auf eine Weise, die von der Pilzvakuole abhängig ist. Des Weiteren, unterstreichen die Ergebnisse, dass die Physiologie der Vakuole ein wesentlicher Faktor ist, welcher die Interaktion zwischen C. albicans und dem Wirt beeinflusst.

OBJECTIVE:: To examine peripheral leukocyte Dectin-1 regulation in clinically relevant models of fungal and polymicrobial sepsis. DESIGN:: Prospective animal study. SETTING:: University medical school research laboratory. SUBJECTS:: Age, weight, and sex matched ICR/HSD mice. INTERVENTIONS:: Mice were infected with Candida albicans (1 × 10, intravenously) or were subjected to cecal ligation and puncture to induce polymicrobial sepsis. MEASUREMENTS:: Blood, spleen, and peritoneal exudate were harvested and leukocytes were isolated. Leukocytes were evaluated for membrane-associated Dectin-1 expression and cell phenotype by flow cytometry. MAIN RESULTS:: In C. albicans infection, Dectin-1-positive blood and splenic leukocytes were increased from 23.5% to 58.9% over the course of infection. The increased percentage of Dectin-1-expressing cells was primarily attributable to neutrophilia. However, the amount of Dectin-1 expressed by blood and splenic neutrophils in C. albicans-infected mice was decreased by a range of 49.0% to 53.3%. C. albicans infection also resulted in an infiltration of Dectin-1-positive macrophages and neutrophils into the kidney. In contrast, polymicrobial sepsis decreased blood leukocyte Dectin-1-expressing cells by up to 51.4%. This reduction was due to a decrease in Dectin-1-positive neutrophils in the periphery. However, the percentage of Dectin-1-expressing cells in the peritoneal cavity increased by 774% with cecal ligation and puncture. Treatment of isolated neutrophils with three soluble glucans, mannan, lipopolysaccharide, or a variety of cytokines revealed that glucans, alone or in combination, were the only treatment that resulted in a decrease in Dectin-1-positive neutrophils. CONCLUSIONS:: We conclude that peripheral leukocyte Dectin-1 expression is differentially regulated in fungal vs. polymicrobial sepsis. These data demonstrate that leukocyte Dectin-1 levels are modulated in response to infections of fungal and nonfungal origin.

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.

Candida albicans is the most common human fungal pathogen. Its relationship with various bacterial species has been documented, showing an increase in host mortality in some cases and a decrease in others. The need for new antibiotics and antifungal treatments have led to studies on polymicrobial interactions and how those interactions impact host health. Interactions between microbes within the human body are inevitable, and exploring these relationships can aid in the development of novel antimicrobials and can deepen our understanding of the complex human microbiome. The relationship between C. albicans and Enterobacter bacteria have yet to be explored. Both are commensal organisms, living asymptomatically in immunocompetent individuals. The hypothesis of this study is that C. albicans and both E. aerogenes and E. cloacae have a positive relationship and work together to infect the host. In this study, the physical cell-to-cell interaction was analyzed by utilizing planktonic (free-floating) and biofilm co-cultures, performing live/dead staining, observing the effect of morphology on the interaction, and examining if Enterobacter alters C. albicans morphology. C. albicans was cultured with Enterobacter lysate to determine if Enterobacter can inhibit C. albicans without physical contact. Enzyme-linked immune assays (ELISAs) were performed on C. albicans ALS deficient mutants to ascertain their potential involvement in cell-to-cell adherence. ELISAs were also utilized to screen E. cloacae mutants for deficiency in attachment to C. albicans. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to compare expression of the HWP1 gene in C. albicans when mono-cultured versus co-cultured. Caenorhabditis elegans were used as a host model to examine the effect that co-culture has on survival and microbial burden. It was found that Enterobacter can inhibit C. albicans growth, no matter its morphology, and it is not necessary for Enterobacter cells to be present for inhibition to occur. C. albicans appears to have more hyphae when incubated with Enterobacter for 24 hours than incubated alone. Research involving ELISAs, RT-PCR, and C. elegans is ongoing.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19774号 / 農博第2170号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4990(農学部図書室) / 32810 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 栗原 達夫, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Objectives: Candida glabrata is the second most common cause of oral candidiasis, second only to C. albicans. Incidence of antifungal resistance has shown a steady increase for C. glabrata. Iron has shown to modulate C. albicans pathogenesis and affect drug-susceptibility. Here, we assess the effect of iron on the growth, antifungal-susceptibility, biofilms, and cell wall of C. glabrata.Methods: Growth, minimal inhibitory concentration (MIC), and biofilm experiments were conducted using 96-well polystyrene plates. Yeast Nitrogen Base medium was used for growth experiments. Cultures of C. glabrata and C. albicans were grown over two nights in respective media containing varying iron concentrations. Rosewell Park Memorial Institute medium was used for MIC and biofilm experiments. Serial dilution was performed to obtain desired concentrations of antifungal drugs. For all experiments, growth was assessed at OD600nm over 24 hours using BioTek Synergy Multi Mode Reader. Paraformaldehyde treated cells and specific stains were used for cell wall studies. Results: Growth of C. glabrata declined significantly below 5μM iron, while C. albicans continued to grow at decreasing iron concentrations, up to 0.5μM. MIC experiments revealed 1.562μM, 1.562μM, and 4μM, as the MIC for Deferasirox, Nystatin, and Fluconazole, respectively. Drug synergy experiments revealed a 128-fold reduction in the amount of Nystatin and Fluconazole needed, with the addition of 1/8th of Deferasirox concentration. The biofilm experiments were inconclusive and the cell wall studies showed decreased levels of mannan, chitin, and an increased β-glucan exposure in high iron conditions. Conclusion: C. glabrata is more sensitive to alterations in environmental iron when compared to C. albicans. Drug synergy experiments underscore the importance of Deferasirox in lowering the MICs of Nystatin or Fluconazole. This can allow use of classical antifungals at lower doses, thereby limiting their side effects. Cell wall studies discuss the effect of iron on the virulence of the C. glabrata. / Oral Biology

Unlike their bacterial counterparts, diseases caused by fungal pathogens are harder to treat due to a lack of discrete targets. Current antifungals are very broad spectrum and fall into three major classes: polyenes which target the cell membrane, azoles which target sterol biosynthesis and the echinocandins which target the cell wall. Recently a novel macrolide antibiotic produced by WAC 2288 was discovered in a co-culturing screen between various actinomycetes and pathogenic fungi. The active compound, a large type I polyketide compound called ibomycin, was specifically able to inhibit the growth of Cryptococcus neoformans but not Candida albicans. A combination of traditional and genetic approaches were used to identify the mode of action of ibomycin. Despite having characteristics associated with membrane perturbing agents such as fungicidal activity, causing hemolysis and even membrane localization in vivo, it does not seem that ibomycin disrupts the membrane in a sterol-dependent manner. We found evidence to suggest that ibomycin is not involved in disruption of cell wall biosynthesis based on localization in vivo and absence of viability rescue in presence of sorbitol. The results of haploinsufficiency and homozygous profiling of yeast deletion strains suggest that is no single protein target for ibomycin, but rather that membrane perturbation of ibomycin leads to downstream effects that impair vesicular trafficking and protein transport. Based on preliminary evidence, it is predicted that C. albicans is able to bind ibomycin but evades the induced toxic effects by barring access to its cell membrane. / Thesis / Master of Science (MSc)