Developmental Regulation of an Adhesin Gene during Cellular Morphogenesis in the Fungal Pathogen Candida albicans

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Transcriptional regulation of morphogenesis in 'Candida albicans'

ALS3 encodes a hypha-specific cell wall protein of the Agglutinin-Like Sequence family of adhesins.  The first aim of this project was to define the region of the ALS3 promoter required for its hypha-specific activation. Previous work in our laboratory defined a region of 190 bp between -496 and -306 that is important for ALS3 activation.  Furthermore, two sequences that bind the transcriptional repressor Nrgl (NREs), were shown to be required for repression under yeast inducing conditions.  In this study, the importance of these NREs for ALS3 repression was confirmed.  Between the two studies, eight 13-68 bp overlapping sequences of the ALS3 promoter were placed upstream of the basal -306 to +4 region of ALS3 promoter (previously shown to cause no activation).  None of these induced expression of the reporter.  Further deletions downstream from -471, or upstream from -321 disrupted the hypha-specific regulation of ALS3. These results suggest that no short region or known response element, in isolation, is sufficient for hypha-specific activation of ALS3.  During this study an efficient Streptococcus thermophilus lacZ reporter was developed for C. albicans.  This was adopted to enable a high throughput mutagenesis screen on the ALS3 promoter. Another aim of this study was to contribute to the annotation of the C. albicans genome sequence with the EU Galar Fungail Consortium.  This was achieved by annotating 6.5% (401 ORFs) of the predicted open reading frames annotated by the Consortium as a whole.  These data were used by Christophe d’Enfert to design whole genome C. albicans microassays and to generate the database CandidaDB (http://genolist.pasteur.fr/CandidaDB/).  Protocols for the use of these microarrays and for transcript profiling data analysis were established.  Validation of the microarrays was carried out by the transcript profiling of cells which had undergone a temperature shift from 25°C to 37°C.  This showed the up-regulation of some heat shock and stress response genes, as expected.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Global Roles of Ssn6 in Tup1- and Nrg1-dependent Gene Regulation in the Fungal Pathogen, Candida albicans

In budding yeast, Tup1 and Ssn6/Cyc8 form a corepressor that regulates a large number of genes. This Tup1-Ssn6 corepressor appears to be conserved from yeast to man. In the pathogenic fungus Candida albicans, Tup1 regulates cellular morphogenesis, phenotypic switching, and metabolism, but the role of Ssn6 remains unclear. We show that there are clear differences in the morphological and invasive phenotypes of C. albicans ssn6 and tup1 mutants. Unlike Tup1, Ssn6 depletion promoted morphological events reminiscent of phenotypic switching rather than filamentous growth. Transcript profiling revealed minimal overlap between the Ssn6 and Tup1 regulons. Hypha-specific genes, which are repressed by Tup1 and Nrg1, were not derepressed in ssn6 cells under the conditions studied. In contrast, the phase specific gene WH11 was derepressed in ssn6 cells, but not in tup1 or nrg1 cells. Hence Ssn6 and Tup1 play distinct roles in C. albicans. Nevertheless, both Ssn6 and Tup1 were required for the Nrg1-mediated repression of an artificial NRE promoter, and lexA-Nrg1 mediated repression in the C. albicans one-hybrid system. These observations are explained in models that are generally consistent with the Tup1-Ssn6 paradigm in budding yeast

The Early Transcriptional Response of Human Granulocytes to Infection with Candida albicans Is Not Essential for Killing but Reflects Cellular Communications

Candida albicans is a polymorphic opportunistic fungus that can cause life-threatening systemic infections following hematogenous dissemination in patients susceptible to nosocomial infection. Neutrophils form part of the innate immune response, which is the first line of defense against microbes and is particularly important in C. albicans infections. To compare the transcriptional response of leukocytes exposed to C. albicans, we investigated the expression of key cytokine genes in polymorphonuclear and mononuclear leukocytes after incubation with C. albicans for 1 h. Isolated mononuclear cells expressed high levels of genes encoding proinflammatory signaling molecules, whereas neutrophils exhibited much lower levels, similar to those observed in whole blood. The global transcriptional profile of neutrophils was examined by using an immunology-biased human microarray to determine whether different morphological forms or the viability of C. albicans altered the transcriptome. Hyphal cells appeared to have the broadest effect, although the most strongly induced genes were regulated independently of morphology or viability. These genes were involved in proinflammatory cell-cell signaling, cell signal transduction, and cell growth. Generally, genes encoding known components of neutrophil granules showed no upregulation at this time point; however, lactoferrin, a well-known candidacidal peptide, was secreted by neutrophils. Addition to inhibitors of RNA or protein de novo synthesis did not influence the killing activity within 30 min. These results support the general notion that neutrophils do not require gene transcription to mount an immediate and direct attack against microbes. However, neutrophils exposed to C. albicans express genes involved in communication with other immune cells