Steffen Rupp

MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene.

In Saccharomyces cerevisiae, two major signal transduction pathways, the Kss1 MAPK pathway and the cAMP‐regulated pathway, are critical for the differentiation of round yeast form cells to multicellular, invasive pseudohyphae. Here we report that these parallel pathways converge on the promoter of a gene, FLO11, which encodes a cell surface protein required for pseudohyphal formation. The FLO11 promoter is unusually large, containing at least four upstream activation sequences (UASs) and nine repression elements which together span at least 2.8 kb. Several lines of evidence indicate that the MAPK and cAMP signals are received by distinct transcription factors and promoter elements. First, regulation via the MAPK pathway requires the transcription factors Ste12p/Tec1p, whereas cAMP‐mediated activation requires a distinct factor, Flo8p. Secondly, mutations in either pathway block FLO11 transcription. Overexpression of STE12 can suppress the loss of FLO8, and overexpression of FLO8 can suppress the loss of STE12. Finally, multiple distinct promoter regions of the FLO11 promoter are required for its activation by either Flo8p or Ste12p/Tec1p. Thus, like the promoters of the key developmental genes, HO and IME1, the FLO11 promoter is large and complex, endowing it with the ability to integrate multiple inputs.

A microarray enzyme‐linked immunosorbent assay for autoimmune diagnostics

In order to quantify autoantibodies in the sera of patients with autoimmune disease, we have created a microarray‐based immunoassay that allows the simultaneous analysis of 18 known autoantigens. The microarrays contain serial dilutions of the various antigens, thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. The assay is very sensitive and highly specific: as little as 40 fg of a known protein standard can be detected with little or no cross‐reactivity to nonspecific proteins. The signal intensities observed from serial dilutions of immobilized antigen correlate well with serial dilutions of autoimmune sera. Miniaturized and highly parallelized immunoassays like these will reduce costs by decreasing reagent consumption and improve efficiency by greatly increasing the number of assays that can be performed with a single serum sample. This system will significantly facilitate and accelerate the diagnostics of autoimmune diseases and can be adapted easily to any other kind of immunoassay.

The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans

The temporal and spatial expression of stage‐specific genes during morphological development of fungi and higher eukaryotes is controlled by transcription factors. In this study, we report the cloning and functional analysis of the Candida albicans TEC1 (CaTEC1) gene, a new member of the TEA/ATTS family of transcription factors that regulates C. albicans virulence. The promoters of the type 4, 5 and 6 proteinase isogenes (SAP4–6) contain repetitive TEA/ATTS consensus sequence motifs. This finding suggests a possible role for a homologue of Saccharomyces cerevisiae TEC1 during the activation of proteinase gene expression in C. albicans. CaTEC1 is predominantly expressed in the hyphal form of C. albicans. In vitro, serum‐induced hyphal formation as well as evasion from MΦ after phagocytosis is suppressed in catec1/catec1 mutant cells. Furthermore, expression of the proteinase isogenes SAP4–6 is no longer inducible in these mutant cells. The deletion of the CaTEC1 gene attenuates virulence of C. albicans in a systemic model of murine candidiasis, although both mutant and revertant cells that were prepared from infected tissues or the vaginal mucosa grew in a hyphal morphology in vivo. CaTEC1 complements the pseudohyphal and invasive growth defect of haploid and diploid S. cerevisiae tec1/tec1 mutant cells and strongly activates the promoter of FLO11, a gene required for pseudohyphal growth. This study provides the first evidence pointing to an essential role for a member of the TEA/ATTS transcription factor family that had so far only been ascribed to function during development as a virulence regulator in microbial pathogenesis.

EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays

Cell wall dynamics in Candida albicans, the most common fungal pathogen in man, underlie regulatory processes during the yeast‐to‐hyphae transition. To analyse this regulation at the transcriptional level, we have established a DNA microarray representing genes implicated in cell wall biogenesis. Using these microarrays, we were able to identify YWP1 and HWP2 that are specifically transcribed in the yeast or hyphal growth form respectively. Cluster analysis revealed at least two major clusters of genes: cluster I comprised genes that were upregulated under at least one hyphae‐inducing condition. Three as yet not further characterized genes were attributed to cluster II. These genes were transcribed in the yeast form of C. albicans and were downregulated in an EFG1‐dependent manner under specific hyphae‐inducing conditions. We show further that, in contrast to CPH1, EFG1 plays a major role in the transcriptional regulation of cell wall proteins under the conditions investigated. EFG1 was essential for the transcription of both hyphae‐specific genes such as HWP1 and HWP2 as well as the yeast form‐specific gene YWP1. Moreover, we found that, under various conditions, EFG1 also can act as a strong repressor for the transcription of RBE1, another not yet characterized cell wall protein. Overall, our data show that EFG1 plays a major role in the induction and repression of cell wall genes, not only in the hyphal form but also in the yeast form of C. albicans.

In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion.

The individual and synergistic contributions of two transcription factors, EFG1 and CPH1, have been characterized with regard to adhesion to, and invasion of, human epithelia by Candida albicans. For this purpose two in vitro reconstructed tissue models were developed. A multi-layered model of human epidermis was used to simulate superficial infections of the skin, whereas a reconstructed human intestinal model was used to mimic the first steps of systemic infections. It was shown that C. albicans deleted for both transcription factors CPH1 and EFG1, in contrast to the congenic clinical isolate Sc5314, was neither able to adhere to, nor to penetrate, either of the model systems. A strain deleted for EFG1 alone showed significant reduction in adhesion and was not able to penetrate through the stratum corneum. However, strains deleted for CPH1 showed phenotypes paralleling the phenotypes of the clinical isolate Sc5314. Using different types of multi-layered human tissues reconstructed in vitro the individual contributions of Efg1p and Cph1p to two important virulence factors of C. albicans, namely adhesion and invasion, could be defined.

Identification of cell surface determinants in Candida albicans reveals Tsa1p, a protein differentially localized in the cell.

To identify cell surface proteins of Candida albicans, the predominant fungal pathogen in humans, we have established an approach using a membrane impermeable biotin derivative in combination with affinity purification. We were able to identify 29 different proteins under two distinct conditions. Among mannoproteins, heat shock proteins and glycolytic enzymes we found thiol‐specific antioxidant‐like protein 1 (Tsa1p) to be differentially localized depending on the conditions applied. Only in hyphally grown cells Tsa1p was localized to the cell surface whereas in blastospores no surface but mainly nuclear localization was found. This indicates that cell surface expression of at least some proteins is mediated by differential translocation.

Immune evasion of the human pathogenic yeast Candida albicans: Pra1 is a Factor H, FHL-1 and plasminogen binding surface protein

The pathogenic yeast Candida albicans utilizes human complement regulators, like Factor H and Factor H like protein-1 (FHL-1) for immune evasion. By screening a C. albicans cDNA expression library, we identified the pH-regulated antigen 1 (Pra1) as a novel Factor H and FHL-1 binding protein. Consequently Pra1 was recombinantly expressed in Pichia pastoris and purified from culture supernatant. Recombinant Pra1 binds Factor H, FHL-1 and also plasminogen. Attached to Pra1, the three human proteins are functionally active. Factor H and FHL-1 inactivate complement and plasminogen can be activated to plasmin which then degrades the extra-cellular matrix component fibrinogen. Polyclonal Pra1 anti-serum was generated and used to localize Pra1 on the surface and also in the culture supernatant of both yeast cells and the hyphal form of C. albicans. Furthermore Pra1 expression was up-regulated upon induction of hyphal growth. Pra1, released by Candida cells binds back to the surface of Candida hyphae and in addition enhances the complement regulatory activity of Factor H in the fluid phase. A Pra1 overexpression strain, with about twofold higher levels of Pra1 on the surface binds more Factor H, and plasminogen. In summary, C. albicans Pra1 is a yeast immune evasion protein that binds host immune regulators and acts at different sites. As a surface protein, Pra1 acquires the two human complement regulators Factor H, FHL-1 and plasminogen, mediates complement evasion, as well as extra-cellular matrix interaction and/or degradation. As a released protein, Pra1 enhances complement control in direct vicinity of the yeast and thus generates an additional protective layer which controls host complement attack.

Vacuolar/lysosomal proteolysis: proteases, substrates mechanisms

Proteolysis is an essential post-transcriptional process. The lysosome/vacuole is the central organelle for non-specific proteolysis in eukaryotes. Most proteases that work in the lysosome enter it via the secretory pathway. The bulk of proteins to be degraded enter this proteolytic compartment via endocytosis and autophagocytosis. Our understanding of the mechanisms involved in these processes has increased considerably, and the information obtained in these studies has enabled new, specific proteolytic pathways to be investigated in other cellular compartments, particularly in the cytoplasm.

Candida albicans Sun41p, a Putative Glycosidase, Is Involved in Morphogenesis, Cell Wall Biogenesis, and Biofilm Formation

ABSTRACT The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction.

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.