Sascha Thewes

The Hyphal-Associated Adhesin and Invasin Als3 of Candida albicans Mediates Iron Acquisition from Host Ferritin

Iron sequestration by host iron-binding proteins is an important mechanism of resistance to microbial infections. Inside oral epithelial cells, iron is stored within ferritin, and is therefore not usually accessible to pathogenic microbes. We observed that the ferritin concentration within oral epithelial cells was directly related to their susceptibility to damage by the human pathogenic fungus, Candida albicans. Thus, we hypothesized that host ferritin is used as an iron source by this organism. We found that C. albicans was able to grow on agar at physiological pH with ferritin as the sole source of iron, while the bakers yeast Saccharomyces cerevisiae could not. A screen of C. albicans mutants lacking components of each of the three known iron acquisition systems revealed that only the reductive pathway is involved in iron utilization from ferritin by this fungus. Additionally, C. albicans hyphae, but not yeast cells, bound ferritin, and this binding was crucial for iron acquisition from ferritin. Transcriptional profiling of wild-type and hyphal-defective C. albicans strains suggested that the C. albicans invasin-like protein Als3 is required for ferritin binding. Hyphae of an Δals3 null mutant had a strongly reduced ability to bind ferritin and these mutant cells grew poorly on agar plates with ferritin as the sole source of iron. Heterologous expression of Als3, but not Als1 or Als5, two closely related members of the Als protein family, allowed S. cerevisiae to bind ferritin. Immunocytochemical localization of ferritin in epithelial cells infected with C. albicans showed ferritin surrounding invading hyphae of the wild-type, but not the Δals3 mutant strain. This mutant was also unable to damage epithelial cells in vitro. Therefore, C. albicans can exploit iron from ferritin via morphology dependent binding through Als3, suggesting that this single protein has multiple virulence attributes.

In vivo and ex vivo comparative transcriptional profiling of invasive and non‐invasive Candida albicans isolates identifies genes associated with tissue invasion

The human pathogenic fungus Candida albicans can cause a wide range of infections and invade multiple organs. To identify C. albicans genes that are expressed during invasion of the liver, we used genome‐wide transcriptional profiling in vivo and ex vivo. By analysing the different phases of intraperitoneal infection from attachment to tissue penetration in a time‐course experiment and by comparing the profiles of an invasive with those of a non‐invasive strain, we identified genes and transcriptional pattern which are associated with the invasion process. This includes genes involved in metabolism, stress, and nutrient uptake, as well as transcriptional programmes regulating morphology and environmental sensing. One of the genes identified as associated with liver invasion was DFG16, a gene crucial for pH‐dependent hyphal formation, correct pH sensing, invasion at physiological pH and systemic infection.

Identifying infection-associated genes of Candida albicans in the postgenomic era

The human pathogenic yeast Candida albicans can cause an unusually broad range of infections reflecting a remarkable potential to adapt to various microniches within the human host. The exceptional adaptability of C. albicans is mediated by rapid alterations in gene expression in response to various environmental stimuli and this transcriptional flexibility can be monitored with tools such as microarrays. Using such technology it is possible to (1) capture a genome-wide portrait of the transcriptome that mirrors the environmental conditions, (2) identify known genes, signalling pathways and transcription factors involved in pathogenesis, (3) identify new patterns of gene expression and (4) identify previously uncharacterized genes that may be associated with infection. In this review, we describe the molecular dissection of three distinct stages of infections, covering both superficial and invasive disease, using in vitro, ex vivo and in vivo infection models and microarrays.

Phenotypic screening, transcriptional profiling, and comparative genomic analysis of an invasive and non-invasive strain of Candida albicans

BackgroundInvasion of host tissue by the human fungal pathogen Candida albicans is an important step during the development of candidosis. However, not all C. albicans strains possess the same invasive and virulence properties. For example, the two clinical isolates SC5314 and ATCC10231 differ in their ability to invade host tissue and cause experimental infections. Strain SC5314 is invasive whereas strain ATCC10231 is non-invasive and strongly attenuated in virulence compared to SC5314. In this study we compare the in vitro phenotypic, transcriptional and genomic profiles of these two widely used laboratory strains in order to determine the principal biological and genetic properties responsible for their differential virulence.ResultsIn all media tested, the two strains showed the same metabolic flexibility, stress resistance, adhesion properties and hydrolytic enzyme secretion in vitro. However, differences were observed in response to cell-surface disturbing agents and alkaline pH. Furthermore, reduced hyphal formation in strain ATCC10231 under certain conditions correlated with reduced invasive properties in an in vitro invasion assay and a reduced ability to invade epithelial tissue. Despite these diverse phenotypic properties, no substantial genomic differences were detected by comparative genome hybridisation within the open reading frames. However, in vitro transcriptional profiling displayed major differences in the gene expression of these two strains, even under normal in vitro growth conditions.ConclusionOur data suggest that the reason for differential virulence of C. albicans strains is not due to the absence of specific genes, but rather due to differences in the expression, function or activity of common genes.

Regulation of aggregate size and pattern by adenosine and caffeine in cellular slime molds

BackgroundMulticellularity in cellular slime molds is achieved by aggregation of several hundreds to thousands of cells. In the model slime mold Dictyostelium discoideum, adenosine is known to increase the aggregate size and its antagonist caffeine reduces the aggregate size. However, it is not clear if the actions of adenosine and caffeine are evolutionarily conserved among other slime molds known to use structurally unrelated chemoattractants. We have examined how the known factors affecting aggregate size are modulated by adenosine and caffeine.ResultAdenosine and caffeine induced the formation of large and small aggregates respectively, in evolutionarily distinct slime molds known to use diverse chemoattractants for their aggregation. Due to its genetic tractability, we chose D. discoideum to further investigate the factors affecting aggregate size. The changes in aggregate size are caused by the effect of the compounds on several parameters such as cell number and size, cell-cell adhesion, cAMP signal relay and cell counting mechanisms. While some of the effects of these two compounds are opposite to each other, interestingly, both compounds increase the intracellular glucose level and strengthen cell-cell adhesion. These compounds also inhibit the synthesis of cAMP phosphodiesterase (PdsA), weakening the relay of extracellular cAMP signal. Adenosine as well as caffeine rescue mutants impaired in stream formation (pde4- and pdiA- ) and colony size (smlA- and ctnA- ) and restore their parental aggregate size.ConclusionAdenosine increased the cell division timings thereby making large number of cells available for aggregation and also it marginally increased the cell size contributing to large aggregate size. Reduced cell division rates and decreased cell size in the presence of caffeine makes the aggregates smaller than controls. Both the compounds altered the speed of the chemotactic amoebae causing a variation in aggregate size. Our data strongly suggests that cytosolic glucose and extracellular cAMP levels are the other major determinants regulating aggregate size and pattern. Importantly, the aggregation process is conserved among different lineages of cellular slime molds despite using unrelated signalling molecules for aggregation.

Stress and development in Dictyostelium discoideum: the involvement of the catalytic calcineurin A subunit

Calcium signaling is one of the most important signaling‐pathways in all eukaryotes. One important target activated by an increased intracellular calcium concentration via calmodulin is the protein phosphatase calcineurin, which is composed of a catalytic subunit (calcineurin A) and a regulatory subunit (calcineurin B). The importance of calcium and calcineurin for the differentiation and development of the social amoeba Dictyostelium discoideum has already been shown by pharmacological approaches. However, so far only a RNAi‐silenced calcineurin B mutant has been investigated on a molecular level. Here, we describe the construction and phenotypic investigation of a RNAi‐silenced calcineurin A mutant. Phenotypic aberrations during development resemble those produced by silencing of calcineurin B with ectopic tip formation of the fruiting bodies. Additionally, we tested the response of the mutants under various stress conditions in liquid culture as well as during development. Both, calcineurin A and B RNAi‐mutants, are hypersensitive during development towards cation stress. Besides its role in development, calcineurin is thus also involved in the stress response in D. discoideum. Further, our data imply that many functions of calcineurin are conserved among the eukaryotes.

The calcineurin dependent transcription factor TacA is involved in development and the stress response of Dictyostelium discoideum

Calcineurin is an important signalling protein in a plethora of Ca(2+)-regulated cellular processes. In contrast to what is known about the function of calcineurin in various organisms, information on calcineurin substrates is still limited. Here we describe the identification and characterisation of the transcription factor activated by calcineurin (TacA) in the model organism Dictyostelium discoideum. TacA is a putative zinc-finger transcription factor orthologue of yeast Crz1. In resting unstimulated cells the protein is located in the cytosol and translocates to the nucleus in a calcineurin-dependent manner after Ca(2+)-stimulation. Nuclear export of TacA is partially dependent on GskA, the Dictyostelium orthologue of mammalian GSK3. The expression of tacA is developmentally regulated with its kinetics roughly paralleling calcineurin regulation. Silencing of tacA via RNAi leads to developmental defects and dysregulation of developmentally regulated and Ca(2+)-regulated marker genes. Additionally, TacA is involved in the stress response of D. discoideum during development in a separate pathway to the well-known stress response in Dictyostelium via STATc. Finally we provide evidence that TacA is not only an orthologue of yeast Crz1 but also functionally related to mammalian NFAT.

Dictyostelium discoideum as a Novel Host System to Study the Interaction between Phagocytes and Yeasts

The social amoeba Dictyostelium discoideum is a well-established model organism to study the interaction between bacteria and phagocytes. In contrast, research using D. discoideum as a host model for fungi is rare. We describe a comprehensive study, which uses D. discoideum as a host model system to investigate the interaction with apathogenic (Saccharomyces cerevisiae) and pathogenic (Candida sp.) yeast. We show that Dictyostelium can be co-cultivated with yeasts on solid media, offering a convenient test to study the interaction between fungi and phagocytes. We demonstrate that a number of D. discoideum mutants increase (atg1−, kil1−, kil2−) or decrease (atg6−) the ability of the amoebae to predate yeast cells. On the yeast side, growth characteristics, reduced phagocytosis rate, as well as known virulence factors of C. albicans (EFG1, CPH1, HGC1, ICL1) contribute to the resistance of yeast cells against predation by the amoebae. Investigating haploid C. albicans strains, we suggest using the amoebae plate test for screening purposes after random mutagenesis. Finally, we discuss the potential of our adapted amoebae plate test to use D. discoideum for risk assessment of yeast strains.

Calcineurin Silencing in Dictyostelium discoideum Leads to Cellular Alterations Affecting Mitochondria, Gene Expression, and Oxidative Stress Response

Calcineurin is involved in development and cell differentiation of the social amoeba Dictyostelium discoideum. However, since knockouts of the calcineurin-encoding genes are not possible in D. discoideum it is assumed that the phosphatase also plays a crucial role during vegetative growth of the amoebae. Therefore, we investigated the role of calcineurin during vegetative growth in D. discoideum. RNAi-silenced calcineurin mutants showed cellular alterations with an abnormal morphology of mitochondria and had increased content of mitochondrial DNA (mtDNA). In contrast, mitochondria showed no substantial functional impairment. Calcineurin-silencing led to altered expression of calcium-regulated genes as well as mitochondrially-encoded genes. Furthermore, genes related to oxidative stress were higher expressed in the mutants, which correlated to an increased resistance towards reactive oxygen species (ROS). Most of the changes observed during vegetative growth were not seen after starvation of the calcineurin mutants. We show that impairment of calcineurin led to many subtle, but in the sum crucial cellular alterations in vegetative D. discoideum cells. As these alterations were not observed after starvation we propose a dual role for calcineurin during growth and development. Our results imply that calcineurin is one player in the mutual interplay between mitochondria and ROS during vegetative growth.