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

<p>Abstract</p> <p>Background</p> <p>Multicellularity in cellular slime molds is achieved by aggregation of several hundreds to thousands of cells. In the model slime mold <it>Dictyostelium discoideum</it>, 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.</p> <p>Result</p> <p>Adenosine 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 <it>D. discoideum </it>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 (<it>pde4^-</it>and <it>pdiA^-</it>) and colony size (<it>smlA^-</it>and <it>ctnA^-</it>) and restore their parental aggregate size.</p> <p>Conclusion</p> <p>Adenosine 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.</p

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

<p>Abstract</p> <p>Background</p> <p>Invasion of host tissue by the human fungal pathogen <it>Candida albicans </it>is an important step during the development of candidosis. However, not all <it>C. albicans </it>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 <it>in vitro </it>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.</p> <p>Results</p> <p>In all media tested, the two strains showed the same metabolic flexibility, stress resistance, adhesion properties and hydrolytic enzyme secretion <it>in vitro</it>. 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 <it>in vitro </it>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, <it>in vitro </it>transcriptional profiling displayed major differences in the gene expression of these two strains, even under normal <it>in vitro </it>growth conditions.</p> <p>Conclusion</p> <p>Our data suggest that the reason for differential virulence of <it>C. albicans </it>strains is not due to the absence of specific genes, but rather due to differences in the expression, function or activity of common genes.</p

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 baker's 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

Candida albicans Scavenges Host Zinc via Pra1 during Endothelial Invasion

The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution

Molecular investigation of invasive properties of Candida albicans

Titel und Inhalt Einleitung Material und Methoden Ergebnisse Diskussion Zusammenfassung / Summary Literaturverzeichnis AnhangCandida-Arten gehören beim Menschen zur normalen mikrobiellen Flora der Haut und Schleimhäute und können in bis zu 71 % der Gesamtbevölkerung nachgewiesen werden. Der häufigste Vertreter ist dabei C. albicans, der als Kommensale normalerweise harmlos ist. Wird jedoch das Gleichgewicht der mikrobiellen Flora verändert oder das Immunsystem des Menschen geschwächt, so kann es neben oberflächlichen Infektionen mit C. albicans auch zu invasiven Infektionen kommen, bei denen der Pilz in tiefere Gewebeschichten vordringt und in das Blutsystem gelangt. Daneben kann der Pilz aber auch bei z.B. operativen Eingriffen direkt in die Bauchhöhle gelangen und von dort parenchymale Organe infizieren. Es haben aber nicht alle C. albicans Stämme die gleichen invasiven Eigenschaften. So konnte für Stamm SC5314 gezeigt werden, dass er hoch virulent und stark invasiv ist. Stamm ATCC10231 hingegen war in allen bisher getesteten Infektionsmodellen avirulent und nicht-invasiv. In der vorliegenden Arbeit konnte gezeigt werden, dass sich die beiden C. albicans Stämme SC5314 und ATCC10231 außer in einem unterschiedlich schnellen Wachstum nur in wenigen phänotypischen und genotypischen Punkten unterschieden. Dabei konnte keine Korrelation zwischen den unterschiedlichen in vitro Phänotypen bzw. den Unterschieden auf genomischer Ebene und der unterschiedlichen Invasivität der beiden Stämme beobachtet werden. Es konnte jedoch auf einer ex vivo perfundierten Schweineleber gezeigt werden, dass Stamm ATCC10231 auch in diesem Infektionsmodell nicht-invasiv ist. Weiterhin konnte mit Hilfe von Transkriptionsprofilen der beiden Stämme, welche aus Organproben nach einer intraperitonealen Infektion der Maus gewonnen wurden, gezeigt werden, dass sich die unterschiedlichen Invasionseigenschaften der beiden Stämme auf transkriptioneller Ebene widerspiegeln. So waren die Transkriptionsprofile der frühen Infektionsphasen bei beiden Stämmen noch recht ähnlich, zu Beginn der Invasion unterschieden sich die Profile jedoch deutlich. Durch die Einbeziehung von Transkriptionsprofilen, welche aus Organproben der ex vivo Infektion gewonnen wurden, konnten Gene identifiziert werden, von denen angenommen wurde, dass ihre Expression direkt im Zusammenhang mit der Invasion steht. Während die putative Metalloproteinase Axl1 keinen direkten Einfluss auf die Invasion in vitro hatte, konnte ein solcher Zusammenhang für den putativen pH-Sensor Dfg16 nachgewiesen werden. Eine Deletion des kodierenden Gens DFG16 führte demnach zu Defekten in der Hyphenausbildung bei neutralen bis alkalischen pH-Werten. Weiterhin sind vor allem die fehlende Invasivität in vitro und die Avirulenz der Mutante in einem intravenösen Infektionsmodell hervorzuheben. Außerdem spiegelte sich bei der Mutante der Defekt in der Wahrnehmung des extrazellulären alkalischen pH- Wertes auch auf transkriptioneller Ebene wider. Unterstützt durch eine Lokalisationsstudie, bei der gezeigt wurde, dass Dfg16 in der Plasmamembran von C. albicans lokalisiert ist, kann somit postuliert werden, dass die korrekte Wahrnehmung des extrazellulären pH-Wertes durch den putativen pH-Sensor Dfg16 eine entscheidende Vorraussetzung für eine erfolgreiche Invasion von C. albicans in Wirtsgewebe darstellt.Fungi of the genus Candida belong to the normal microbial flora of the skin and mucosa of humans. They can be found in up to 71 % of the total population. The major representative of this genus is Candida albicans which is harmless in its commensal lifestyle. But if the balance of the microbial flora is changed or if the immune system of the host is weakened C. albicans is able to produce superficial and invasive infections. During invasive infections C. albicans penetrates into deeper tissue finally reaching the bloodstream resulting in disseminated candidosis. Furthermore the fungus can infect the intraperitoneal cavity during e. g. surgery resulting in infection of parenchymal organs. But not all C. albicans strains have the same invasive properties. Strain SC5314 for example is fully virulent and invasive whereas strain ATCC10231 is avirulent and non-invasive in all animal models tested so far. And also in this study it was shown that the two strains differ in their ability to invade into a newly established ex vivo infection model based on perfused pig liver. Furthermore it was shown in this study that the two C. albicans strains SC5314 and ATCC10231 differ only in a few phenotypic and genotypic points. No correlation was found between the observed phenotypes in vitro and the differences on the genomic level. However, analysing transcriptional profiles of both strains after intraperitoneal infection of mice it could be shown that the different invasive properties of both strains correlate with the transcriptional profiles. At early stages of infection the profiles of both strains were very similar. But at later time points when strain SC5314 began to invade the liver the profiles were differed significantly. Including transcriptional profiles from ex vivo infected organ samples into the analysis, genes were identified which were only expressed during invasion. Whereas the putative metalloproteinase Axl1 had no direct influence on the invasion of C. albicans such a relationship was observed for the putative pH sensor Dfg16. Deletion of the coding gene DFG16 resulted in (1.) defects in hyphae formation at neutral to alkaline pH values, (2.) loss of invasiveness in vitro, and (3.) significant reduced virulence of the mutant in an intravenous infection model. In addition, defects in recognition of the extracelluar pH were also seen on the transcriptional level of the mutant. Supported by localisation studies in which it was shown that Dfg16 is located in the plasma membrane of C. albicans it can be postulated that correct sensing of the extracellular pH by the putative pH sensor Dfg16 is essential for successful invasion of C. albicans into host tissue

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

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 baker's 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 Deltaals3 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 Deltaals3 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

The thyroxine inactivating gene, type III deiodinase, suppresses multiple signaling centers in Dictyostelium discoideum

Thyroxine deiodinases, the enzymes that regulate thyroxine metabolism, are essential for vertebrate growth and development. In the genome of Dictyostelium discoideum, a single intronless gene (dio3) encoding type III thyroxine 5′ deiodinase is present. The amino acid sequence of D. discoideum Dio3 shares 37% identity with human T4 deiodinase and is a member of the thioredoxin reductase superfamily. dio3 is expressed throughout growth and development and by generating a knockout of dio3, we have examined the role of thyroxine 5′ deiodinase in D. discoideum. dio3− had multiple defects that affected growth, timing of development, aggregate size, cell streaming, and cell-type differentiation. A prominent phenotype of dio3− was the breaking of late aggregates into small signaling centers, each forming a fruiting body of its own. cAMP levels, its relay, photo- and chemo-taxis were also defective in dio3−. Quantitative RT-PCR analyses suggested that expression levels of genes encoding adenylyl cyclase A (acaA), cAMP-receptor A (carA) and cAMP-phosphodiesterases were reduced. There was a significant reduction in the expression of CadA and CsaA, which are involved in cell–cell adhesion. The dio3− slugs had prestalk identity, with pronounced prestalk marker ecmA expression. Thus, Dio3 seems to have roles in mediating cAMP synthesis/relay, cell–cell adhesion and slug patterning. The phenotype of dio3− suggests that Dio3 may prevent the formation of multiple signaling centers during D. discoideum development. This is the first report of a gene involved in thyroxine metabolism that is also involved in growth and development in a lower eukaryote