Budi Tunggal

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal–fungal lineage after the plant–animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Dictyostelium transcriptional host cell response upon infection with Legionella.

Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co‐regulated genes, an enrichment of preferentially up‐ or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co‐incubation with Legionella hackeliae and L. pneumophilaΔdotA. One hundred and thirty‐one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host‐pathogen interactions and for further studies on the Dictyostelium response to Legionella infection.

Phylogeny-wide analysis of social amoeba genomes highlights ancient origins for complex intercellular communication

Dictyostelium discoideum (DD), an extensively studied model organism for cell and developmental biology, belongs to the most derived group 4 of social amoebas, a clade of altruistic multicellular organisms. To understand genome evolution over long time periods and the genetic basis of social evolution, we sequenced the genomes of Dictyostelium fasciculatum (DF) and Polysphondylium pallidum (PP), which represent the early diverging groups 1 and 2, respectively. In contrast to DD, PP and DF have conventional telomere organization and strongly reduced numbers of transposable elements. The number of protein-coding genes is similar between species, but only half of them comprise an identifiable set of orthologous genes. In general, genes involved in primary metabolism, cytoskeletal functions and signal transduction are conserved, while genes involved in secondary metabolism, export, and signal perception underwent large differential gene family expansions. This most likely signifies involvement of the conserved set in core cell and developmental mechanisms, and of the diverged set in niche- and species-specific adaptations for defense and food, mate, and kin selection. Phylogenetic dating using a concatenated data set and extensive loss of synteny indicate that DF, PP, and DD split from their last common ancestor at least 0.6 billion years ago.

Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting growth, development, phagocytosis and clearance and replication of Legionella pneumophila

Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell‐autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP‐tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9‐GFP‐containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9‐deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild‐type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.

STATc is a key regulator of the transcriptional response to hyperosmotic shock

BackgroundDictyostelium discoideum is frequently subjected to environmental changes in its natural habitat, the forest soil. In order to survive, the organism had to develop effective mechanisms to sense and respond to such changes. When cells are faced with a hypertonic environment a complex response is triggered. It starts with signal sensing and transduction and leads to changes in cell shape, the cytoskeleton, transport processes, metabolism and gene expression. Certain aspects of the Dictyostelium osmotic stress response have been elucidated, however, no comprehensive picture was available up to now.ResultsTo better understand the D. discoideum response to hyperosmotic conditions, we performed gene expression profiling using DNA microarrays. The transcriptional profile of cells treated with 200 mM sorbitol during a 2-hour time course revealed a time-dependent induction or repression of 809 genes, more than 15% of the genes on the array, which peaked 45 to 60 minutes after the hyperosmotic shock. The differentially regulated genes were applied to cluster analysis and functional annotation using gene GO terms. Two main responses appear to be the down-regulation of the metabolic machinery and the up-regulation of the stress response system, including STATc. Further analysis of STATc revealed that it is a key regulator of the transcriptional response to hyperosmotic shock. Approximately 20% of the differentially regulated genes were dependent on the presence of STATc.ConclusionAt least two signalling pathways are activated in Dictyostelium cells subjected to hypertonicity. STATc is responsible for the transcriptional changes of one of them.

31P NMR spectroscopy for in vitro viability testing of porcine pancreatic islets

The quality of pancreatic islets prepared by an intraductal pancreas collagenase perfusion technique was tested using three independent methods: 31P NMR spectroscopy, an insulin secretion test, and a staining method. The viability of pancreatic islet tissue was evaluated using the ratio of phosphate diester to phosphate monoester (PDE/PME) as a new criterion obtained by 31P NMR spectroscopy. According to this criterion, three types of tissue fragments could be characterized: vital (PDE/PME 0.5-0.9), damaged (PDE/PME less than 0.2), and necrotic (no PDE, no PME). The findings in the three different groups could be correlated to three trends of insulin secretion of the preparations following glucose challenge: good response to the glucose challenge, continuous decrease of insulin production, and no insulin secretion. We feel that 31P NMR spectroscopy offers a rapid and suitable method for classifying the viability of isolated pancreatic islets.

STUDIES ON THE REGIOSPECIFICITIES IN THE BINDING OF COMPLEX LIPIDS

Publisher Summary This chapter discusses the studies on the regiospecificities in the binding of complex lipids. A thorough understanding of the molecular events in the function of lipid requiring proteins, for example, lipid transporting particles such as serum lipoproteins or intrinsic membrane proteins, demands the detailed knowledge of the molecular basis of the association of these specific apoproteins with their naturally occurring lipid classes and species or if model systems are being studied the lipid species should mimic as closely as possible the naturally associated ones. 13C-NMR-spectroscopy can measure two parameters accurately: the longitudinal relaxation time T1 and the nuclear overhauser enhancement (NOE). Their functional dependence on the correlation times is substantially different.

The binding of lysolecithin to human serum high density apoprotein A-I. A13C-NMR study.

l-[14,15-H2;16-C]Palmitoyl-, 1-[9,10-H2 ;11-C]and 1-[9,10,12,13-H4;14-C]stearoyl-, 1-[9,10-H2 ;l l-C]oleoyl-, l-[9,10,12,13-H4;14-C]linoleoyl-sw-glycero-3phosphocholine and 1 -palmitoyl-sn-glycero-3phospho-[7V-CH3,CH3]choline have been prepared by chemical synthesis. They recombine with apolipoprotein A-I of human serum high density lipoproteins. This binding leads only to minor changes in circular dichroism with an increase of a-helicity </H) from 0.44 to 0.54 on the average. The lipid-apoprotein A-I-complexes remain stable during agarose chromatography. 60 70 lysolecithin molecules were bound to the apolipoprotein, approximately the number associating with the apolipoprotein A-I during equilibrium dialysis (70 lysolecithins per apolipoprotein A-I molecule).